Sarcoplasmic Reticulum Function in Smooth Muscle - Physiological ...

Sarcoplasmic Reticulum Function in Smooth Muscle 

SUSAN WRAY AND THEODOR BURDYGA 

Physiol Rev 90: 113–178, 2010; 

doi:10.1152/physrev.00018.2008. 

Department of Physiology, School of Biomedical Sciences, University of Liverpool, Liverpool, United Kingdom 

I. Introduction and Brief Historical Overview 114 

II. Structure and Location of Sarcoplasmic Reticulum in Smooth Muscle 116 

A. Introduction 116 

B. Electron microscopic studies 116 

C. Confocal imaging of the SR 117 

D. SR, caveolae, microdomains, and superficial buffer barrier 119 

E. SR and the mitochondria 120 

F. SR and the nucleus 121 

G. Lysosomes 121 

III. Sarco/Endoplasmic Reticulum Calcium-Adenosinetriphosphatase 122 


B. Enzyme type and activity 122 

C. SERCA structure and catalytic cycle 122 

D. SERCA isoforms and expression in smooth muscles 123 

E. Regulators of SERCA expression and activity 124 

F. Summary 125 

IV. Proteins Associated With the Sarcoplasmic Reticulum 125 


B. The SR membrane 125 

C. Phospholamban 126 

D. Sarcolipin 128 

E. Calsequestrin 128 

F. Calreticulin 129 

G. Triadin and junctin 130 

H. Summary 130 

V. Calcium Release Channels 130 


B. Ryanodine receptors 130 

C. Ip 3-sensitive Ca release channels 132 

D. Modulation of SR Ca release 133 

E. SR Ca leak 135 


VI. Luminal Calcium 136 


B. Fluorescent indicators for SR luminal Ca measurement 136 

C. Luminal Ca buffers 137 

D. Measurements 137 

E. Effects of SR Ca load 138 

F. Conclusions 139 

VII. Calcium Reuptake Mechanisms 139 


B. SERCA uptake of Ca and Ca homeostasis 139 

C. SERCA and Ca signaling and contractility 139 

D. SERCA Ca efflux and relaxation 140 

E. Store-operated Ca entry 140 

VIII. Common or Separate Calcium Pools 144 


B. One store with one type of receptor 144 

C. One store with two types of receptors 146 

D. Two stores, two types of receptors 146 

E. Role of Ca release channels and store type in Ca signaling 146 

www.prv.org 0031-9333/10 $18.00 Copyright © 2010 the American Physiological Society 

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114 SUSAN WRAY AND THEODOR BURDYGA 


IX. Elemental Calcium Signals From Smooth Muscle Sarcoplasmic Reticulum 148 

A. Ca sparks 148 

B. Ca puffs 152 

X. Sarcoplasmic Reticulum and Plasmalemmal Ion Channels 153 

A. Overview of Ca-activated ion channels 153 

B. BK channels 153 

C. SK and IK channels 154 

D. Ca-activated Cl channels 155 

XI. Sarcoplasmic Reticulum and Development and Aging 156 

XII. Gender 156 

XIII. pH, Metabolism, and Sarcoplasmic Reticulum Function 157 


B. pH 157 

C. Metabolism 158 

XIV. Mathematical Modeling 158 

XV. Conclusions and Future Directions 158 

Wray S, Burdyga T. Sarcoplasmic Reticulum Function in Smooth Muscle. Physiol Rev 90: 113–178, 2010; 

doi:10.1152/physrev.00018.2008.—The sarcoplasmic reticulum (SR) of smooth muscles presents many intriguing 

facets and questions concerning its roles, especially as these change with development, disease, and modulation of 

physiological activity. The SR’s function was originally perceived to be synthetic and then that of a Ca store for the 

contractile proteins, acting as a Ca amplification mechanism as it does in striated muscles. Gradually, as investigators 

have struggled to find a convincing role for Ca-induced Ca release in many smooth muscles, a role in controlling 

excitability has emerged. This is the Ca spark/spontaneous transient outward current coupling mechanism which 

reduces excitability and limits contraction. Release of SR Ca occurs in response to inositol 1,4,5-trisphosphate, Ca, 

and nicotinic acid adenine dinucleotide phosphate, and depletion of SR Ca can initiate Ca entry, the mechanism of 

which is being investigated but seems to involve Stim and Orai as found in nonexcitable cells. The contribution of 

the elemental Ca signals from the SR, sparks and puffs, to global Ca signals, i.e., Ca waves and oscillations, is 

becoming clearer but is far from established. The dynamics of SR Ca release and uptake mechanisms are reviewed 

along with the control of luminal Ca. We review the growing list of the SR’s functions that still includes Ca storage, 

contraction, and relaxation but has been expanded to encompass Ca homeostasis, generating local and global Ca 

signals, and contributing to cellular microdomains and signaling in other organelles, including mitochondria, 

lysosomes, and the nucleus. For an integrated approach, a review of aspects of the SR in health and disease and 

during development and aging are also included. While the sheer versatility of smooth muscle makes it foolish to 

have a “one model fits all” approach to this subject, we have tried to synthesize conclusions wherever possible. 

I. INTRODUCTION AND BRIEF 

HISTORICAL OVERVIEW 

For an excellent general historical overview of the 

sarcoplasmic reticulum (SR) and endoplasmic reticulum 

(ER), the recent review by Verkhratsky (728) can be 

consulted. For other general references to the SR (or ER), 

Ca homeostasis and SR/ER Ca-ATPase (SERCA), the following 

reviews are recommended: Laporte et al. (384), 

Pozzan et al. (573), Karaki et al. (336), Strehler and 

Treiman (665), Floyd and Wray (184), Rossi and Dirksen 

(597), Carafoli and Brini (106), Carafoli (105), and Endo 

(167). 

An internal store of Ca in smooth muscle cells was 

postulated following demonstrations of Ca remaining in 

the cells after immersion in Ca-free solutions. This followed 

older observations that contractile responses, 

which were known to be Ca dependent, could continue 

for varying periods in Ca-free solutions, and that if external 

[Ca] had been elevated before the switch to Ca-free 

Physiol Rev VOL 90 JANUARY 2010 www.prv.org 

solution, this period was extended (278). Although we 

now have a more sophisticated view of Ca handling and 

Ca sensitivity (e.g., Refs. 13, 201, 271, 612, 769), which 

could also account for contraction in Ca-free solutions, 

the conclusion reached, i.e., there must be an internal Ca 

store, was correct. Electron microscopy (EM) of smooth 

muscle revealed a membranous system of tubules and 

sacs, the SR, that was both close to the periphery and 

caveolae as well as deep within the cell. Close apposition 

of the SR to mitochondria and the nucleus was also noted. 

This membrane system excluded extracellular markers 

such as ferritin and horseradish peroxidase, but was contiguous 

with the lumen of the nuclear envelope. Although 

originally described as sparse, following improvements in 

fixation and microscopy, these earliest accounts were 

replaced with terms such as “well developed” and “rich 

reticulum.” It is now appreciated that the SR’s distribution, 

amount, and shape is not only smooth muscle specific, 

but also changes with physiological stimuli and developmental 

stage and in disease.

One of the earliest suggestions that the SR in smooth 

muscle was the major intracellular Ca store and that it 

acts as in striated muscle was made by Giorgio Gabella 

and was based on EM studies (194–196). In the same year, 

Avril and Andrew Somlyo demonstrated that strontium, 

used as a surrogate for Ca, was accumulated in the SR of 

smooth muscle (aorta and pulmonary artery), and therefore, 

it followed that the SR could store Ca and be an 

activator of contraction (656). Many further important 

contributions were made by the Somlyos, including the 

demonstration using electron-probe X-ray microanalysis 

that [Ca] decreased in the SR following agonist stimulation 

(e.g., Refs. 95, 371) and that Ca cycling to and from 

the SR occurred during contraction and relaxation (69). 

The demonstration of a rich reticular formation in 

smooth muscle renewed interest in examination of the 

role of the SR in excitation-contraction (E-C) coupling. 

Much of this work was looking for or assuming similarity 

of mechanisms to those of striated muscle. It is only in the 

last decade that the distinct role of the SR in smooth 

SMOOTH MUSCLE SR 115 

muscle has been elucidated. Work from Nelson’s group 

(70, 306, 507) and others (76, 682) showed that local Ca 

release from the SR can control plasma membrane excitability. 

We built on this and showed that there is a fundamental 

relationship between SR Ca release and the 

action potential in ureteric smooth muscle (87). 

We now appreciate that the SR can have the following 

roles as demonstrated in Figure 1: 1) contributing to 

Ca homeostasis by maintaining low resting [Ca], via action 

of the SERCA; 2) restoration of [Ca] and relaxation 

following stimulation, via the action of SERCA; 3) augmenting 

contraction through release of Ca and producing 

or modifying global Ca signals in response to agonist 

stimulation; 4) modulating membrane excitability through 

activation of Ca-sensitive ion channels; 5) contributing to 

the efficacy of plasma membrane Ca extrusion mechanisms 

by vectorially releasing Ca to them; 6) contributing to the 

maintenance of signaling microdomains around the plasma 

membrane and other organelles; and 7) influencing development, 

aging, and the health of smooth muscle tissues. 

FIG. 1. Functions of the SR in smooth muscle. A cartoon to demonstrate the functions of the SR. 1: Contributing to Ca homeostasis and 

maintenance of low resting level of intracellular [Ca] via sarco/endoplasmic reticulum Ca-ATPase (SERCA) activity. 2: Contributing to relaxation 

of the smooth muscle cell by taking up Ca via SERCA. 3: Contributing to Ca signals and contraction by Ca release via IP 3 receptor (IP 3R)- or 

ryanodine receptor (RyR)-gated Ca release channels and Ca puffs and Ca sparks. 4: Contributing to excitability via Ca-activated K and Cl channels. 

5: Facilitation of Ca efflux on plasma membrane Ca-ATPase (Ca pump) or Na/Ca exchanger, via vectorial release of Ca. 6: Contributing to subcellular 

microdomains and organellar Ca homeostasis via Ca uptake and release between organelles. 7: Correct SR function needed for normal development 

and health, and SR functional change is associated with disease and aging. 

Physiol Rev VOL 90 JANUARY 2010 www.prv.org


This is an extensive job description for an organelle only 

occupying 5% of the cell. We highlight these physiological 

and pathological functions inter alia. 

Our approach in this review has been to synthesize a 

holistic view of how the SR works in smooth muscle and 

then discuss how these components are put together in 

different smooth muscles leading to tissue specificity. 

There is a daunting amount of literature in some areas, 

not all of which can be cited, and for this we apologize. 

There are areas of the literature where clarity is slowly 

emerging and others where unfortunately it remains elusive. 

Thus the conclusions we make to clarify, summarize, 

and hopefully provide insight will include a degree of 

speculation and author bias, and perhaps not all will stand 

the test of time. We offer them as discussion points in the 

quest of understanding SR function in smooth muscle. We 

have also focused wherever possible on literature obtained 

on intact tissues, intact muscle strips, and native 

smooth muscle cells, i.e., freshly dissociated and isolated. 

The effects of cell culturing on almost every aspect of 

smooth muscle E-C are so well known and long standing 

that investigators ignore them at their peril (116, 533). 

II. STRUCTURE AND LOCATION OF 

SARCOPLASMIC RETICULUM IN 

SMOOTH MUSCLE 

A. Introduction 

Although it may sound out-moded to be concerned 

with the structure and location of the SR, recent work, for 

example, on membrane microdomains and SR compartmentalization, 

make it important that we have a clear 

factual understanding of SR structure and distribution, to 

better judge what is feasible both within the SR and in its 

relation to other organelles and the plasma membrane. 

B. Electron Microscopic Studies 

Much of our structural information concerning smooth 

muscle has come from EM studies. These studies addressed 

the size of the SR, usually compared with striated 

muscle and in terms of cell volume, and SR location 

within myocytes. By revealing these features EM made a 

significant contribution not just to the understanding of 

SR structure, but also to E-C coupling in smooth muscle. 

Before fluorescent probes sensitive to ion concentration 

were developed, electron X-ray microprobe analysis provided 

spatial information about the elemental composition 

of different areas in the myocytes, in relaxed and 

contracted stages. Determinations of SR Ca content are 

considered in section VI. 

Consistent between different workers and smooth 

muscles is the location of the SR: close to, but not touch- 


ing the plasma membrane. The SR was noted to encircle 

the invaginations of the surface membrane, the caveolae. 

Sheets of reticulum running along the longitudinal axis of 

the cell are a feature of all the tissues examined, e.g., 

vascular (152), airway (375), gastrointestinal tract and 

bladder (197), human blood vessels (677), developing 

blood vessels (693), and portal vein (69). The SR is described 

as peripheral (or superficial) when it is close to 

the plasma membrane and central (or deep) when it is 

located away from this membrane. The peripheral SR has 

been associated with Ca homeostasis, local Ca release, 

and interactions with plasma membrane ion channels and 

hence excitability, whereas the central SR has been suggested 

to be more directly involved in contraction by 

supplying Ca to the myofilaments. This is discussed further 

in section V. 

Given the different activity and mechanisms of excitation 

between phasic and tonic smooth muscles, differences 

in the amount and localization of the SR have been 

sought. In vas deferens, a phasic muscle, the SR at EM 

level was described as being located predominantly at the 

periphery, whereas the tonic aorta had proportionally 

more SR in a central location (515). This paper also noted 

a central distribution for the tonic pulmonary artery and 

trachealis. 

The smooth muscle cell’s interior is packed with myofilaments 

and intermediate filaments. It is estimated that 

they, along with associated dense bodies, occupy up to 90% 

of the cell’s volume. The intracellular organelles share the 

remaining volume (195). Although the earliest EM accounts 

described the SR of smooth muscles as being poorly developed 

(e.g., Refs. 194, 548, 577), this view gradually changed 

and is borne out by recent studies using confocal microscopy 

(described later). In one of the best comparative studies, 

Devine et al. (152) compared rabbit smooth muscles and 

reported that 5% of the cell volume was accounted for by 

the SR in aorta and pulmonary artery and that this fell to 

2% in mesenteric vein and artery and portal vein. These 

findings also correlated with the varying ability of these 

preparations to continue contracting in zero-Ca solutions, 

i.e., longer in aorta than mesenteric and portal vein. However, 

when low values for SR volume were reported in some 

(vas deferens and taenia coli, 1.7%) but not all (uterus, 7%) 

(596) phasic smooth muscles, the correlation with contraction 

time in zero Ca fell away. Somlyo’s group pointed out 

that osmium fixation can damage smooth muscle SR and 

that this could have contributed to reports of being sparse 

(652). Thus the view gradually changed from “However, we 

must bear in mind that a sarcoplasmic reticulum, as we 

understand the structure from skeletal muscle work, is definitely 

not present in smooth muscles, and it seems most 

unlikely that the minute fraction of reticulum present in 

some smooth muscles is capable of modulating calcium 

levels” (275) to “Our major conclusion is that there is in 

mammalian smooth muscles a sarcoplasmic reticulum that,

while being variable in different smooth muscles, is sufficiently 

well developed and organised.......tofunction as a 

Ca store in the process of excitation-contraction and inhibition-relaxation 

coupling” (152). 

Notwithstanding the fact that smooth muscle SR is 

well developed, its volume is less than that of striated 

muscles (10% of cell volume). Therefore, there are reasonable 

grounds to question whether the SR in smooth 

muscle can have the same role as in striated muscles, 

particularly with respect to it being the amplifier of a 

small Ca entry signal to provide the necessary rise of [Ca] 

required for contractile activation by Ca-induced Ca release 

(CICR). This questioning is also suggested by the 

generally smaller SR volume in visceral smooth muscles, 

where Ca entry is predominant, compared with vascular 

smooth muscles, where SR Ca release is more prominent. 

It seems to us that the pendulum swung from initially 

dismissing the SR’s role in modulating Ca signals 

and CICR in smooth muscle to viewing the SR as playing 

a very similar role to that occurring in striated 

muscles. We are perhaps now able to readjust the 

pendulum and see more clearly that smooth muscle SR 

is important to E-C coupling, but in its own distinctive 

way. 

C. Confocal Imaging of the SR 

Confocal imaging can show the extent and three-dimensional 

distribution of the SR in smooth muscle and identify 

Ca release and uptake sites, as well as allowing changes in 

its Ca content to be determined. Following from the development 

of Ca-sensitive fluorescent indicators to monitor 

cytoplasmic [Ca] changes, efforts were directed at developing 

similar probes for intracellular organelles. One of the 

earliest methods was to use dicarbocyanine dye, DiOC6. 

This has been used in a variety of cell types as a label for 

SR/ER (696). Although some authors have noted its specificity 

for the ER (605), others considered that DiOC6 probably 

labels other intracellular membranes (696). Another 

approach to SR imaging is to target specific components 

associated with the SR membrane using antibodies, e.g., 

against ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate 

(IP 3) receptors (IP 3Rs), or SERCA pumps. The Ca within 

the SR can also be targeted by fluorescent indicators. If 

these can be preferentially loaded into the SR lumen (as 

opposed to other organelles and/or cytoplasm), then 

these too can provide a picture of the SR, assuming Ca is 

not compartmentalized within the lumen. Another useful 

range of markers for the SR/ER has been developed 

from ceramide analogs bearing the fluorophore boron 

dipyrromethene difluoride (BODIPY) (540). To confer 

SR organellar specificity, this backbone is added to 

fluorescent ryanodine or thapsigargin (an inhibitor of 

SERCA) (635). 



Techniques of molecular biology to specifically target 

the SR proteins and fuse them with fluorescent proteins 

such as aequorin can also be used to image the SR, 

although these are rarely suitable or successful in smooth 

muscle cells. Unlike fluorescent indicators that can be 

used directly in intact tissues or single cells, using genetically 

manufactured SR-targeted probes requires transfection 

of the recombinant proteins in the cells, and hence 

culturing. This poses two problems for smooth muscle 

studies. First, the well-appreciated problems of phenotypic 

change that occur with even short periods of culturing. 

Second, conventional transfection has proven difficult 

in most smooth muscles. However, methods based 

on organ-culturing and nonviral delivery, such as reversible 

permeabilization, get around both these difficulties. 

They have been shown to work well in some smooth 

muscles, to introduce plasmids, antisense oligonucleotides, 

and small interfering RNAs (siRNA), e.g., focal adhesion 

kinase depletion with antisense intracheal myocytes 

(690), calponin depletion with antisense in aorta 

(311), Rho knockdown in cerebral arteries (131), and 

siRNA against arterial TASK-1 K channel (234). These 

methods have added to our understanding of the complex 

signaling pathways in smooth muscle, e.g., downregulation 

of profilin with antisense oligonucleotides was shown 

to inhibit force in carotid smooth muscle (691), and transfection 

with plasmids encoding a paxillin mutant was 

employed to explore its role in tension development in 

tracheal myocytes (529). However, these and related 

methods have been little used to target the SR and its 

regulators. There appears to be only one study, which 

used adenoviral vectors and targeting of apoaequorin in 

native tail artery cells (588). This paper should also be 

consulted for an informed discussion and evaluation of 

methods. The use of fluorescent probes to measure luminal 

SR content is discussed in section VIB. 

Several studies using fluorescent BODIPY linked to 

ryanodine or thapsigargin and DiOC6 have been used in 

native smooth muscle: stomach (753), portal vein (221), 

and uterus (635, 636). Recently, the value of BODIPY 

ryanodine has been questioned as it may not block RyR 

activity, perhaps due to the presence of the fluorophore 

(231, 455). Using fluorescently labeled thapsigargin and 

ryanodine in uterine myocytes, we found that both 

were distributed nonhomogeneously, but in the same 

pattern; there was abundant labeling around the nucleus 

and peripherally. The close apposition of the SR 

to plasma membrane leads to the cell shape being 

clearly outlined. 

Figure 2 shows examples of the SR in live smooth 

muscle cells, imaged using a variety of techniques. 

The SR in uterine myocytes can be loaded with the 

low-affinity Ca indicator mag-fluo 4. With the use of this 

area of increased [Ca], “hot spots” can be seen. This 

distribution was similar to that of SERCA and RyRs


FIG. 2. Morphological and functional demonstration of the SR in smooth muscle. A and B: in freshly isolated smooth muscle cells from guinea 

pig ureter, 3-dimensional live staining of the SR with BODIPY FL-X ryanodine (A) and 2-dimensional pseudo-color image of the SR loaded with 

Mag-fluo 4 in a -escin chemically skinned myocyte (B). C and D: pseudo-color images of fluo 4-loaded myocyte showing a spontaneous Ca spark 

(C) and a Ca wave (D) induced by 10 mM caffeine. Images were obtained on Ultraview confocal system with Hamamatsu CCD camera. (From T. 

Burdyga, L. Borisova, and S. Wray, unpublished work.) 

(635). With the use of three-dimensional image reconstruction 

techniques, a network of interconnecting, spirally 

shaped tubules, mainly running along the longitudinal 

axis of the cell, can be seen. This is reminiscent of 

earlier descriptions in fixed tissue with EM. It is notable 

that when uterine cells in culture were studied with 

antibodies to IP 3Rs and RyRs, a different distribution 

was found; they had an homogeneous (diffuse) distribution 

and hot spots detected by fluo 3FF, were scattered 

throughout the cytoplasm (792). In stomach myocytes 

from Bufo marinus, Steenbergen and Fay (661) 

used mag-fura 2 and found the SR to be distributed in a 

punctate manner, but again with a concentration peripherally 

and around the nucleus. Other studies describing 

more or less similar structures, i.e., interlacing 

tubules, sacs, and cisternae, and location of the SR, 

include cultured A7r5 (706), mesenteric artery (214), 

and portal vein (221). It appears that when different 

fluorescent indicators are used, the same pattern of SR 

distribution in smooth muscle myocytes is observed. 

As found with EM studies, these images of the SR 

show it to be abundant around mitochondria and continuous 

with the nucleus (221, 635). There may be more 

SR in a peripheral location in phasic muscles (portal 

vein and uterus), compared with mesenteric artery and 

A7r5 cells, again consistent with suggestions from EM 

studies. Immunostains and immuno-EM of RyRs in vas 

deferens and urinary bladder myocytes also revealed a 


largely discrete, peripheral distribution for the SR, assuming 

that the RyRs represent the entire SR (394, 525). 

Ohi et al. (525) also noted colocalization with largeconductance, 

Ca-activated K (BK) channels, discussed 

further in section XB. In a study of aortic SR, Lesh et al. 

(394) found a patchy distribution of RyR throughout the 

cell but absent from the nuclear region. The occurrence 

of frequent discharge sites or hot spots on the SR and 

their relation to Ca release events are discussed in 

section IX. 

In summary, recent data obtained by live staining of 

native smooth muscle cells have added confidence to 

earlier data on SR structure and distribution obtained 

with EM. There is no certainty about what the distinction 

between peripheral and central SR means in terms of SR 

components or functional consequences for any particular 

smooth muscle. Perhaps because of the considerable 

interest in local Ca signals from the SR and their relation 

to plasma membrane ion channels, the literature 

tends to emphasize the peripheral SR, and few studies 

have addressed the role of central SR. All phasic 

smooth muscles examined with confocal techniques 

appear to have predominantly peripheral SR. As well as 

the interactions between the SR and plasma membrane 

and caveolae, there is also interest in elucidating the 

functional importance for smooth muscles of having 

their SR enveloping mitochondria and appearing con-

tinuous with the nuclear envelope, and these aspects 

are discussed next. 

D. SR, Caveolae, Microdomains, and Superficial 

Buffer Barrier 

1. Introduction 

An excellent review of Ca microdomains in smooth 

muscle was provided recently by McCarron et al. (451). 

This topic in other cell types has also been recently 

reviewed by Rizzuto and Pozzan (592). 

Many fine structural studies have noted the close 

(15–30 nm) apposition of the SR to the plasma membrane, 

particularly around caveolae (565, 677). The close contact 

between caveolae and the SR membrane (195, 197) has 

led some investigators to consider if caveolae also possess 

Ca release mechanisms, i.e., IP 3R or RyRs. In mouse 

ileal smooth muscle, Fujimoto et al. (191) reported IP 3 

type 1 receptors on caveolae. This observation has, however, 

never been confirmed to the best of our knowledge. 

It has been reported that there is a regularity in the close 

apposition of the two membranes (565), leading to the 

suggestion that there may be a structural anchor between 

the two (688). However, it is generally concluded from 

EM studies that the foot structures composed of clusters 

of RyR reaching to the plasma membrane and found in 

skeletal muscles are not a feature of smooth muscle (652), 

although a case for this has been made by some (490). No 

structural rearrangements of the plasma-SR membranes 

have been observed with stimulation (although this has 

been reported for SR-mitochondrial junctions, see sect. 

IIE; Ref. 140). 

2. Caveolae 

That the membrane invaginations that are caveolae 

approach very close to the SR membrane remained 

largely a structural note of little interest to physiologists, 

until it became clear that caveolae were specialized regions 

of the plasma membrane and could facilitate cell 

signaling and transduction pathways (85). It is now appreciated 

that the protein caveolin is responsible for the 

invagination of the surface membrane and that caveolae 

are a form of membrane lipid rafts. Lipid rafts, rich in 

cholesterol (and sphingomyelin), are less fluid than surrounding 

areas (hence “raft”) and have certain proteins 

concentrated or excluded from them, in a dynamic fashion. 

For a recent review of caveolae in smooth muscle, 

see References 298 and 520. That these biochemical specializations 

of the raft/caveolar membrane are functionally 

important has now been shown in several smooth 

muscles by disrupting caveolae, usually by extracting cholesterol 

[rat uterus (648) and ureter (26), human myometrium 

(798), blood vessels (155), and bladder (273)]. A link 


with obesity, through elevated cholesterol and other 

smooth muscle pathologies, has recently been demonstrated 

(186, 797). 

A variety of techniques have shown that BK channels 

localize in caveolae (26, 80, 634). As will be discussed in 

section XB, BK channels are targeted by local SR Ca 

release, Ca sparks, and their activation can cause hyperpolarization 

and therefore changes in excitability in 

smooth muscles. Thus any disruption of the close link 

between the SR and caveolae would be expected to 

have functional significance (406). The Na-K-ATPase 

(Na pump) is also preferentially located in caveolae 

(491). As this has profound effects on setting ionic 

gradients for Na and K and thereby also membrane 

potential (11), anything which modulates its activity 

will be expected to influence smooth muscle excitability 

(e.g., Refs. 56, 183, 399). Recently, it has been shown 

that the 2 isoform may directly regulate Ca transport 

and signaling, due to its spatial proximity to the Na/Ca 

exchanger (NCX) (184, 326, 445). In vascular smooth 

muscles, a link between the 2 Na pump subunit and 

relaxation pathways has been demonstrated (801). As 

with the BK channels, these components of ionic homeostasis 

and Ca signaling are located in caveolae and 

close to the SR (57). From these close appositions of SR 

and caveolae, and congregation of signaling components 

associated with (but not exclusively limited to) 

Ca regulation, it has been proposed that the SR in this 

region is part of a specialized signaling complex, sometimes 

referred to as a signalosome (e.g., Refs. 280, 423). 

3. Microdomains 


Although it is unlikely that there is a structural anchor 

between caveolae and SR, or that there is a Ca 

release function in caveolae, there is evidence that these 

regions in the smooth muscle myocytes form a functional 

unit affecting Ca signaling. This microdomain arises between 

the plasma membrane and SR and has the conditions 

and components necessary to 1) have higher [Ca] 

than the bulk cytoplasm, 2) vectorially release Ca, and 

3) act as a barrier or buffer to the free diffusion of Ca in 

this domain. 

The existence of Ca microdomains in smooth muscle, 

where the [Ca] may be several orders of magnitude 

greater than in the bulk cytoplasm, overcomes objections 

to the NCX and Ca-activated membrane channels having 

too low a Ca affinity to contribute to Ca homeostasis 

during physiological processes. If [Ca] can also change 

rapidly in these microdomains, then those proteins with 

high association constants will be activated preferentially, 

even when they have similar K d values (451). The relatively 

slow diffusion constant of Ca in smooth muscle 

cytoplasm 2.2 10 6 cm 2 /s (37), compared with water, 

will contribute to the maintenance of the microdomain.


Many of the features that led to the postulation of a 

specialized space and environment between the SR and 

plasma membrane will also hold for regions between the 

SR and other organelles. 

4. Superficial buffer barrier 

Van Breemen and colleagues (713, 715) hypothesized 

in the 1980s that there is a subplasmalemmal domain that 

is functionally separated from the cytosolic constituents 

by the superficial SR and that this would be important in 

modulating Ca signaling and force production in smooth 

muscle. This peripheral SR, being so close to the sites of 

Ca entry, would act to scavenge and buffer extracellular 

Ca entry. This buffering ability would be overcome during 

agonist stimulation or depolarization but could act to help 

maintain resting [Ca] low between stimuli. Following 

stimulation, the superficial SR will release Ca back to the 

plasma membrane Ca extrusion mechanisms. Because of 

the difficulty of directly measuring [Ca] in microdomains, 

direct experimental support for them was lacking. However, 

with the advent of near-membrane Ca indicators 

(170, 809), evidence supporting or consistent with the 

superficial buffer barrier role of the SR in smooth muscles 

has been provided in a variety of preparations [vascular 

(587, 713), gastric (754), bladder (789), uterus (631, 793)]. 

The sites on the SR of Ca release and uptake during these 

processes may be functionally and spatially separate 

(635). The estimates of subplasmalemma [Ca] have been 

between 10 and 50 M [gastric (170), colonic (77), arterial 

(809)], and the elevations of [Ca] last up to 100–200 ms 

(339). 

Inhibition of SERCA increases bulk Ca signals produced 

in response to agonists, which is consistent with 

the superficial buffer barrier hypothesis. Bradley et al. 

(77) calculated that the buffer accounted for binding of 

50% of the Ca entering colonic myocytes. Recently however, 

this group has argued that the SR should be viewed 

more as a “Ca trap” and that SERCA activity cannot 

curtail the rise of cytosolic [Ca] but rather contributes to 

its decline when efflux ends (451; see also Ref. 635). 

Although not generally considered in accounts of 

smooth muscle microdomains, a role for calmodulin in 

contributing to localizing Ca signals has been proposed 

(764). This arises from the finding that, even at low [Ca], 

a specific “contractile” pool of calmodulin with zero or 

two of its four Ca-binding sites occupied is tightly bound 

to the contractile machinery. Local changes of [Ca] near 

the myofilaments could then activate this specific calmodulin 

pool by saturating the four Ca-binding sites to activate 

myosin light-chain kinase (MLCK) and cause contraction, 

while more distant local [Ca] changes need not 

be associated with contraction (764). 

E. SR and the Mitochondria 


Both EM and confocal imaging studies have shown 

the SR and mitochondrial membranes to be in very close 

proximity (175, 677). The distance between them may be 

as small as 10 nm (515). As with caveolae, the SR may 

appear to engulf mitochondria, and thereby create a specialized 

space for Ca signaling, discussed below. Although 

close association between ER and mitochondria has been 

described in other cell types (489, 561), few have reported 

the almost complete enveloping, which appears to be a 

feature of smooth muscles. This aspect has been examined 

in detail at the EM level by Dai et al. (140) in pig 

tracheal muscle. At rest, all (99.4%) mitochondria were 

within 30 nm of SR membrane, and the average distance 

was 22 nm. The majority (82%) of mitochondria were 

completely encircled or enwrapped by the SR network. 

The regions of close contact lacked the regular spacing 

sometimes seen between SR and plasma membrane, but 

specific anchors have been suggested in other cell types 

(175, 241). 

What makes the above data particularly interesting is 

that the association of mitochondria and SR changed with 

acetylcholine (ACh) stimulation. The number of mitochondria 

surrounded by SR fell to 12%, and fewer were in 

close contact. The authors suggest the SR unwraps from 

mitochondria with stimulation and extends more into the 

cytoplasm. These structural arrangements between SR 

and mitochondria have been reported in Madin-Darby 

canine kidney (MDCK) II cells, but it is elevated [Ca] that 

keeps the close association and between them and low 

[Ca] causes dissociation (735). Lateral movements of mitochondria 

relative to the SR of up to 3 m/s have been 

reported in neuroblastoma cells (405). 

1. Mitochondria and smooth muscle Ca signaling 

There is now a large amount of literature on mitochondrial 

Ca signaling and the interactions between the 

SR and mitochondria. It is beyond the scope of this review 

to assess all the literature on mitochondrial Ca signaling, 

but we will give an account of recent data investigating 

SR-mitochondrial signaling in smooth muscle. The following 

reviews provide a good perspective on this area (296, 

451, 565). 

Mitochondrial Ca uptake has enjoyed a resurgence of 

interest (53) largely accounted for by new techniques 

allowing for monitoring their role in Ca signaling and the 

weight of accumulated evidence showing that Ca uptake 

occurs during physiological conditions (e.g., Refs. 49, 159, 

590). Due to the low affinity of their Ca transporter (46), 

the capacity of mitochondria to rapidly accumulate Ca 

was considered only relevant during pathologically high 

[Ca]. That there could be microdomains of high [Ca] 

around mitochondria, to which the SR (or ER) would

contribute, removed the “problem” of the mitochondrial 

transporter having such a low affinity for Ca (591). 

In smooth muscles, the following studies have shown 

that mitochondria can contribute to Ca signaling (114, 

156, 454, 488, 515, 589). Elevations of mitochondrial [Ca] 

with stimulation have been demonstrated in pulmonary 

arterial cells and considered to curtail the cytosolic [Ca] 

increases (157, 488). If the mitochondrial membrane potential 

(usually about 180 mV with respect to cytoplasm) 

is dissipated to disable the mitochondria, the decay 

of the cytosolic Ca transient was slowed in myocytes 

from portal vein (225), tail artery (675), pulmonary artery 

(157), and cultured aortic cells (678). Although not extensively 

studied in smooth muscles so far, it may be that 

mitochondrial uptake of SR Ca is a mechanism of promoting 

maximal Ca release from the SR, perhaps by removing 

Ca-induced feedback on the SR Ca release (128, 

603, 675). In their study of anococcygeus smooth muscle, 

Restini et al. (589) describe cross-talk between the SR and 

mitochondria and show EM figures of the close contact 

between the two. Interactions have also been found with 

smooth muscle mitochondria when SR Ca release is 

through RyR (124, 603). These studies found that when 

mitochondria were inhibited there was a slowing of the 

rate of Ca release and decay of the Ca transient, and a 

decrease in Ca sparks. The work of Chalmers and McCarron 

(114) suggests that in colonic myocytes mitochondrial 

activity promotes Ca release through IP 3Rs and that some 

mitochondria are situated particularly close to these receptors. 

They also addressed the important issue of 

whether the Ca entry into mitochodria during smooth 

muscle activity causes significant mitochondrial inner 

membrane depolarization, which would impair their function 

and ATP production. They found no change during 

normal Ca transients but suggest depolarization may occur 

during repetitive Ca oscillations or oxidative stress. 

Further work is required in smooth muscles to determine 

if the above data can be generalized to other smooth 

muscles, and if cross-talk or functional outcomes differ, 

depending on whether Ca is released via RyR or IP 3R. 

More work is also needed to establish the effects of 

mitochondria on smooth muscle local and global Ca signals. 

Finally, there is emerging data indicating that mitochondria 

may also play a role in pacemaking in smooth 

muscle cells (345, 374, 741, 742), which deserves further 

attention (see also Refs. 160, 456, 503). 

F. SR and the Nucleus 

The nucleus and the SR are commonly associated in 

smooth muscle cells. For example in aorta, Lesh et al. 

(394) describe the SR forming a continuous network and 

being contiguous with the outer nuclear envelope. Over 

the last decade, the evidence has shown that the nucleus 


can regulate its own [Ca] and that the nuclear pores are 

regulated (208, 353, 436). Furthermore, identification of 

IP 3Rs, RyRs, and SERCA on the nuclear membrane has 

added to our understanding of how it may generate and 

regulate its Ca signals (2, 276, 415). 

The SR may contribute to Ca-regulated gene transcription, 

via CREB or NFAT (31, 110, 217, 662). The term 

excitation-transcription coupling has been used to highlight 

that the changes in [Ca] occurring during E-C coupling 

will also be involved in regulating gene expression 

and phenotypic modulation (733). 

There is only a sparse literature on interactions between 

the SR and nucleus in smooth muscles. In cultured 

aortic cells using BODIPY-RY and thapsigargin, Abrenica 

et al. (3) reported a discrete localization of SERCA and 

RyR perinuclearly and suggested that they could contribute 

to regulating nucleoplasmic [Ca]. In turn, a role for the 

nucleus as a Ca sink was also suggested by the authors. In 

their study of Ca sparks in portal vein myocytes, Gordienko 

et al. (221) reported that the majority of spark 

initiation sites were within 1–2 m of the nuclear envelope 

(mitochondria were not particularly associated with 

sparks in this study). The frequency of sparks in these 

perinuclear regions often increased to produce a Ca wave 

and contraction. 

In cultured aortic (A7r5) cells, Missiaen et al. (482) 

noted Ca release and uptake in a thapsigargin-insensitive 

and nonmitochondrial store, which could have been the 

Golgi or nucleus. However, there seems to be little or no 

information on such interactions in native smooth muscles, 

so nothing further can be said at present. 

G. Lysosomes 


Interest in lysosomes as Ca storage organelles was 

stimulated by the finding that in sea urchin eggs a recently 

identified new Ca mobilizer, nicotinic acid adenine dinucleotide 

phosphate (NAADP), was stored in organelles 

equivalent to lysosomes in mammalian cells (126). 

NAADP is discussed further in section V. In smooth muscle, 

a close association between the SR and perinuclear 

lysosomes has been shown (350, 650), and this region is 

described as a “trigger zone.” Lysosomes are acidic organelles 

dependent on vacuolar H-ATPase activity. Drugs 

that inhibit the transporter, such as bacliomycin, have 

therefore been used to determine the importance of 

this Ca store to cells in which this type of Ca mobilizer 

has been found [myometrial (650), pulmonary (61), and 

coronary myocytes (800)]. In these studies a decrease 

in the Ca signal to agonists (endothelin-1, histamine) 

was found. To date, no studies in smooth muscle have 

tried to assess the size of the lysosomal Ca pool or 

looked at the dynamics of its interaction with the SR. It 

appears at least in some cells that the NAADP release


can trigger SR Ca release from RyRs, but not IP 3R, 

suggesting a clustering of RyRs with the release channels 

on the lysosomes (61). 

III. SARCO/ENDOPLASMIC RETICULUM 

CALCIUM-ADENOSINETRIPHOSPHATASE 


SERCA is the major protein associated with the SR. It 

is a P-type ATPase responsible for transporting Ca into 

the SR at the expense of ATP. Calcium binding proteins 

enable this process to continue with an SR free ionized Ca 

content of 500 M, only 0.5–1% of the total Ca stored 

in the SR (463). This sequestering of luminal Ca also 

means that inhibitory feedback of Ca on the ATPase is 

minimized. Auxiliary proteins, most notably phospholamban, 

as well as metabolites and pH, regulate SERCA activity. 

Several isoforms of SERCA exist, and expression 

varies between smooth muscles. 

Functionally SERCA creates an internal Ca reservoir. 

As a by-product of this activity it will also contribute to Ca 

homeostasis and basal [Ca] increases within the cytoplasm 

when its activity is inhibited. SERCA may be considered 

a Ca buffer, transferring bound Ca from the cytosol 

to the lumen of the SR (105). SERCA also functions 

to expedite the removal of Ca from the cytoplasm following 

stimulation and thereby contributing to relaxation. 

Pharmacological inhibitors of SERCA allowed progress to 

be made in understanding its structure and roles in 

smooth muscle, and fluorescent indicators have permitted 

the monitoring of its effects on intracellular [Ca] and 

direct measurements of luminal [Ca]. Molecular studies 

have given a precise understanding of many aspects of 

how SERCA structure relates to function and the consequences 

of genetic mutations. The structure of SERCA 

will be described next, followed by its catalytic cycle and 

then isoform types and function in smooth muscle. Features 

of its regulation and function are shown in Figure 3. 

B. Enzyme Type and Activity 

Active transport is required to drive Ca into the SR. 

This is brought about by the P-type (ion-motive) ATPase 

of the SR membrane, SERCA, which is in the same enzymatic 

class as the Na-K-ATPase and the plasma membrane 

Ca-ATPase (PMCA). Amino acid sequence similarity 

to the Na pump is more extensive than with PMCA. 

Recent studies using chimeras have begun to elucidate 

how SERCA and PMCA are targeted to their respective 

membranes (36, 509); SERCA has an ER retention signal 

close to the NH 2 terminus, which is cytoplasmic facing. 

SERCA transports two Ca for every ATP hydrolyzed. This 


FIG. 3. Summary of SERCA functions and regulation. SERCA is 

known to be regulated by changes in Ca, pH, ATP/ADP ratio, phospholamban, 

and hormones, e.g., thyroxine. SERCA activity plays a role in Ca 

homeostasis, establishment and maintenance of Ca microdomains, determining 

luminal [Ca], buffering Ca, transporting Ca and countertransporting 

H , regulating IP 3R and RyR Ca release, and relaxation via Ca 

uptake. 

also distinguishes it from PMCA, which has a 1:1 stoichiometry 

(290). For further details on the structure of 

SERCA and its similarity to other type ATPases, see the 

following reviews (106, 676). 

The demonstration that SR membrane fractions 

could accumulate Ca and hydrolyzed ATP was the first 

evidence of SERCA (161). Such investigations were 

helped by the abundance of SERCA in striated muscle SR, 

as were the subsequent structural studies. The uptake and 

release of Ca into the SR by SERCA occurs as a cycle of 

reactions and conformational changes. 

C. SERCA Structure and Catalytic Cycle 

The following account is taken largely from several 

excellent reviews (487, 573, 777, 791). The pump was purified 

in 1970 (428) and the crystal structure resolved in 2000 

by Toyoshima (701), and the conformational changes during 

Ca release were elucidated 2 years later by the same 

group (702). The structural determination of SERCA (isoform 

1a) was the first for any active transporter. The 

SERCAs are complex proteins with single-stranded, 

polypeptide chains of 110 kDa and distinct domains 

(676). These domains undergo considerable movement as 

the enzyme performs its function, which has the effect of 

limiting the amount of regulation that can occur without

compromising function. Folded into the molecule are 

four domains: M, transmembrane domain; P, phosphorylation 

domain; N, nucleotide-biding domain; and A, the 

actuator domain. Figure 4 shows diagrammatically 

SERCA structure and its catalytic cycle. The Ca binding 

sites are in the M domain, with the two Ca ions being 5.7A˚ 

apart. There are 10 transmembrane sections in the M domain 

(M1-M10), and this domain forms the channel through 

which Ca moves. The interaction site for phospholamban, a 

major regulator of SERCA1 (see sect. IVC) is located in the N 

domain, around Lys-397, although other binding sites on the 

M domain have been described (19, 20). Thapsigargin and 

cyclopiazonic acid (CPA), specific inhibitors of SERCA, bind 

to the M domain (Phe-256 of M3) (521) and helped with the 

elucidation of SERCA structure and function (102). From 

topological models, the structure of SERCA can also be 

described as having three parts: a cytoplasmic head, a stalk 

domain, and a transmembrane domain (442). The cytoplasmic 

head region constitutes around half of the total SERCA 

mass, and it encompasses the A, N, and P domains. The A 

and P domains are connected to the M domain, and the N 

domain is connected to the P domain. 

The simplest catalytic cycle for SERCA can be described 

as follows: E 132Ca-E 1,32Ca-E 1-ATP32Ca-E 1P i32Ca-E 2- 

P i3E 2-P i3E 23E 1 (see also Fig. 4). 

The two Ca binding domains exist in either a high- or 

low-affinity form, known as E 1 and E 2 states, and allow 

access only from the cytosolic and luminal side, respec- 


tively. Their K d values are 10 7 and 10 3 M, respectively. 

Work has suggested that two Ca ions from the cytosolic 

side bind, and the enzyme is subsequently phosphorylated 

by ATP at Asp-351. The formation of an “energy-rich” 

aspartyl-phosphorylated intermediate is why SERCA is 

classified as a P-type pump. The phosphorylation reaction 

is highly pH sensitive and H facilitate the reaction of 

inorganic phosphate (P i) with the Ca pump (291). Phospholamban 

binds preferentially to the E 2 state, lowering 

its apparent Ca affinity. It is as this high-energy intermediate 

(2Ca-E 1P i) that the translocation of Ca occurs. 

Following translocation and release of the two Ca ions 

into the SR lumen, the enzyme becomes a low-energy 

intermediary. Two or possibly three (395) protons are 

countertransported during the step E 2-P i3E 2, which aids, 

but does not complete, electroneutrality across the SR 

membrane. The protons are released into the cytoplasm 

following binding of the two Ca ions. 

D. SERCA Isoforms and Expression 

in Smooth Muscles 

Three genes (ATP2A1, ATP2A2, and ATP2A3) encode 

the three mammalian isoforms of SERCA known as 

SERCA1, -2, or -3. In addition, alternative splicing occurs 

giving rise to SERCA1a, SERCA1b, SERCA2a, and SERCA2b. 

SERCA1a is found in adult fast-twitch skeletal muscle, and 

FIG. 4. Schematic representation of the structural changes in SERCA and the accompanying catalytic cycle. Folded into the molecule are four 

domains: M, transmembrane domain; P, phosphorylation domain; N, nucleotide-binding domain; A, actuator domain. The two Ca binding domains 

exist in a either a high- or low-affinity form, known as E 1 and E 2 states, and allow access only from the cytosolic and luminal side, respectively. 

(Adapted from Petsko GA, Ringe D. Protein Structure and Function. New Science Press, 2004; on-line update 2006–2007.) 

Physiol Rev VOL 90 JANUARY 2010 www.prv.org


SERCA1b is in neonatal muscles. Brody’s disease is a rare 

inherited muscle disorder in which relaxation rates of 

skeletal muscle are impaired, leading to muscle stiffness. 

Patients have high rates of mutation in the SERCA1 gene 

which lower its Ca transport activity (43). SERCA3 appears 

to be the most diverse form, and an ever-growing 

number of isoforms are being discovered (15, 58, 534, 

566). The first was SERCA3a, found in nonmuscle cells 

and which has a low apparent Ca affinity, leading to 

questioning of its functional role in Ca signaling (573), 

although it may be that it maintains high [Ca] in microdomains. 

Subsequently, seven additional SERCA3 gene 

products have been described (58, 441). SERCA3 has 

been found in epithelial and endothelial cells, platelets, 

megakaryocytes, many endocrine glands, cerebellum, 

pancreas, and spleen (59). It seems that SERCA3 is not 

found in smooth muscle cells but could appear as a 

contaminant from other cell types when whole smooth 

muscle tissues are analyzed. For example, SERCA3-deficient 

mice have impaired vascular and tracheal relaxation, 

but the effects are mediated via endothelial and 

epithelial cells, respectively, which express SERCA3b. 

SERCA2 is the isoform found in smooth muscles, and 

this will be discussed in detail. 

1. SERCA2 

SERCA2 is 84% identical in sequence to SERCA1a. 

The gene for SERCA2 is located on human chromosome 

12 at 12q24.11. SERCA2b has a longer (luminal) COOHterminal 

tail than that of SERCA2a, which is cytoplasmic 

(726). SERCA2a is expressed in heart, slow-twitch skeletal 

muscle, and many types of smooth muscles (25). 

SERCA2b appears be ubiquitously expressed, leading to it 

being labeled as the housekeeping form of SERCA. This 

designation, while also consistent with it being the phylogenetically 

earliest isoform, is likely to be too simplistic, 

as different tissues express different levels of SERCA2b 

(777). From expression studies in COS and cardiac cells it 

has been suggested that, compared with SERCA2a, the 2b 

isoform has a lower turnover rate of ATP hydrolysis, a 

10-fold lower vanadate sensitivity and Ca transport rate, 

but a higher apparent affinity for Ca (425, 723, 724). 

Removal of the last 12 amino acids from SERCA2b in 

mouse cardiac cells abolished these differences, suggesting 

the long tail in 2b is affecting SERCA activity (723). 

Another difference between SERCA2a and -2b is the 

greater stability of 2a’s mRNA, perhaps explaining its 

increased expression levels. Both isoforms have the same 

sensitivity to phospholamban and thapsigargin (426). 

Although the differences between 2a and 2b are not 

extreme, their functional and developmental importance, 

at least in cardiac muscle, is great. Mice engineered to 

have SERCA2a replaced with SERCA2b suffer from embryonic/neonatal 

mortality due to malformations of their 

hearts (40%), and if the switch is made in adults, they 

develop cardic hypertrophy (723). Heterozygous mice developed 

squamous cell tumors (560). The human disease 

Darier-White condition, in which there are foci of separation 

between keratinocytes, has been associated with mutations 

of the SERCA2 gene, but no cardiac symptoms are 

apparent (608). 

In smooth muscles, when comparisons have been 

attempted, SERCA2b expression is often greater than that 

of 2a. Both 2a and 2b have been described in most smooth 

muscles: stomach (162), aorta (15, 434, 774), uterus (94, 

707), mesenteric artery (15), trachea (14, 458), ileum 

(162), and pulmonary (162). 

Following an investigation of splice variants, a third 

isoform, SERCA2c, has been described in a number of cell 

types: epithelial, mesenchymal, hematopoietic, and cardiac 

(141, 207), but its expression in, or relevance to, 

smooth muscle is unknown. 

E. Regulators of SERCA Expression and Activity 

Given the importance of SERCA to SR function and 

hence cellular Ca homeostasis, Ca signaling, and ultimately 

function, it was anticipated that its function would 

be modulated under physiological conditions and perhaps 

altered in pathophysiological states. However, the structure 

of SERCA has evolved to translocate Ca, and this is 

accomplished by large conformational changes in the 

molecule. This requirement for substantial conformational 

change to function is considered to impose limitations 

upon how much modification, protein-protein interactions, 

and other coupling to effectors can occur. 

1. Accessory proteins 

The best known regulator of SERCA is phospholamban, 

a small membrane protein discussed in section IVC, 

along with other protein modulators that work from the 

luminal side of the SR. Other cytoplasmic regulators of 

SERCA activity are emerging and were well reviewed by 

Wuytack and colleagues (719). The list includes the insulin-receptor 

substrate IRS1/2, the Ca binding protein 

S100A1, acylphosphatase, and the antiapoptotic protein 

Bc1-2. IRS1/2 has been found to interact with SERCA2 in 

an insulin-dependent manner in cultured aortic cells (9). 

2. Calcium and protons 


In smooth muscles (and other cell types), regulation 

of SERCA expression has been linked to cytoplasmic 

(774) and luminal [Ca] (376). This latter study noted the 

coordinated regulation of SERCA and the PMCA, e.g., in 

response to thapsigargin. It is not apparent that the linked 

regulation of SERCA and PMCA are functionally synergistic, 

i.e., increased plasmalemmal Ca extrusion on PMCA

will not aid SR Ca refilling, although cytoplasmic [Ca] will 

fall when both pumps act. Further studies are required to 

confirm this. Protons (2 or 3) are translocated from the SR 

to the cytoplasm during each cycle of SERCA activity. 

Cytoplasmic pH can also affect SERCA activity. Although 

pH alteration has not been studied very much in smooth 

muscle, it appears that alkalinization will decrease and 

acidification will increase SERCA activity (22, 645, 772). 

There appears to be no data on which SERCA isoforms 

are most sensitive to pH alteration. 

3. Development, hormones, and gestation 

While there is considerable developmental regulation 

of SERCA1 and SERCA2a in striated muscle (559), and 

SERCA3 also shows clear ontological differences in expression, 

SERCA2b, which predominates in smooth muscle, 

appears not to be developmentally regulated. 

Culturing has been shown to change SERCA isoform 

expression, as the cells become less differentiated (387, 

534). Thus the relative increase in SERCA2a with development 

in aorta is reversed with culturing (387). It is 

worth noting here that more work is required on native 

smooth muscle cells if clarity concerning modulators of 

SERCA expression and activity is to be obtained. 

Thyroxine is the best recognized hormonal modulator 

of SERCA expression, and the SERCA2 gene has promoter 

elements that bind thyroid receptors. In neonatal hypothyroidism, 

SERCA2a is downregulated. SERCA2a and -2b were 

reported to be increased in pregnant compared with nonpregnant 

rat myometrium (344) and in laboring compared 

with nonlaboring human myometrium (707). The mechanism 

for this upregulation is unclear, although endocrine 

changes might well be suspected. A recent paper by Liu et al. 

(401) showed that 17-estradiol could upregulate SERCA2a 

in cultured cells, along with the Na-K-ATPase subunit. 

Tamoxifen, the anti-estrogen drug, decreased SERCA activity. 

In the male urogenital system, testosterone may influence 

SERCA activity, given that castration in rabbits decreased 

its activity in corpus cavernosum (325). However, 

these authors also reported no change in SERCA activity in 

urethral myocytes. 

F. Summary 

There is a very large amount of literature concerning 

many aspects of SERCA, but it is often not obtained on 

smooth muscle. We have a detailed understanding of 

SERCA structure and catalytic mode of action. We are 

also aware of the different SERCA isoforms and their 

expression in smooth muscle. However, we can still say 

very little about how these differences influence the finetuning 

of smooth muscle Ca stores and hence Ca signaling. 

Further work in smooth muscles on how SERCA 


activity is affected by developmental or hormonal 

changes is needed. 

IV. PROTEINS ASSOCIATED WITH THE 

SARCOPLASMIC RETICULUM 


SERCA, a protein associated with the SR and of 

pivotal importance to its functioning, has been described 

above. We will now discuss other proteins associated 

with the SR and SERCA, especially phospholamban, and 

those proteins whose prime role is to bind Ca within the 

SR lumen, i.e., calreticulin and calsequestrin. We also 

briefly mention other proteins that may have a role to play 

such as junctin. We have not considered proteins whose 

role is not specific to the SR. However, it is important that 

we know about the environment in which these proteins 

(and SERCA) are operating; therefore, the SR membrane 

and the gradients across it, a topic that has received scant 

attention in many discussions of the SR, will be discussed 

first. 

B. The SR Membrane 


While much attention is given to the plasma membrane 

surrounding the smooth muscle cell, e.g., its composition, 

fluidity, and microdomains, less attention has 

been given to that of the SR. However, it is reasonable to 

suggest that the activity of SR channels, transporters, and 

associated proteins will also be affected by the same 

parameters. 

The SR (and ER) has a unique lipid membrane. It is 

the most fluid of all membranes, a direct consequence of 

it also being cholesterol-poor and having many unsaturated 

phospholipids (719). Thus SERCA has evolved to 

function in this specific membrane, and its optimal activity 

in artificial lipid bilayers, for example, occurs with a 

different composition than that required for maximal Na 

pump activity (390). The high fluidity of the SR/ER membrane 

presumably helps nascent proteins to fold correctly. 

The low cholesterol content also ensures that the 

SR membrane is relatively thin. 

The SR membrane is notably unsaturated in nature. 

The major phospholipids of the SR are choline (65%), 

ethanolamine (15%), and inositol (7%) phospholipids; 

together they constitute 80% of total lipids. Of the remaining 

neutral lipids, cholesterol makes up 95%. Cholesterol 

is excluded from the phospholipid annulus around 

SERCA (743). The informative reviews by Tada et al. 

(681), Lee et al. (390), and Vangheluwe et al. (719) should 

be consulted for further reading on SR lipid and protein 

composition.


SERCA is in direct contact with several SR lipids but 

in a cholesterol-poor environment, and this is essential for 

its function, as a highly fluid lipid membrane is needed for 

the large conformational changes required for SERCA 

function. Maximal activity of SERCA is affected by the 

thickness and head group composition of the SR phospholipids; 

phosphocholine containing 18C fatty acids 

yielded maximal ATPase activity, while longer or shorter 

fatty acids lowered its activity (261, 499). Dietary fatty 

acid changes have been shown to change the composition 

of the SR membrane (674), and its cholesterol content 

may increase with age (373) and contribute to the decreased 

SERCA function found in older animals (198) as 

the SR membrane becomes increasingly rigid. 

Variation in the lipid composition of the SR from 

different skeletal muscles has been described and related 

to differences in SERCA activity (73), but nothing is 

known about how composition may differ between 

smooth muscles. In a study of macrophage SERCA, Li 

et al. (397) showed that enrichment with cholesterol inhibited 

SERCA2b activity. This loss of fluidity may explain 

SERCA decreased activity, as the large conformational 

changes required for Ca transport are limited. In terms of 

macrophage cell function, the authors noted that dysfunction 

of SERCA could lead to protein unfolding in the ER 

and contribute to atherosclerotic lesions in foam cells 

(cholesterol-loaded macrophages). 

C. Phospholamban 


Phospholamban is a 52-amino acid homopentameric 

protein, which, depending on its phosphorylation state, 

reversibly binds to SERCA and affects its activity (431, 

639, 680). The phosphorylation of phospholamban occurs 

by both cAMP-dependent protein kinase (PKA) at Ser-16 

and Ca/calmodulin-dependent protein kinase II (CaM kinase 

II) at Thr-17, and the name phospholamban was 

given to mean “phosphate receptor” (679; see also Katz, 

1998 and associated articles in the same volume). Phospholamban 

inhibits SERCA by lowering its apparent affinity 

for Ca through direct protein-protein interactions, 

probably of a single phospholamban molecule associated 

with two SERCA molecules, and thus phospholamban 

may restrict the large domain movements needed for 

SERCA activity (21, 790). Upon phosphorylation there is a 

large change in phospholamban charge, and this greatly 

decreases its inhibitory effect on SERCA. It is clear in 

cardiac muscle that phosphorylation of phospholamban is 

a highly important part of the mechanism whereby -agonists 

via cAMP increase cardiac contractility; by relieving 

the inhibition of phospholamban on SERCA, more Ca can 

enter the SR and contribute to an increased Ca release on 

the next heart beat, and relaxation occurs at a faster rate. 


In heart, chronic inhibition of SERCA, as can occur with 

some human phospholamban null mutations (447, 621), is 

associated with dilated cardiomyopathy, as contractility 

is diminished. 

2. Distribution in smooth muscle 

Phospholamban is expressed in smooth muscle, although 

at lower levels than occur in cardiac myocytes 

(378, 643). Raeymaekers and Jones (584) first reported the 

presence of phospholamban in smooth muscle. They 

found it in pig stomach and rabbit and dog aorta but not 

pig aorta. This group then used pig stomach to cDNA 

clone and sequence phospholamban (725) and concluded 

it was 100% sequence identical to cardiac phospholamban 

and was the product of the same gene. Thus any differences 

in effects of phospholamban between cardiac and 

smooth muscle cannot be explained by isoform diversity. 

An immunogold EM study of phospholamban showed a 

patchy SR distribution of it in a variety of smooth muscles 

(ileum, iliac artery, and aorta), again suggesting that its 

density was lower than that of cardiac SR (172). Interestingly, 

phospholamban labeling of the outer nuclear envelope 

was also seen, although this observation does not 

appear to have been further investigated. These early 

studies concluded that whereas SERCA expression was 

approximately comparable between smooth muscles, 

phospholamban mRNA levels varied 12-fold, with aorta 

expressing low levels compared with ileum and stomach 

(162). 

Given the importance of PKA and CaM kinase II to 

smooth muscle force modulation, phosphorylation of 

phospholamban would be predicted to be one of their 

targets. It has also been suggested that in smooth muscle 

cGMP is an important mediator of phospholamban phosphorylation 

(129, 583) acting at Ser-16. A recent report 

(363) has found that in tracheal smooth muscle phospholamban 

is associated with a PKA signaling complex, in 

addition to its expected association with SERCA. If this 

were also to be the case in other smooth muscles, then 

this may lead to facilitated IP3-induced Ca release, rather 

than effects via SERCA Ca pumping, i.e., phospholamban 

may have multiple functions and effects in smooth muscles. 

As the target for several second messenger-dependent 

kinases, phospholamban may also play a role in Ca 

signaling events associated with vascular smooth muscle 

migration and hyperplasia, or influence endothelial Ca 

signals, but a discussion of this is beyond the scope of this 

review (117, 563, 672). 

3. Functional effects 

The earliest studies performed in smooth muscle to 

investigate phospholamban’s role involved vesicle studies 

of Ca uptake (582, 745). Sarcevic et al. (615), using cultured 

aortic smooth muscle cells, suggested that the

cGMP-mediated atrial natriuretic peptide transduction 

pathway involved phosphorylation of phospholamban. In 

1992, Karczewski et al. (338) suggested a role for phospholamban 

in the nitric oxide (NO)/endothelium-derived 

relaxing factor (EDRF)-induced relaxation of rat aorta. 

This work was followed up with a paper demonstrating 

more directly phosphorylation of phospholamban and 

coronary artery relaxation (337). However, no difference 

in blood pressure was reported when phospholamban 

knockout animals were made (418), and little effect of 

modulators of cGMP on the kinetics of aortic force was 

seen (379). Thus phospholamban may play a role in the 

actions of endogenous vasodilators, but its effects appear 

to be specific to different vascular beds. 

The development of the phospholamban knockout 

mouse has led to investigation of its role in smooth muscles. 

An increase in the rate of force development to 

phenylephrine stimulation in aorta was observed, compared 

with wild-type controls (378). However, these authors 

reported no differences when KCl was the stimulant 

and, importantly, no significant difference in relaxation 

rates. As noted above, this was also the case when cyclic 

nucleotides were used (379). Given the role of SERCA in 

facilitating Ca efflux from smooth muscle myocytes, these 

data do not support a prominent role for phospholamban 

in this process, with two caveats. First, as with all knockout 

studies, other compensatory mechanisms can occur, 

e.g., changes in plasma membrane Ca transporters or 

other SR proteins such as sarcolipin (see below). Second, 

force measurements can be an unreliable surrogate for 

[Ca] changes, and no simultaneous [Ca] and force measurements 

have been made. 

The study by Lalli et al. (378) on aorta of knockout 

mice described above did however note that greater phenylephrine-induced 

force was produced, compared with 

controls suggesting, but not proving, that an increase in 

SR Ca uptake may be occurring. In subsequent studies, 

the same group has examined portal vein, a phasic, blood 

vessel, which may be considered to have its [Ca] changes 

controlled and regulated in a different manner from the 

tonic aorta (671). This study found an alteration of the 

contractile pattern in the knockout mice, but no difference 

in sensitivity to ACh, and no difference in relaxation 

rate to isoproterenol. 

It appears then that there are some differences in 

blood vessels from phospholamban knockout mice compared 

with wild type, but there is no consistent finding 

and perhaps only subtle differences in the response to 

agonists or relaxation rates. One drawback to conclusions 

drawn from the studies to date is that no measurements of 

SR Ca uptake or release have been made on smooth 

muscle cells taken from these animals to directly test 

suggestions, such as increased SR Ca uptake or faster Ca 

release. Functionally no differences in blood pressure 

have been found in these studies. 



Several visceral smooth muscles have also been used 

to help elucidate phospholamban’s functional contribution 

to Ca signaling and contractility. In a series of studies 

on gastric smooth muscle, Kim and colleagues (346–348) 

have investigated the role of phospholamban in NO-mediated 

relaxation. Their work suggests that CaM kinase II 

is responsible for the phosphorylation of phospholamban 

in this tissue. They conclude that the major mechanism 

involved in phospholamban’s effect on gastric relaxation 

is an increase in SERCA activity influencing Ca removal, 

rather than an increase of spark-spontaneous transient 

outward current (STOC) coupling mechanism, although 

neither was measured. PKA-stimulated phosphorylation 

of phospholamban is postulated as one of the mechanisms 

whereby 2-adrenergic receptors ( 2-AR) act to 

relax airway smooth muscle (263). Stimulated by the 

observation that asthmatics chronically treated with 

2-AR agonist develop enhanced sensitivity to bronchoconstrictors 

(372), transgenic mice overexpressing the 

2-AR were studied (458). The authors noted that phospholamban 

transcripts and protein were markedly reduced 

in airway smooth muscle cells from these mice. 

These mice also had reduced constriction to methacholine. 

Thus they suggest that downregulation of phospholamban 

is a target of 2-AR agonists and protects against 

airway hyperactivity. 

We have preliminary data on knockout mice indicating 

that in the uterus phospholamban can influence the 

pattern of contractility. Thus the pattern of spontaneous 

contractions compared with controls was less frequent 

but longer lasting, and with many of the contractions 

having a spiked appearance (518). This pattern was also 

found in portal vein from knockout mice (671). 

Phospholamban is expressed in bladder smooth muscle 

(517). Measurements of force and [Ca] have been 

made in phospholamban knockout (KO) and phospholamban 

overexpressing (OE) mice. When stimulated by carbachol, 

compared with wild types, maximal increases of 

force and [Ca] were depressed in KOs and increased in 

OE mice (517). These differences were abolished when 

SERCA was inhibited by CPA. Resting [Ca] was also 

elevated in the OE mice. It was concluded that Ca uptake 

into the SR, rather than content, is the mechanism by 

which phospholamban is affecting force. Interestingly, 

the phospholamban OE mice also had a significant decrease 

in SERCA expression. 

In summary, it appears to us that despite some elegant 

studies there are insufficient mechanistic and functional 

studies to date to draw many firm conclusions 

about the role of phospholamban in smooth muscle. Some 

of the expected findings of knocking out phospholamban, 

e.g., slowing of Ca relaxation rates, do not occur and the 

reported changes in force are subtle rather than obvious; 

certainly the profound effects on contractility seen in 

cardiac muscle with phospholamban alteration appear


not to occur in most smooth muscles. Few studies on 

smooth muscle have considered the more complex role of 

the SR in this tissue, e.g., providing negative feedback on 

Ca entry via Ca sparks, as opposed to it acting as a Ca 

store to augment contraction. We tentatively conclude 

that phospholamban is not a major physiological regulator 

of SERCA in most smooth muscles; its effects on 

SERCA, for example, are not as large as those of luminal 

[Ca] alteration. This difference between smooth and cardiac 

muscles may be due to the relative expression of 

SERCA isoforms between the two muscles, but we suggest 

it also reflects the differences in the role of SERCA 

between them. 

D. Sarcolipin 

This 31-amino acid protein appears to be a homolog 

of phospholamban (522); it associates with the SR membrane, 

and its transmembrane domains are structurally 

similar. Sarcolipin inhibits SERCA by lowering its apparent 

Ca affinity (increasing K m) and V max (27, 205, 435, 

523). It has been found in greatest abundance in fast- (199, 

425, 511) and slow-twitch muscles (523) as well as cardiac 

muscles (720). To date, we can find no data describing 

sarcolipin presence in any smooth muscle. 

E. Calsequestrin 


Calsequestrin and calreticulin (discussed next) are 

the two most important luminal buffers of Ca in the SR. 

They share many protein properties, as well as fulfilling 

the requirement of being a Ca buffer, i.e., binding Ca with 

a high capacity and low affinity. Calsequestrin was the 

first SR Ca binding protein to be identified (103, 429, 432). 

Its crystal structure has been elucidated from rabbit skeletal 

muscle (734). These studies suggest that three very 

negative thioredoxin-like domains underlie the high Ca 

binding capacity of calsequestrin. 

2. Isoforms and expression 

There is evidence for calsequestrin expression, at 

varying levels, in the following smooth muscles: stomach 

(585, 730, 778), vas deferens (729, 730), aorta (730), ileum 

(585), and trachea (585). It may not be present in bladder 

(730) or pulmonary artery (585) and is either absent or in 

trace amounts in uterus (472). 

It is now appreciated that two genes exist for calsequestrin 

expression, leading to two isoforms which are 

65% identical (622). These isoforms are often referred to 

as the fast-twitch skeletal and cardiac forms (182, 622), 

but slow skeletal muscles express the fast form (181) and 

it appears that many smooth muscles express both iso- 


forms (730). The functional significance of smooth muscles 

expressing the two isoforms of calsequestrin or neither 

remains to be established. 

Calsequestrin in skeletal muscle binds 40–50 Ca 

ions with a binding constant of 1 mM and a high off-rate 

(430). The cardiac isoform binds 20 Ca ions with an 

affinity of 0.5 mM (51). In striated muscles calsequestrin 

binds to other proteins, including triadin and junctin (see 

sect. IVG), leading to a physical association with RyRs 

(233, 443). It may be that this arrangement enables the Ca 

of the SR to be in the right place, i.e., close to the release 

channel (187). Regular arrays of calsequestrin with Ca 

attached appear as crystalline structures in the SR lumen 

(607). Calsequestrin has been shown to increase the RyR 

open probability in skeletal muscle (343). 

Wuytack et al. (778) were the first to report that 

calsequestrin was present in SR from smooth muscle. 

They examined pig antrum and concluded it contained 

the cardiac form of calsequestrin in low amounts compared 

with heart, although commensurate with the lower 

SERCA expression in smooth muscle compared with 

heart. These authors also noted that calsequestrin in portal 

vein smooth muscles had first been proposed, on 

morphological grounds, by Somlyo (652) and Franzini- 

Armstrong (187). Vas deferens expresses both calsequestrin 

isoforms in about equal amounts (730). Its distribution 

was reported to be clustered at discrete luminal sites 

and on SR located superficially below the plasma membrane 

and rich in IP 3R (729). Thus a specific distribution 

of calsequestrin in smooth muscle as reported also for 

striated muscles may occur (322, 323). This contrasts with 

the nondiscrete distribution of calreticulin (see below). 

The colocalization of IP 3R with calsequestrin presumably 

aids rapid release of messenger Ca in smooth muscles 

expressing both these elements. Moore et al. (491) demonstrated 

in toad stomach that calsequestrin was not only 

present in the SR in a discrete manner, but that it was 

associated with the superficial SR. They also demonstrated 

a close association between calsequestrin and the 

NCX and Na pump distribution on the plasma membrane, 

in caveolae. Thus not only is calsequestrin close to SR Ca 

release channels but also to exchangers important to Ca 

homeostasis in smooth muscle, and contributing to microdomains 

(as discussed in sect. IID). 

Despite these several publications from a number of 

different laboratories, a paper appeared stating that calreticulin 

and not calsequestrin was the major Ca binding 

protein in smooth muscle SR (472). This study however 

only examined one type of smooth muscle, pig uterus, and 

actually reported that calsequestrin could be detected in 

uterine tissue extracts but not SR vesicles. They also used 

calsequestrin antibodies on cultured uterine cells and 

reported negligible staining, but given the culturing and a 

switch to a noncontractile, synthetic state, these data are 

difficult to interpret. This publication appears to have

stimulated Meldolesi’s laboratory to reexamine, confirm, 

and extend their earlier findings of calsequestrin expression 

in vas deferens (730). They again demonstrated both 

isoforms of calsequestrin are present in vas deferens and 

also expressed in stomach and aorta, particularly the 

skeletal muscle isoform. In another study combining ultrastructure 

and antibody labeling, the presence of calsequestrin 

in guinea pig vas deferens was confirmed, as was 

its absence from aorta (515). Again the calsequestrin distribution 

was similar to that of IP 3R. 

3. Function 

Although calsequestrin has been demonstrated in 

many smooth muscles, there appears to have been no 

recent work investigating its function and role in Ca signaling. 

Thus its functions, as a binding protein for Ca, as 

a part of an SR complex with Ca release channels (particularly 

RyRs), and triadin and junctin (see sect. IVG), and 

as a sensor of luminal Ca, can largely only be assumed by 

analogy to studies in striated muscles, with all the caveats 

that this requires. 

In summary, it seems reasonable to conclude that 

many, but not all, smooth muscles express calsequestrin 

and that those that do express the two different isoforms 

in a tissue-specific manner. It is not possible to do anything 

other than speculate about how these differences 

may relate to differences in Ca signaling or function and 

pathology in different smooth muscles. 

F. Calreticulin 


Calreticulin is a 46-kDa protein, the product of a 

single gene, and has been the subject of recent reviews 

focused on its role within the ER and skeletal muscle 

(164, 320, 419, 468, 761). It was first described in 1974 as 

a high-affinity Ca binding protein of the SR binding 25 mol 

Ca/mol protein (531) and its cDNA code was revealed in 

1989 (180, 647). Unlike calsequestrin it contains an ER 

retention signal. It was initially isolated from the SR and 

its Ca binding ability noted, which led to Ca binding being 

viewed as its major function. As discussed in the reviews 

cited above, it is now appreciated that calreticulin has 

many functions, including chaperoning of ER proteins, 

and that it is also expressed in other cellular organelles 

and at the cell surface. A list of its functions would now 

include cell adhesion (e.g., Ref. 320), antithrombotic activity, 

induction of NO in endothelial cells, long-term 

memory in Aplysia, and development (see Refs. 320 and 

469 for review of these other functions of calreticulin). 

Our focus is on its role in Ca homeostasis within the SR. 



2. Expression 

In smooth muscle, calreticulin has been found to be 

expressed in the SR lumen of several different tissues. In 

1992 Tharin et al. (697) reported its widespread distribution 

in rabbit tissues, especially smooth muscle (aorta, 

uterus). Calreticulin has been described as the major Ca 

buffer in smooth muscle (472), but as already noted, this 

study only examined uterine tissue, which expresses calreticulin 

and only trace amounts of calsequestrin. In their 

study of vas deferens, Villa et al. (729) found both Cabinding 

proteins present and reported that calreticulin 

was widely distributed throughout the SR, whereas calsequestrin 

had a more discrete distribution and overlapped 

with IP3Rs. A recent study of human esophageal smooth 

muscle has detected a particularly strong calreticulin signal 

(stronger than for cardiac muscle), as well as calsequestrin 

but not phospholamban (177). Both Ca binding 

proteins decreased in patients with reduced gastrointestinal 

motility and elevated esophageal pressures. 

3. Function 

Calreticulin may specifically affect the activity of 

SERCA2b, suggesting some specificity of function in 

smooth muscles (318). SERCA2b has an additional transmembrane 

segment (see sect. IIIC) that is thought to contain 

a calreticulin binding site. It has been suggested that 

when SR [Ca] is low, calreticulin is not bound to SERCA 

and thus Ca enters the SR as SERCA is maximally active. 

When SR [Ca] is high, calreticulin binds to the luminal tail 

of SERCA2b and decreases its activity (468). 

In vascular smooth muscle (aorta), calreticulin increased 

when glucose concentration was elevated, both 

in vitro and in vivo (699). These effects however may be 

due to calreticulin’s chaperone role and a destabilization 

of GLUT1 mRNA, rather than direct effects on Ca signaling 

in the myocytes. Daniel’s group working on airway 

and esophageal smooth muscle (142, 143) have presented 

evidence that calreticulin and calsequestrin are colocalized 

and precipitated in the detergent-resistant membrane 

fraction indicative of caveolae, along with L-type Ca channels. 

While these data await confirmation with additional 

techniques to ensure that the immunoprecipitation with 

caveolin is not due to their loss from the SR during 

membrane isolation, they indicate that these Ca-binding 

proteins may influence caveolar function and interactions 

with the peripheral SR and may contribute to Ca microdomains 

within the smooth muscle cell. The finding of calreticulin 

outside the SR/ER has been reported in other cell 

types and explains its role in processes such as cell 

adhesion (320, 468). 

Using a Xenopus oocyte expression system, calreticulin 

was found to suppress IP3-induced Ca oscillations, 

suggestive of it acting to inhibit Ca uptake by SERCA2b 

(318). It has been estimated that 50% of Ca in the ER is


bound to calreticulin (501), and if its expression is deficient, 

then the ER Ca storage capacity is decreased and 

agonist-evoked Ca release is inhibited (467, 501). Conversely, 

overexpression leads to increased Ca storage 

capacity and a decrease in store-operated Ca influx (466). 

Emptying of the ER Ca store stimulates expression of 

calreticulin (744). Thus changes in calreticulin concentration 

may be expected to have a direct effect on Ca signaling, 

as well as indirect ones, as its chaperone function 

is also affected (470). Cardiac development is so impaired 

in calreticulin knockout mice that they die at the embryonic 

stage (467). However, in adult heart, the level of 

calreticulin expression is low, and its overexpression produces 

arrhythmias, heart block, and death (500). 

G. Triadin and Junctin 

While many proteins have been identified in the SR 

lumen (51, 424), two that are abundant and intrinsic to the 

SR membrane are triadin and junctin (38, 340). They 

appear to anchor calsequestrin to the junctional face of 

the SR membrane, perhaps to keep it close to Ca release 

channels. The binding to calsequestrin is disrupted by low 

(10 M) and high (10 mM) [Ca] (803). It is also possible 

that these two proteins are SR luminal Ca sensors 

and modulators of RyR open probability (51, 359, 803). To 

date, triadin and junctin do not appear to have been 

described in smooth muscle. This may be a consequence 

of the lack of triad and diad arrangement of the SR in 

smooth muscle, but it may also be due to lower expression 

levels, methodological difficulties (443), or that different 

isoforms occur. For example, recent data have 

identified new triadin isoforms, which colocalize with 

IP 3R (721). Thus there may be an IP 3R-specific complex 

that could be in smooth muscle. Similarly triadin and 

junction by mediating interactions with calsequestrin 

could have function in smooth muscle if expressed there 

(237). 

H. Summary 

In summary, while it is clear that for the SR to 

function as a Ca store the Ca binding proteins calreticulin 

and calsequestrin are required, little data are available 

investigating the functional effects of altering their expression 

in smooth muscle. Nor is it clear what advantages 

or specificity is conferred on smooth muscles by 

their expressing the different proteins or their isoforms. 

Little recent data have been obtained in this field despite 

the increased interest in how luminal Ca content affects 

Ca signaling and functions in smooth muscle. Triadin and 

junctin may not be expressed in smooth muscle, but 

verification of this would be helpful. We concur with the 

words of Volpe et al. (730) written more than a decade 

ago on this issue that “it is therefore possible that what at 

present appears to be no more than a complex pleiotropism 

could ultimately be attributed to specific physiological 

characteristics of the various smooth muscles.” We 

have however come no closer to examining this possibility 

in the intervening years. 

V. CALCIUM RELEASE CHANNELS 


Ca release from the SR in smooth muscle cells occurs 

though activation of two families of Ca release channels: 

RyR and IP 3R channels, which have substantial similarities 

in structure (113, 657). These channels are involved in 

control of various functions in smooth muscle cells including 

contraction, relaxation, proliferation, and differentiation 

(31). The functional role of RyR and IP 3R channels 

in these processes critically depends on molecular 

identity, level and proportion of expression, subcellular 

distribution, and the types of functional units they form 

with other cellular structures. Smooth muscles differ by 

types (vascular and visceral), location (different vascular 

beds or various hollow organs), size (resistance versus 

conduit arteries), orientation (circular versus longitudinal), 

and function (phasic versus tonic), and it is not 

surprising that the data obtained so far indicate marked 

differences in expression, spatial distribution, and subcellular 

location of different isoforms of RyRs and IP 3Rs and 

that these underlie the generation of tissue- or cell-specific 

Ca responses (224, 292, 493, 765, 794). NAADP is an 

agonist at RyRs, but there may also be a third Ca release 

channel, which is discussed in the section VD. 

B. Ryanodine Receptors 


The RyR is a homotetramer with the (565 kDa) 

subunits surrounding a central Ca pore. The subunits act 

in a coordinated way to gate the Ca channel and are also 

each associated with an important 12-kDa regulatory 

binding protein FKBP (427, 461). Cryo-EM and threedimensional 

reconstruction reveal that RyR is a symmetrical 

mushroom like structure with a large cytosolic assembly 

and a short region that traverses the SR membrane 

(610, 624). The ion channel-forming, membranespanning 

regions are highly conserved between different 

RyR isoforms and are localized to the COOH terminus. 

The cytosolic domain consists of 80% of the mass of the 

RyRs. It assumes a quatrefoil shape and is the modulatory 

region of the molecule, containing binding sites for Ca, 

adenine nucleotides, calmodulin, FKBPs, as well as phosphorylation 

sites (624). The massive size of RyRs makes 

them physically the largest ion channels (almost twice as

large as the IP 3R, their closest relatives). Because of its 

short and wide channel region, the RyR is suitable for 

sudden and large release of Ca from intracellular stores. 

The Ca conductance of RyR is on the order of 100 pS, 

and Ca is their principal endogenous effector. Studies 

describing the Ca dependence of channel activity in isolated 

preparations showed that there are sites within the 

protein where Ca can bind and inhibit as well as activate 

the channels (460). Data obtained using purified channels 

reconstituted into planar lipid bilayers and from [ 3 H]ryanodine 

binding studies, demonstrate that RyR1 and RyR2 

are activated by sub- to low micromolar levels of [Ca] and 

inactivated by micromolar to millimolar [Ca] in the cytoplasm. 

The RyR2 isoform is less sensitive to inactivation 

by calcium than RyR1 (130, 460). The steady-state Ca 

dependencies of isolated single RyRs are nearly identical 

(238). Similar to RyR1, RyR3 was shown to also be sensitive 

to caffeine, adenine nucleotides, and ryanodine. 

However, it has the lowest Ca sensitivity of the RyR 

family. This led to the suggestion that it serves as a 

high-threshold Ca release channel (687). The luminal sensitivity 

of RyR3 to Ca was also lower than that of RyR1 

and RyR2 (669). 

1. Expression 

The RyR specifically binds the plant alkaloid ryanodine, 

which is the reason for its name. Each of the three 

isoforms of RyRs are encoded by a distinct gene (670). 

RyR1 and RyR2 are predominantly expressed in skeletal 

(812) and cardiac (532, 599) muscle, respectively, while 

RyR3 appears to be expressed in a variety of tissues (211, 

243, 389). Unlike cardiac and skeletal muscle, which express 

only one RyR isoform, all three RyR isoforms can be 

expressed in smooth muscles, with the type and the relative 

proportion of expression of each being tissue and 

species dependent. Expression of all three isoforms has 

been reported for smooth muscles of the pulmonary and 

systemic vasculatures (133, 475, 511, 787, 805) and in 

pregnant human and rat myometrium (439, 440). Some 

types of smooth muscles coexpress only two RyR isoforms: 

RyR1 and RyR3 are subtypes identified in airway 

(158), and RyR2 and RyR3 are subtypes found in rat aortic 

myocytes (340, 351) and duodenum (138). Expression of 

just the RyR2 isoform was reported for smooth muscle 

cells of urinary bladder (115, 490), toad stomach (781), 

and cerebral artery (213), and only the RyR3 isoform is 

expressed in rat ureteric (60) and human nonpregnant 

(24) and mouse pregnant uterine (358) smooth muscle 

cells. An alternative splicing of RyR3 subtype was recently 

reported for some types of smooth muscles (138, 

139). In mouse, spliced short isoform (RyR3s), without 

channel function, are coexpressed with nonspliced functional 

full-length isoforms of RyR3 (RyR3L) (139). Mouse 

duodenum myocytes express RyR2 and spliced and non- 



spliced RyR3 isoforms (138). The functional significance 

of these nonfunctioning RyRs is discussed below. 

Culturing of smooth muscle cells (132, 444, 711) or 

tissues (154) alters the patterns and levels of RyR expression 

in smooth muscle cells. Changes in RyR expression 

have been reported in some smooth muscle pathologies, 

e.g., a loss of RyR2 expression was reported for hyperactive 

detrusor smooth muscle (316). 

2. Localization 

The spatial arrangement of RyRs in smooth muscle 

cells is in marked contrast to the regular distribution of 

RyRs in sarcomeric muscles. A helical arrangement of 

well-developed superficial SR was reported in live smooth 

muscle cells double stained with fluorescently labeled 

indicators for the SR and RyRs (221). Immunofluorescence 

and immuno-EM studies also show that the subcellular 

distribution of RyRs closely follows that of the SR 

(394, 490). Both a peripheral and cytoplasmic distribution 

of RyRs is found in tonic smooth muscle (394), whereas 

the distribution is largely peripheral in phasic smooth 

muscles [vas deferens (394), urinary bladder (490)]. A 

transition from a diffuse cytoplasmic distribution of functional, 

but nonsparking, RyR2 in neonatal cerebral arteries, 

to a distinct distribution in peripheral clusters in adult 

arteries, with the production of Ca sparks, indicates that 

Ca spark-generating units of RyRs appear late in tissue 

differentiation and are confined to peripheral regions of 

the cell (213). Distinct RyR2 staining in close proximity to 

the plasma membrane colocalized with voltage-gated Ltype 

Ca channels and BK has been reported for guinea pig 

urinary bladder myocytes (490) and vas deferens (525). 

Rat pulmonary artery myocytes that express all three 

subtypes of RyRs have RyR2, their predominant isoform 

located mainly in peripheral sites, and RyR3 in the perinuclear 

region and RyR1s in both regions (787). In rat portal 

vein, the antibody-staining pattern of RyR3 is diffuse, 

unlike that of RyR1 and RyR2 which exhibit discrete areas 

of higher density staining (475). A diffuse distribution of 

RyR3 was also reported for smooth muscle cells of duodenum 

(138) and nonpregnant mouse myometrium (476). 

The existing histological data would fit a model of 

peripherally located collections of RyR2 and RyR1 and 

more diffuse distribution of RyR3 in smooth muscle cells. 

3. RyR function 

The physiological contribution of different RyR subtypes 

to Ca signaling in smooth muscle cells has been 

recently addressed using RyR knockout mice (314, 805) 

and antisense oligonucleotides specifically targeting each 

of the subtypes (138, 139, 407, 476). In portal vein, injection 

of myocytes with either RyR1 or RyR2 antisense 

oligonucleotides abolished Ca sparks, suggesting RyR1 

and RyR2 may colocalize to form Ca spark sites, while


suppression of RyR3 did not alter Ca spark activity. In 

urinary bladder myocytes, RyR2s are required for generation 

of Ca sparks (314). Native RyR3s are either not 

involved (133, 314) or may even have an inhibitory effect 

on Ca sparks (407). However, when both RyR1 and RyR2 

are inhibited with antisense oligonucleotides, and under 

conditions of increased SR Ca loading, RyR3 can be activated 

by caffeine or agonists (475, 476). RyR3 gene knockout 

significantly inhibits the contractile response to hypoxia 

but not the norepinephrine-induced Ca and contractile 

responses, in pulmonary artery smooth muscle cells 

(805). 

The function of RyR3 in Ca signaling is complicated 

by alternative splicing of RyR3. The expression of a short 

isoform of RyR3 (RyR3s) in HEK293 cells can inhibit both 

the full-length RyR3 (RyR3L) and RyR2 subtypes (315). In 

native smooth muscle, the dominant negative effect of the 

spliced isoform of RyR3 inhibits RyR2 activation and Ca 

signals in duodenum (138) and the activity of RyR3 in 

mouse pregnant myometrium (139). These observations 

reveal a novel mechanism by which a splice variant of one 

RyR3 isoform may heteromerize with the other RyR variants 

and suppress the activity of these RyR isoforms via a 

dominant negative effect. Thus tissue-specific expression 

of RyR3 splice variants is likely to account for some of the 

pharmacological and functional heterogeneities of RyR3, 

for example, the lack of Ca sparks and caffeine sensitivity 

in rat myometrium, despite expression of all three forms 

of RyRs (88). 

On the basis of the available information, RyR2 appears 

to be the most important component of Ca sparks in 

smooth muscle, and recent findings on RyR3 splice variants 

have helped unravel some of the perplexity in Ca 

signaling in smooth muscle. Calcium sparks are discussed 

in detail in section IX. 

C. IP 3-Sensitive Ca release channels 

Binding of G protein-coupled receptor agonists to their 

receptors in smooth muscles leads to activation of phosphatidylinositol-specific 

phospholipase C (PLC), which catalyzes 

the hydrolysis of phosphatidylinositol 4,5-bisphosphate 

(PIP 2) to yield IP 3 and diacylglycerol (DAG) (48). The 

IP 3 releases Ca from intracellular stores by binding to IP 3R, 

which are IP 3-gated Ca release channels, while DAG activates 

protein kinase C (PKC). Activation of IP 3Rs via activation 

of L-type Ca channels independent of Ca entry, socalled 

metabotropic Ca channel-induced Ca release, has 

been suggested in arterial myocytes but requires confirmation 

by other studies (149), and is not discussed further. 

1. Expression 

Three distinct IP3R gene products (types 1–3) have 

been identified in different mammalian species (283, 546). 

All three isoforms of IP 3R are structurally and functionally 

related (193). The IP 3R isoforms differ in their sensitivities 

to breakdown by cellular proteases (IP 3R2 relatively 

more resistant that IP 3R1 and IP 3R3) (765). These 

receptors exist as tetrameric structures, with a monomeric 

molecular mass of 300 kDa. Unlike RyRs, IP 3Rs 

are poorly expressed in skeletal and cardiac muscle but 

extensively expressed in brain and other tissues including 

smooth muscles (317, 471, 498, 546, 695, 760, 794). In 

smooth muscles, the density of IP 3Ris100 times less 

than in brain (437, 802). In visceral smooth muscles (356, 

379–381), IP 3Rs are expressed in greater quantity than 

RyRs with an overall stoichiometric ratio of IP 3Rs to RyRs 

of 10–12:1, while in vascular smooth muscles the ratio 

is 3-4:1 (62). 

Smooth muscle cells express multiple IP 3R isoforms, 

although as with RyRs, the level of expression of different 

subtypes is tissue and species dependent. Using reversetranscriptase 

PCR analysis, Morel et al. (493) showed that 

rat portal vein expressed IP 3R1 and IP 3R2, whereas rat 

ureter expressed predominantly IP 3R1 and IP 3R3 (493), 

although in an earlier study it was reported that rat ureteric 

myocytes expressed all three isoforms of IP 3Rs (60). 

In the thoracic aorta and mesenteric arteries, IP 3R1 was 

the only isoform found, while IP 3R1 and weak expression 

of IP 3R2 were reported for smooth muscle cells of basilar 

artery (324). The type 1 isoform predominates in guinea 

pig intestinal smooth muscle cells (222) as well as in adult 

porcine (297) and rat aorta (693), whereas type 3 predominates 

in neonatal aorta (693). Subtype-specific IP 3R antibodies 

revealed that the expression of IP 3R1 was similar 

in cultured aortic cells and aorta homogenate, but expression 

of IP 3R2 and IP 3R3 types was increased threefold in 

cultured cells (693). Immunostaining and functional studies 

performed on isolated cells and cells in situ in rat 

portal vein showed two subpopulations of cells, one 

which predominantly expressed IP 3R1 and a second predominantly 

expressing IP 3R2. Distinct patterns of the Ca 

responses were generated by the two subpopulations of 

myocytes in response to agonist (493). 

2. Localization 


In some smooth muscles, IP 3R1 has been localized 

throughout the cell, i.e., central and peripheral SR, as 

visualized using EM (191, 515, 693, 729), and in the others 

it had a mainly subplasmalemmal location (222). IP 3R2 

has a diffuse cytoplasmic distribution, similar to IP 3R1 

(and IP 3R3), but also occurs as dense patches in the 

peripheral cytoplasm, a pattern that IP 3R1 did not exhibit 

(668). IP 3Rs were also arranged in clusters in rat ureteric 

myocytes, as well as throughout the cell (60). In vascular 

myocytes, IP 3R2 is distributed peripherally and associated 

with the nucleus in proliferating cells (668, 694). In 

portal vein, IP 3R2 was also found closely associated with

the nucleus and at the plasma membrane, whereas the 

IP 3R3 was distributed predominantly around the nucleus 

(209). 

Interestingly, IP 3Rs are also localized in structures 

other than the ER/SR. For instance, they have been found 

in the nuclear envelope in many cell types, including 

vascular (694) and visceral (729) smooth muscles. This 

may be important in regulating intracellular calcium release 

around the nucleus when vascular smooth muscle 

cells switch to a more proliferating phenotype. In myocytes 

from basilar artery, IP 3R1 staining was detected 

close to both the nuclear and plasma membranes. IP 3R1 

was also observed throughout the cytoplasm intermingled 

with the actin filaments. This observation suggests that an 

extensive SR network spreads from the nuclear region, 

throughout the cell, and comes into close association with 

the plasma membrane. Localization of IP 3Rs close to the 

cell membrane may account for the activation of Cainduced 

Ca entry via nonselective cation channels (381), 

calcium-dependent potassium (34), or chloride channels 

(239). 

3. Role of IP 3Rs 

Interaction of IP 3 with the IP 3R is a principal mechanism 

for mediating the release of Ca from the ER/SR in 

response to agonist stimulation. The IP 3R has a single 

high-affinity binding site (K D value of 80 nM), and it is 

estimated that half-maximal release of Ca from the SR 

requires 40 nM InsP 3. The InsP 3 sensitivities of the isoforms 

vary to a limited extent and are ranked IP 3R1 

IP 3R2 IP 3R3 (K D values of 1.5, 2.5, and 40 nM, respectively) 

(766). IP 3R activity not only depends on the concentration 

of IP 3 but also on the cytosolic concentration 

of Ca (283, 546). This property allows Ca release activated 

by the IP 3R to regulate further Ca release. IP 3R1 activity 

can be stimulated at [Ca] below 300 nM, while at 300 nM 

Ca inhibits activity. This type of biphasic Ca dependence 

has been reported in smooth muscle cells (52, 282) in 

which the IP 3R1 isoform is predominant (283). The IP 3R2 

and IP 3R3 do not exhibit such marked Ca concentrationdependent 

inhibition (240, 586, 586). The positive regulation 

of the IP 3R by Ca may be largely due to a direct 

binding of Ca to the receptor (484). The sensitivity of IP 3R 

is also controlled by the SR luminal Ca content such that 

a reduced content also reduces IP 3-induced Ca release 

(481), although recent data point to the cytoplasmic aspect 

of the channel being important (114). 

Recent studies have shown that Ca oscillations in 

cultured rat portal vein myocytes and rat adrenal chromaffin 

cells depend on IP 3R2 or an interaction between 

IP 3R1 and IP 3R2 (323, 326). It has been shown that coexpression 

of IP 3R types 1 and 2 facilitates Ca oscillations, 

whereas expression of IP 3R3 alone generates monophasic 

Ca transients (493). Since IP 3R types 1 and 2 are more 


sensitive to IP 3 and Ca, it was postulated that they are 

required for generation of spontaneous SR Ca release 

known as Ca puffs (i.e., local Ca release through IP 3Rs) 

and Ca oscillations (60, 493). In contrast, the IP 3R3 is 

activated by higher [Ca] and may participate in agonistinduced 

Ca waves (493). 

The clustering of IP 3Rs provides spatially discrete 

release sites and enables Ca signals that arise from IP 3Rs 

to exist with various amplitudes and locations throughout 

the cell (60). These localized increases produce an increase 

in [Ca] throughout the cell when puffs coalesce to 

form transient [Ca] increases (Ca waves) that propagate 

through the cell in a regenerative manner (60, 72). Calcium 

puffs are discussed further in section IX. 

A cautionary note on using pharmacological inhibitors 

to study SR release mechanisms was made by Mac- 

Millan et al. (433). They found that dantrolene and tetracaine, 

thought to be specific RyR blockers, could affect 

IP 3R Ca release, i.e., they may not be as specific as previously 

thought, and this will have implications when 

interpreting such data. 

4. Conclusions 

The variety, distribution, and combination of IP 3Rs 

and RyRs in smooth muscle and their contribution to 

different types of signals will underlie the specificity of 

mechanisms, as well as the spatial and temporal parameters 

that are required for specificity of function in different 

smooth muscles. These functions include not just 

tissue-specific contraction and relaxation parameters, but 

also secretion, proliferation, and phenotypic change. In 

the following section we address how these SR release 

processes can be modulated. 

D. Modulation of SR Ca Release 

Recently several excellent reviews have been published 

on the pharmacology of the RyRs and IP 3R channels 

(231, 384, 670); thus here for completeness we will 

briefly overview the basic pharmacology of the Ca release 

channels in smooth muscle cells. We will then consider in 

a little more detail the roles of cADP ribose and NAADP, 

as evidence grows for their physiological role in smooth 

muscle. 

1. RyRs 


RyRs have binding sites for many agents, reflecting 

the huge size of these channels. Methylxanthines, imidazoles 

and imidazolines, perchlorates, suramin, and volatile 

anesthetics all activate RyR. Inhibitors of RyRs include 

ryanodine, ruthenium red, procaine, tetracaine, 

dantrolene, and octanol (for more details, see references 

in Ref. 384).


A) CAFFEINE. Caffeine, an alkaloid methylxanthine, increases 

the RyR Ca sensitivity by increasing its open 

probability (P o) without changing its conductance, as 

shown in single-channel experiments on RyR purified 

from cardiac (601), skeletal (600), and smooth muscles 

(258) reconstituted into planar lipid bilayers. The threshold 

caffeine concentration for Ca release is 250 M (404). 

The caffeine-induced Ca release is a two-step process: a 

small Ca release and then a second regenerative phenomenon, 

in which released Ca acts on a cluster of RyRs, 

triggering further Ca release. Caffeine has long been used 

to deplete SR Ca stores in smooth muscle; this is frequently 

done in a Ca-free medium containing Ca chelators 

such as EGTA. The transient contractile response obtained 

under such conditions is a qualitative estimate of 

the average size of the Ca stores in the SR. Caffeine, 

however, does not deplete the SR Ca store in those tissues 

expressing the RyR2 dominant negative splice variant, 

e.g., the uterus. 

In the guinea pig ureter, which expresses effectively 

only RyR (91, 92), concentrations of caffeine for Ca release 

ranged from 0.5 to 20 mM (89). The use of caffeine 

in smooth muscle comes with several concerns: 1) it 

inhibits phosphodiesterase and therefore raises cAMP 

concentration (341), which at concentrations exceeding 1 

mM inhibits voltage-gated Ca channels; 2) it has the potential 

to augment capacitative Ca entry by depletion of 

the SR; and 3) it inhibits the SR Ca release induced by IP 3 

(480) and inositol phosphate formation (576). 

B) RYANODINE. Ryanodine is a poisonous alkaloid found 

in the plant Ryania speciosa (670). The compound has an 

extremely high affinity for the RyR. The pharmacology of 

RyRs has been elegantly reviewed (231). Ryanodine has 

complex concentration-dependent effects on the conductance 

and gating of single RyR channels (670). It binds 

with such high affinity to the receptor that it was used as 

a label for the first purification of RyRs and gave its name 

to them. At nanomolar to low micromolar concentrations, 

ryanodine locks the receptors in a half-open state, 

whereas it closes them at micromolar concentration, irreversibly 

inhibiting channel opening. There is an agreement 

that high-affinity binding results in channel activation 

or subconductivity, whereas low-affinity binding 

leads to channel inhibition (670). Ryanodine at low concentration 

is frequently used to deplete the SR by causing 

the Ca release channels to remain in a semiconducting 

state. This leak from the SR has several consequences, 

such as preventing the SR from storing any Ca it may 

accumulate, loss of Ca sparks and their effects on Casensitive 

ion channels, and loss of ability to generate 

IP 3R-mediated Ca waves and Ca oscillations in many 

types of smooth muscle. This last effect is due to ryanodine 

depleting common Ca stores, i.e., shared by RyR and 

IP 3Rs. The binding of ryanodine to RyRs is use-dependent, 


that is, the channels have to be in the activated state for 

it to be effective. 

C) TETRACAINE. Tetracaine is a potent local anesthetic 

and an allosteric blocker of RyR channel function. At low 

concentrations, tetracaine causes an initial inhibition of 

spontaneous Ca release events (137), while at high concentrations 

it blocks release completely (236). 

D) RUTHENIUM RED. Ruthenium red is an organic polycationic 

dye that tightly binds to tubulin dimers and RyRs. 

It is a potent (nM) inhibitor of RyR (783), but also inhibits 

mitochondrial Ca uptake (242). 

2. IP3Rs Pharmacological inhibitors of IP3Rs are less abundant 

compared with RyRs, and unfortunately, agents used 

to block these receptors are nonselective. The inhibitors 

most commonly used include 2-aminoethoxy-diphenylborate 

(2-APB), heparin, and xestospongins (for a more 

detailed review, see Ref. 384). 

A) 2-APB. 2-APB is a synthetic monomer that can form 

a five-membered boroxazolidine heterocyclic ring (boroxazolidone), 

when an internal coordinate bond is formed 

between the nitrogen in the ethanolamine side chain and 

the tricoordinated boron (664). 2-APB inhibits the IP3R channel opening without affecting IP3 synthesis or binding. 

Despite earlier claims to the contrary, 2-APB is not 

selective for IP3Rs as it reduces capacitative Ca entry and 

Ca efflux from mitochondria by inhibition of the NCX, and 

it may also block gap junctions (reviewed in detail in Ref. 

384). 

B) HEPARIN. Heparin is a cell-impermeant inhibitor of 

IP3R and acts as a competitive inhibitor of IP3 in permeabilized 

smooth muscles (91). At low concentrations (up 

to 2 mM), heparin may be specific, but at higher concentrations 

(20 mM), it chelates Ca and inhibits contraction 

(358). 

C) XESTOSPONGINS. The xestospongins A, C, and D, araguspongine 

B, and demethylxestospongin B are alkaloids 

from the Australian marine sponge Xestospongia sp. 

(199). They are cell-permeant, potent inhibitors of IP3Rs. Xestospongin C is the most potent and widely used in 

smooth muscle studies (34, 45, 169, 483). Although more 

studies are required, accumulated data suggest that the 

various isoforms of IP3Rs differ in their sensitivities to 

xestospongins. Xestospongins are not selective and can 

inhibit voltage-gated Ca channels, which complicates 

their use. 

3. cADPR 

Cyclic ADP ribose (cADPR) is a cellular messenger 

for calcium signaling (235). It is derived from nicotinamide 

adenine dinucleotide (NAD) and ADP-ribosyl cyclase 

(392). It is a physiological allosteric modulator of 

RyRs and helps stimulate CICR at low cytosolic [Ca]. RyR

activation with high concentrations of caffeine is partly 

due to caffeine mimicking the binding of cADPR to RyRs. 

Whether the action is by direct binding to RyR or indirect 

(through binding to FKBP) is debated. Some reports suggest 

that cADPR binding makes FKBP, which normally 

binds RyR2, fall off RyR2. Involvement of cADPR in Ca 

release via RyRs has been suggested for some vascular 

and visceral smooth muscles (262, 757, 776), but not 

colonic myocytes, where Ca efflux by the Ca-ATPase was 

stimulated (78). Firm conclusions are difficult to draw 

until further studies are performed in smooth muscle and 

cADPR synthesis in response to agonist stimulation is 

demonstrated. 

4. NAADP 

NAADP was first suggested as a Ca-mobilizing agent 

after studies in sea urchin eggs (125, 391). While NAADP 

is similar to cADPR and may be synthesized from the 

same enzyme, ADP-ribosyl cyclase CD38 (but see Ref. 

650), it is a distinct Ca mobilizer, using RyR to amplify its 

signals after releasing Ca, rather than acting to modulate 

RyR release as cADPR appears to do. 

It has been shown that the enzymes for its synthesis 

and metabolism are present in smooth muscle (cultured 

A7r5 and mesangial cells) (795, 796). It appears to be 

unimportant in terms of Ca signals linked to contraction 

in some smooth muscles [tracheal, bronchial, and ileum 

(501)] but functionally important in others [coronary 

(799) and pulmonary (61) arterial, myometrial (650)]. Elucidation 

of its signaling pathway in smooth muscle has 

been best worked out in pulmonary myocytes. In elegant 

experiments where myocytes were freshly isolated and 

then cannulated with a micropipette, NAADP was introduced 

into the cytoplasm and produced Ca bursts, which 

on occasion produced global Ca waves and cell shortening 

(61). The full effects of NAADP on Ca signals in 

smooth muscle cells appear to need reinforcement from 

CICR via RyRs, recently identified as being RyR3 subtype, 

at least in pulmonary myocytes (350). 

Recent interest has turned to identifying the cell 

compartment that NAAPD receptors are located within. 

Although initially assumed to be the ER/SR and then 

nuclear envelope, there is growing evidence for a lysosomal, 

acidic organellar source in smooth muscle, and other 

tissues. The first evidence came from sea urchins, where 

lysosomal-related organelles were shown to be the store 

released by NAADP (149). Zhang and Li (799) have since 

demonstrated that the NAADP channel can be reconstituted 

in liver lysosomes. Kinnear et al. (350) in pulmonary 

arterial cells used bafilomycin, an inhibitor of lysosomes, 

and showed that the Ca response to endothelin-1 but not 

PGF 2 was reduced. They suggested that this lysosomal 

store was perinuclear and situated in close proximity (0.4 

m) to RyRs on the SR, a “trigger zone.” NAADP alone 


was unable to generate global Ca waves. Similar findings, 

i.e., bafilomycin decreases NAADP-induced Ca release 

and contraction, were obtained in coronary arterial myocytes 

(800). These authors also used lipid bilayers to 

demonstrate that NAADP does not act directly on RyRs. 

In myometrial cells, the microsomal fraction releasing Ca 

in response to NAADP was inhibited by a drug glycyl-Lphenylalanine--naphthylamide 

(GPN) that lyses lysosomes, 

but SR inhibitors had no effect (650). This study 

also showed close apposition of fluorescent signals for 

lysosomes (lysotracker red) and the SR (BODIPY-TG) in 

myometrial cells. In peritubular myocytes, the NAADP 

signaling pathway has been shown to act via endothelin-1 

receptor subtype B (ET B), and lipid raft integrity is 

needed for the endothelin-NAADP signaling pathway to 

operate (200), possibly due to the location of both ET B 

and CD38 in this compartment. Recently, Calcraft et al. 

(100) identified the NAADP release channel as two pore 

channels (TRCPs) and showed an involvement of IP 3Rs in 

amplifying the NAADP Ca signals. 

E. SR Ca Leak 


As with the plasma membrane, there will be some 

low level leak of Ca through the SR membrane, i.e., a 

constitutive nonregulated process. Once luminal Ca indicators 

had been developed it was possible to directly 

monitor the decrease in SR [Ca] that occurred in the 

absence of stimulation. Before this the leak was assumed 

to be the mechanism of SR Ca depletion following inhibition 

of SERCA activity, and surrogates such as spark 

frequency were used to monitor it. That the leak of Ca 

from the SR or ER is not via Ca release channels (573) has 

been shown by, for example, blocking these channels and 

monitoring a decrease in luminal Ca content following 

SERCA inhibition, often in permeabilized cells. Indeed, it 

is easy to argue that this leak, or passive efflux, must be 

present to balance the basal SERCA activity (266). The 

nature of this leak is still an enigma (101). 

Although the SR Ca leak will not be energy dependent, 

Hofer et al. (266) presented data in a fibroblast cell 

line that shows it to be ATP regulated; more Ca was lost 

at higher ATP concentration. This effect was very slow 

compared with Ca loss stimulated by IP 3. Clearly then, 

there can be a link between the metabolic state of the cell 

and SR [Ca]. We can speculate that if ATP is compromised, 

e.g., during hypoxia and ischemia, then a feedback 

regulator such as ATP, which would reduce leak, would 

also therefore reduce the need for SERCA activity. It has 

been calculated that SERCA activity is responsible for 

1–3% of a cell’s energy demand (573). 

In the smooth muscle cell line A7r5, Missiaen and 

colleagues (478, 480) investigated the kinetics of the leak 

in the absence of IP 3. They found a nonexponential leak


that persisted after reducing SR Ca content to 40% of its 

initial value. At extremely low [IP 3], Ca efflux via the leak 

exceeded IP 3-induced Ca efflux. These authors also speculated 

that there is heterogeneity of Ca stores with respect 

to this leak, perhaps between peripheral and central 

SR. Additional support for a nonrelease channel leak has 

come from experiments in permeabilized pancreatic acinar 

cells, where inhibitors of RyR, IP 3R, and Ca release by 

NAADP were used, and still an unchanged basal leak 

occurred from the ER (408). This group also found a 

consistent leak rate over a broad range of SR Ca loads 

(486). Although it may be SERCA isoform specific, there is 

little reason to think that the leak is due to reverse mode 

of SERCA in smooth muscle cells, as has been suggested 

for cardiac muscle (627, 646). 

To date, there appear to have been but a handful of 

papers examining the nature and extent of the SR Ca leak 

in smooth muscles. The amount of Ca reported upon 

blocking SERCA has varied, with substantial depletion of 

the SR seen in cells from uterus (633), stomach (753), and 

(cultured) aorta (478, 706) and very little depletion in 

bladder (218), (toad) gastric (661), and (cultured) uterus 

(792). These differences are consistent with estimates 

made in other cell types of leak rates from 10 to 200 

mol/min (see Camello et al., Ref. 101). 

For A7r5 cells the rate of loss was, expressed as a 

maximal fractional Ca content loss, 22%/min (478). Thus it 

is clear that in several smooth muscles, the SR leak could 

rapidly (few minutes) deplete the SR of Ca. However, it 

should be cautioned that none of these measurements has 

been made under physiological conditions, as isolated, 

permeabilized cells, with a pharmacopeia of inhibitors 

are required for these studies. Thus, in smooth muscle 

tissues, the contribution of the leak to setting luminal 

Ca levels and its modulation during stimulation to affect 

functional responses remains to be better investigated. 

F. Summary 

In this section we have reviewed SR Ca release mechanisms, 

their modulation by pharmacological and endogenous 

agents, and the process of “leak.” Molecular and 

pharmacological tools along with knockout animals have 

greatly increased our understanding of how Ca is released 

from the SR of smooth muscle cells. With the existence of 

these different Ca-releasing mechanisms, along with the 

different mechanisms of modulation, isoform expression, 

and cellular distribution, smooth muscle myocytes appear 

to be the most complex of all cells. Certainly striated 

myocytes as well as neuronal and secretory cell types are 

not as rich in their Ca release processes. As we have 

noted elsewhere, we consider this to be a reflection of the 

wide range of functions and phenotypic lability in smooth 

muscle cells. 

VI. LUMINAL CALCIUM 



Some of the first evidence that the SR in smooth 

muscle is a Ca storage site was obtained histochemically 

after calcium had been precipitated (147, 259, 567). Better 

spatial resolution and the ability to look simultaneously at 

several ions came with the technique of electron-probe 

microanalysis, used to determine elemental concentrations 

in different regions of the myocyte. This approach 

led to measurements of [Ca] in the SR of 30–50 mmol/kg 

dry wt or 2–5 mM wet wt (69, 371), i.e., considerably 

higher than in the cytoplasm. These studies also detected 

a decrease in SR [Ca] with stimulation (655). There are, 

however, numerous limitations to this approach, such as 

the need for fixation and specialized apparatus and the 

small dynamic range. Determination of 45 Ca efflux can 

provide some indication of SR function (393, 477), but 

large background counts from 45 Ca bound to Ca-binding 

proteins, and nonspecific leaks, severely limit its usefulness. 

In cardiac muscle, 19 F-NMR has been used with a 

difluorinated version of BAPTA. The advantage of this 

technique is that by alternating the obtaining of 19 F data 

with 31 P spectra, near-simultaneous information on ATP 

and phosphocreatine and pH can also be obtained. 

B. Fluorescent Indicators for SR Luminal 

Ca Measurement 

Other methods have been sought for measuring SR 

[Ca], of which fluorescent indicators appear the most 

promising. As Zou et al. (813) put it, “there is a strong 

need to develop Ca sensors capable of real-time quantitative 

Ca concentration measurements in specific subcellular 

environments without using natural Ca binding proteins 

such as calmodulin, which themselves participate as 

signaling molecules in cells.” In this paper they describe 

the development of one such set of sensors with K d values 

ranging from 0.4 to 2 mM. The [Ca] in the SR is such that 

K d values in this range are required. This along with 

selectivity (for Ca and the SR) and considerations of what 

is bound and what is free Ca, make measuring SR/ER Ca 

nontrivial. For a commentary on these issues around SR 

[Ca] determination, see Bygrave and Benedetti (96), and 

for a general review of [Ca] measurement, see Takahashi 

et al. (684). 

Luminal [Ca] measurements to date in smooth muscles 

have only been reported for a limited number of 

tissues. Most of the studies have used the approach of 

fluorescent indicators such as Mag-fura 2 (K d 49 M; 

Ref. 667), which was originally developed as a probe for 

magnesium, and fluo 5-N, Furaptra (e.g., Ref. 667). Some 

indicators originally considered for cytoplasmic studies

were found to preferentially enter organelles, and do so in 

a reasonably selective manner in some cell types. For 

example, rhod 2 will accumulate in mitochondria. In the 

case of the SR indicators originally developed to monitor 

Mg, they load relatively easily into the SR and can be used 

to report luminal [Ca]. These indicators include Mag-fura 

5, Mag-fura 2, Mag-indo 1, and Furaptra, and it may be 

because of the particular efficiency of hydrolysis within 

the SR that they are accumulated within it (667). Indicators 

with less Mg dependence include fluo 3FF and fura 

2FF, both of which have been used in smooth muscles 

(214, 792). Little is known about SR [Mg] in smooth 

muscle or indeed many other cell types; Sugiyama and 

Goldman (667) estimated it to be 1 mM. These authors 

also concluded that Furaptra (another probe sensitive to 

Mg, with a K d of 6 mM and for Ca of 50 M), could be 

used to determine SR [Ca], without Mg impacting on its 

usefulness. In the aortic cell line A7r5, they estimated 

luminal [Ca] to be 75–130 M. 

Another approach used to investigate luminal Ca levels 

is to modulate its buffer capacity by using compounds 

that can quickly diffuse into the SR and bind Ca. As with 

Ca indicators for the SR, the K d for such compounds has 

to be in the high micromolar range, i.e., low affinity. The 

heavy metal chelator TPEN with a K d around 200–500 M 

is such a compound (108, 571). However, TPEN may also 

increase the P o of RyRs (327). 

C. Luminal Ca Buffers 

Calcium binding proteins are present in the cytosol 

and intracellular organelles (703). Their role in maintaining 

a low cytosolic [Ca] and compartments capable of 

storing Ca at relatively high concentration, respectively, is 

crucial to Ca acting as a ubiquitous second messenger. 

Soluble (nonmembranous) Ca-binding proteins buffer Ca 

but are limited by their finite amount. Calcium pumps 

such as SERCA may also be viewed as membrane-bound 

Ca-bindings proteins, as they act to bind Ca, transport it 

across their constituent membrane, release it, and are 

then ready to complex Ca again. As such, SERCA makes 

a large contribution as a Ca-binding protein and a very 

important and efficient regulator of [Ca] inside cells (105). 

It has been estimated that cytoplasmic Ca buffering takes 

up 84 of every 85 Ca ions entering gastric smooth muscle 

cells (230) and 30–46:1 in bladder myocytes (145, 204). 

Although cells contain millimolar [Ca], only 0.01% 

is free ionized Ca. High-affinity Ca binding proteins, such 

as calmodulin, are present in large amounts in the cytoplasm 

of all mammalian cells and contribute to Ca sensitizing 

and exchange. Calmodulin is the best studied of the 

endogenous Ca sensors (proteins with high affinity and 

low capacity) in smooth muscle (653). It binds Ca with a 

K d of 2 M (319) and is present at 40 M (336). In 


early studies of [Ca] within smooth muscle cells (rabbit 

portal vein) using electron-probe microanalysis, it was 

concluded that there was not sufficient calmodulin to 

account for the [Ca] measurements made (66). In a recent 

study of cytosolic Ca buffering in single smooth muscle 

cells from guinea pig bladder, it was concluded that the 

Ca buffers present have a low affinity (EF-hand) for free 

Ca, are present at 530 M, and have a calculated binding 

ratio of 40 (145). In this study the volume of the SR was 

taken to be 2%, which may be an underestimate. Other 

studies in smooth muscles have yielded similar values for 

the ratio of Ca binding: airways (178), bladder (204), 

larger portal vein (329), stomach (349). 

Within the SR lumen, specific proteins are expressed 

to bind Ca. These SR luminal Ca storage proteins can be 

considered to act as buffers, as they keep Ca relatively 

loosely bound and thus available for quick release, and 

help prevent insoluble calcium phosphate precipitating. 

By reducing the amount of free or ionized Ca in the SR, 

they also reduce inhibition by Ca of SERCA activity. Thus 

the expression of these Ca binding proteins is critical to 

the SR’s ability to act as a Ca store, in all cells. 

Although many proteins capable of binding Ca may 

be expressed in the SR, the consideration here is of those 

which bind considerable quantities of Ca and which appear 

to have this as their prime role within the SR lumen. 

There are just two protein families that fulfil this description: 

calreticulin and calsequestrin. As described already 

in section IV, E and F, they are both high-capacity (25–50 

mol/mol), low-affinity (1–4 mM) Ca binding proteins that 

function only or primarily to dynamically store Ca (384, 

597). These two proteins have similar Ca-binding properties, 

molecular weights, and other protein properties, 

such as acidic isoelectric points (573). As noted above, 

specific data for either protein in smooth muscle are not 

abundant, or there have been controversies in the literature 

(472, 730) and little recent work. 

D. Measurements 


Not withstanding caveats around indicator specificities, 

K d values, Mg sensitivity, and calibration methods, a 

few investigators have estimated/measured the SR luminal 

[Ca] (214, 636, 667, 755, 811). Not unexpectedly, there 

is a range of values, from 60 to 150 M, but this range is 

perhaps surprisingly small given the difficulties involved. 

It would therefore appear that the SR free [Ca] is less 

in smooth muscles compared with other cell types, where 

values of 200–1,500 M have been reported (33, 118, 121, 

268). However, until more measurements in native 

smooth muscle cells are made, it is unproductive to draw 

further conclusions.


E. Effects of SR Ca Load 


How luminal SR Ca content relates to a change in 

cytosolic [Ca] is not easy to predict or determine. This is 

because there is feedback from cytosolic [Ca] onto the SR 

release channels (282) and a difference in buffering capacity 

between the SR and the cytoplasm (230). In cardiac 

muscles where a proper quantitative study can be made, 

the nonlinearity between the two can be steep (704). This 

has not been as well studied in smooth muscle, but the 

following study also points to a steep relation. 

In rat uterine cells we have shown that increasing 

external [Ca] from 2 to 10 mM increases cytosolic [Ca], 

from 120 to 270 nM, and is associated with a substantial 

rise (17% increase compared with steady-state value) in 

SR [Ca] (636). This elevation of SR [Ca], however, did not 

lead to any potentiation of the Ca transient elicited in 

response to the IP3-generating agonist ATP. If the SR load 

was decreased (but not emptied) from its steady-state 

level, the ATP-induced Ca transients fell. If the SR load 

was decreased below 80% of normal, no Ca transients to 

ATP could be elicited. Thus there is a steep dependence 

of IP3-induced SR Ca release with SR load as it depletes, 

but not as it overloads, at least in uterine myocytes. 

2. Relation between SR Ca load and Ca signals 

The effects of altering SERCA activity on global Ca 

signals and function in smooth muscle cells are discussed 

in section VII, C and D. Here we will focus on how SERCA 

activity affects SR luminal [Ca] and then Ca release 

events. Blocking SERCA, e.g., with thapsigargin or CPA, 

has been shown by us and others to produce a decrease in 

SR Ca content, which is accelerated with agonist stimulation 

(16, 633, 661). In our study of uterine cells, we 

simultaneously measured cytosolic and luminal [Ca] 

changes and compared the depletion of SR Ca in response 

to agonist stimulation, with and without SERCA activity 

(633). We found that the depletion was greater when 

SERCA was inhibited and the intracellular Ca transients 

were smaller. In a follow up study we found that the rise 

in cytosolic [Ca] seen with increasing external [Ca] was 

significantly increased, from 270 to 320 nM, when 

SERCA was inhibited with CPA (636). Removal of external 

Ca produced a fall in cytosolic [Ca] and SR [Ca]. The 

effects of repetitive agonist applications under conditions 

of low and high SR [Ca] and with and without SERCA are 

also described by Shmygol and Wray (636). 

From these and other studies we can confidently say 

that agonists lower SR [Ca] and SERCA activity is required 

to restore, and presumably maintain, the ability of 

smooth muscle cells to respond to agonists. As a rundown 

of luminal [Ca] would be anticipated to rapidly curtail 

agonist-induced Ca signals, it can be assumed that under 


normal physiological conditions SERCA is active during 

stimulation to refill the store, although as noted by Gomez- 

Viquez et al. (218), the turnover rate of SERCA is slower 

than that of the release channels, being 3.5 Ca/s for 

SERCA2b compared with 1 10 6 Ca/s through RyR 

(462). Given the available data on SERCA and RyR expression, 

the number of SERCA pumps is not sufficient to 

compensate for the RyR Ca efflux. 

In their interesting study, Gomez-Viquez et al. (218) 

explored the relation between SERCA pump activity and 

SR Ca release channels in urinary bladder myocytes. They 

investigated whether SERCA activity per se rather than 

luminal [Ca] could influence Ca release events, or if uptake 

and release operate independently of each other, 

assuming there is sufficient [Ca] in the SR lumen. They 

rapidly and completely inhibited SERCA (thapsigargin) so 

that luminal [Ca] would be maintained (as verified from 

the Mag-fura 2 signals). With SERCA inhibited, the AChor 

caffeine-induced Ca release into the cytoplasm, as 

measured with fura 2, was reduced in both amplitude and 

rate of rise. Thus SERCA2b in bladder smooth muscle 

cells can modulate both IP 3R and RyR Ca release events; 

SERCA activity is required for optimal Ca release. The 

authors speculate that SERCA2b pumps are modulating 

Ca availability in the SR. As described earlier, the 

SERCA2b isoform contains a carboxy tail extension that 

interacts with luminal Ca binding proteins, suggesting a 

molecular and structural mechanism for this effect. 

SERCA activity can indirectly modulate Ca release 

events through setting the [Ca] load in the SR. Both IP 3Rs 

and RyRs have been shown, in lipid bilayer experiments, 

to have their activity affected by luminal SR [Ca] (477, 

642; see sect. V). In turn, it is SERCA activity that loads the 

SR with Ca (661). ZhuGe et al. (811) working on gastric 

myocytes noted the effects of luminal load by detecting 

Ca sparks and STOCs at different Ca loads. They found 

the frequency of both increased with load. A similar relation 

between the SR Ca load (altered by knocking out 

phospholamban) and Ca sparks had previously been reported 

in cardiac muscle (614). The effects of SR Ca load 

on STOCS was also confirmed by others (123, 452). It was 

found that a relatively small fall in SR [Ca] (16%) had a 

profound effect on STOC frequency (decreased by 70%) 

(452). As mentioned earlier, in uterine myocytes, overloading 

the SR with Ca did not increase the simultaneously 

measured cytosolic Ca transient in response to 

ATP stimulation (636). In marked contrast, partial depletion 

of the SR Ca substantially reduced the amplitude of 

the ATP-induced Ca release: a reduction to 80% of normal 

luminal SR Ca content abolished responses. Previous 

work had shown that uptake of Ca into the SR occurred 

even in the absence of external Ca, although this only 

replenished luminal Ca to 50% of its resting value (633). 

Thapsigargin abolished both subsequent ATP-evoked Ca 

release and SR refilling. Interestingly, this study found no

changes in luminal [Ca] during spontaneous activity, suggesting 

no role for the SR in this process, but rather 

supporting the view that L-type Ca entry is both necessary 

and sufficient for normal phasic activity in the uterus, and 

store-operated Ca influx is not necessary. When the SR 

was overloaded, spontaneous activity was abolished, consistent 

with a negative feedback from the SR on E-C 

coupling in the uterus, despite the lack of Ca sparks (88). 

F. Conclusions 

While it is well accepted that luminal [Ca] is a very 

important control regulator of both SR function and Ca 

signaling, the field would be well served by some additional 

studies in this area. For example, additional calibrated 

measurements of luminal [Ca] would clarify if the 

value in smooth muscles is 100 M, i.e., considerably 

lower than striated muscles. Will values be lower in tissues 

like uterus where the SR may not contribute to 

rhythmic activity, compared for example to blood vessels, 

which rely more heavily on the SR Ca store? Similarly, 

more simultaneous measurements of cytosolic and luminal 

[Ca] would add to our knowledge of how SR Ca load 

affects Ca signaling. Does the SR release Ca during phasic 

activity? Is SR Ca overload inhibitory to contractility in 

smooth muscles? How do changes in the expression of SR 

Ca buffers affect SR Ca load? Although these experiments 

are technically challenging, especially when combined 

with electrophysiological studies, the insight gained will 

be important and significant to the field. 

VII. CALCIUM REUPTAKE MECHANISMS 


In section III, the structure of SERCA and its isoforms 

and catalytic cycle were described. The ability of SERCA 

to take up Ca is of course crucial to the ability of the SR 

to act as a Ca store. In this section we review the effect of 

Ca uptake by SERCA on cytosolic [Ca] levels and Ca 

signals, its role in plasma membrane Ca extrusion, and the 

effects of inhibiting its activity either pharmacologically 

or molecularly. (Discussions of how phospholamban and 

related proteins affect SERCA were dealt with in section 

IV.) This is then followed by a review of store-operated Ca 

entry in smooth muscle, exploring the relation between 

luminal Ca levels and Ca entry mechanisms that replenish 

the SR with Ca. 

B. SERCA Uptake of Ca and Ca Homeostasis 

For the cell to maintain low cytosolic [Ca] with respect 

to both the extracellular fluid and the SR, requires 



not just SERCA activity, but also that of the PMCA and 

NCX. For the cell to be in a steady state, Ca entering the 

cell across the plasma membrane must be removed by 

PMCA and NCX, and Ca released from the SR must be 

retaken up by SERCA. This will also be the case for Ca 

uptake into organelles such as the nucleus and mitochondria. 

There will be basal or “resting” SERCA activity, 

which presumably balances SR Ca leaks, as well as spontaneous 

Ca releases from the SR, Ca puffs, and sparks. 

SERCA activity is stimulated when cytosolic [Ca], from 

whatever source, rises and the presence of the luminal Ca 

buffers, calsequestrin and calreticulin, ensures that inhibition 

of SERCA by luminal Ca does not occur. The SR 

will contribute to plasma membrane Ca efflux by vectorially 

releasing Ca towards efflux sites. As with many 

other aspects of smooth muscle physiology, the exact 

contribution of SERCA, PMCA, and NCX will differ between 

them, depending on expression levels and distribution. 

Perhaps the easiest way to uncover the role of 

SERCA in maintaining cytosolic Ca levels is to determine 

the effects of its inhibition. As mentioned already, there 

are two specific inhibitors of SERCA: thapsigargin, which 

is irreversible, and CPA, which is reversible. When 

SERCA is inhibited in smooth muscle cells there is a rise 

in cytoplasmic [Ca]. This rise of [Ca] can be fast or slow 

and then plateaus, leading to an elevated resting [Ca]. Due 

to the difficulties of calibrating Ca signals, many studies 

have simply reported a rise of [Ca] with SERCA inhibition, 

perhaps expressed relative to high-K depolarization or 

some other calibrator. Some of the first studies to explore 

the effect of CPA were on vascular myocytes (1, 120) and 

used aequorin to monitor [Ca]. With CPA there was a 

slow, i.e., minutes, increase in the Ca signal and basal 

tension. The response to norepinephrine was decreased. 

Rises in [Ca] in response to SERCA inhibition have also 

been shown in bladder myocytes (from 137 to 471 nM; 

Ref. 789) and uterine myocytes (from 120 to 320 nM; Ref. 

636). A study of airway myocytes, however, found no 

significant change in resting [Ca] (130 nM) after 25 min 

in 0-Ca and CPA solution (603), perhaps due to increased 

Ca efflux under these conditions. 

C. SERCA and Ca Signaling and Contractility 

Both pharmaceutical SERCA inhibition and to a more 

limited extent gene ablation have been used to investigate 

the role of SERCA in Ca signaling in smooth muscles. 

When SERCA2 was reduced to 50% and aortic contractility 

measured, no changes from wild-type mice were 

found in response to KCl and phenylephrine (294). This 

contrasts with the significant effects found on cardiac 

contractility, including decreases in Ca transients and cell 

shortening (558). It is not clear whether the lack of effect


in the vascular preparation is due to increased SERCA 

reserve compared with the heart or some form of compensatory 

regulation of other parameters. When CPA is 

used to inhibit SERCA in blood vessels, clear effects are 

seen (504). When SERCA3-deficient mice were examined, 

the relaxation to ACh was reduced in aorta (402) and to 

substance P in trachea (334); however, as discussed earlier, 

these effects are not direct on the myocytes, but 

rather are due to endothelial- and epithelial-dependent 

effects. No overt phenotype is associated with the 

SERCA3 knockout (402). This appears to be the extent of 

the literature on gene targeting studies of SERCA function 

in smooth muscle. There is, however, a more extensive 

literature concerning effects obtained with CPA and thapsigargin. 

In most smooth muscles, a clear elevation of 

baseline tension is found with SERCA inhibition, and 

when measured, this can be seen to be accompanied by 

increased global Ca signals [ileal (710), aorta (417, 629), 

fundus (562), esophageal (640), uterus (377, 446, 682)]. 

Before the link between SR Ca release and BK 

channels and membrane hyperpolarizations were elucidated, 

many authors interpreted their data, i.e., SERCA 

inhibition increases [Ca] and contraction, in terms of 

the SR blocking or buffering the flow of Ca to the 

myofilaments. The spark-STOC coupling mechanisms, 

however, can directly account for the effect of SERCA 

inhibition in many, but importantly, not all smooth 

muscles. The effects of SERCA inhibition on Ca signaling 

and contractility in phasic smooth muscles can be 

very pronounced. For example, in the neonatal uterus 

(519) or term myometrium (707), the normal phasic 

activity becomes tonic-like. 

D. SERCA Ca Efflux and Relaxation 

Although SERCA takes up Ca and aids restoration of 

[Ca] to resting levels following smooth muscle stimulation, 

there is evidence from uterine smooth muscle to 

show that acting on its own, it does not significantly alter 

the decay of the Ca transient, but rather, it enhances 

efflux mechanisms. In isolated uterine myocytes (631) 

when PMCA and NCX were blocked, intracellular [Ca] did 

not fall after stimulation (644). However, if SERCA was 

blocked, but not PMCA and NCX, then the decay of the Ca 

transient was slowed. This was the case whether Ca efflux 

was via PMCA or NCX or both. We concluded that when 

SERCA is active, it takes up a portion of Ca entering the 

cell, and the SR then releases Ca in a directed manner 

towards the plasma membrane extruders, thereby maximizing 

their efficiency (see also Refs. 184, 445). A study in 

airway myocytes also concluded that the decay of the Ca 

transient was not altered by SERCA, although it is active 

(603). These authors suggested that mitochondria take up 

Ca and contribute to the decrease in [Ca]. In an earlier 

study in isolated tracheal myocytes, SERCA had been 

shown to contribute to the decay of agonist-induced Ca 

transients as the decay was slower when SERCA was 

inhibited (640). 

E. Store-Operated Ca Entry 



The Ca content of the SR acts as a powerful factor 

controlling the open probabilities of both RyR and IP 3R 

channels (see sects. IV and VII). Depletion of Ca stores in 

smooth muscle cells by SERCA pump inhibitors results in 

an inhibition of Ca sparks and STOCs in those tissues 

producing them. This in turn can lead to depolarization of 

the cell membrane and activation of Ca entry via L-type 

Ca channels, as was reported for rat cerebral arteries 

(507). However, in addition to activation of L-type Ca 

channels, depletion of SR Ca can also activate nifedipineresistant 

Ca entry, through opening of Ca release-activated 

Ca channels (CRAC). In nonexcitable cells, ER Ca 

depletion opens voltage-independent plasma membrane Ca 

channels through which extracellular Ca enters the cell to 

refill the store. These are termed “capacitative calcium entry” 

(CCE) channels (542, 579, 580). The role of CCE, also 

known as store-operated Ca entry (SOCE) and Ca entry 

mechanism across the sarcolemma of the smooth muscles, 

is still being determined. 

Recent work has identified the specific molecular 

components, Stim and Orai, which constitute the Ca sensor 

in the ER/SR and the Ca channel, respectively, and 

their discovery has helped to clarify the mechanisms of 

CCE. The process and mechanisms of SOCE have been 

best studied in nonexcitable cells, where the process was 

first discovered, and studies in smooth muscle are relatively 

sparse. Therefore, an overview of current knowledge 

taken from the literature on nonexcitable cells will 

be given to set the scene for smooth muscle. 

2. Overview of store depletion, Orai, STIM1, 

and TRPCs 

This account is based on information in several recent 

reviews that have appeared since the discovery of 

Stim (stromal interaction molecule 1) proteins (99, 173, 

189, 260, 524, 569, 581, 649). 

Agonist binding to cell membranes and subsequent 

generation of IP 3 leads not only to ER Ca release, but also 

to activation of Ca channels in the plasma membrane. 

This connected depletion and loading of the ER Ca store 

was termed capacitative Ca entry by Putney in 1986 (578) 

and is synonymous with the term store-operated Ca entry. 

This form of Ca entry is the major route of Ca entry in 

nonexcitable cells, which lack functional voltage-gated Ca 

channels. It was subsequently demonstrated that the Ca

entry could be stimulated without agonist or IP 3, if the ER 

Ca was depleted, e.g., using SERCA inhibitors, particularly 

thapsigargin (545, 686). Detecting the rise of cytoplasmic 

[Ca] and the associated small Ca release-activated 

Ca current (I crac) (272) resulting from this store 

depletion-activated Ca entry was difficult. This led to the 

suggestion from experiments on arterial myocytes that 

the refilling of the SR occurred by-passing the cytoplasm, 

i.e., direct coupling between the plasma membrane and 

SR, or that the Ca was in a restricted/protected space 

between the two, and not accessible by the myofilaments, 

as no contraction occurred (111). However, given the 

existence of I crac, in both excitable and nonexcitable 

cells, Ca clearly is entering the cytoplasm, and the failure 

to detect a rise in [Ca] in most cell types is now attributed 

to the close proximity between ER/SR and the plasma 

membrane and the rapid buffering of the Ca entry by the 

ER/SR. 

Much experimental effort was put into elucidating 

the molecular events between the ER and plasma membrane, 

which could activate the capacitative store-operated 

Ca entry. Until a few years ago, the discussion would 

be focused around canonical TRPCs, which were discovered 

in Drosophila and shown to be activated after an 

increase in PLC activity (245). These channels, expressed 

in a large variety of isoforms in mammalian cells, almost 

became accepted as “the” store-operated channels responsible 

for capacitative Ca entry. However, as discussed 

by Putney (581), these reports were often from 

overexpressing or constitutively active preparations and 

the TRPC channels did not reproduce the known properties 

of I crac and were usually cationic rather than Caselective 

channels. 

While it is too early to be categorical, the evidence 

that TRPC channels are responsible for capacitative Ca 

entry is not strong. It is also the case that since 2006 new 

putative channels in the form of Orai proteins have been 

discovered, which appear to be much better candidates. 

Orai is a plasma membrane protein which when mutated 

results in abrogation of I crac and severe combined 

immunodeficiency syndrome (174). It appears to be unlike 

other previously described Ca channels. Mammalian 

cells can express three types of Orai, 1–3, and all are 

capable of forming Ca channels, probably as homotetramers. 

A recent paper suggests that Orai is primarily a dimer 

in the plasma membrane under resting conditions, and 

upon activation by the COOH terminus of Stim, the 

dimers dimerize, forming tetramers that constitute the 

Ca-selective channel (553). These and other data make it 

hard to propose that TRPC channels may contribute to 

capacitative Ca entry via inclusion in an Orai/TRPC Ca 

channel (153, 398). Orai1 forms the most conductive Ca 

channels, but Orai2 and -3 are also able to perform this 

function (464). Orai proteins have four transmembrane 

domains, and the first of these may be most important for 



channel function (575, 788). When Orai1 is expressed with 

another protein, Stim1, large I crac currents are observed 

(552, 804). 

Stim1 was discovered as a cell surface protein (530) 

and was related to the ER in 2005 (400, 595) as its Ca 

sensor, responsible for activating capacitative Ca entry. 

Thus Stim1 knockout reduces I crac and SOCE. Stim1 is a 

phosphoprotein with a single transmembrane domain. 

The NH 2 terminus of Stim has an EF hand domain in the 

ER lumen that is considered to be the sensor of luminal 

[Ca]. It has been calculated that the EC 50 for Stim1 redistribution 

is between 200 and 400 M luminal Ca (82, 651, 

660) and that oligomerization of about four Stim1 proteins 

occurs with the conformational change in the NH 2 terminus 

as Ca dissociates. This oligomerization is necessary 

for activation of I crac (575, 788), and mutation of this 

region produces constitutive activation of Ca entry (464). 

Both Stim and Orai are required to produce I crac. Although 

not completely clear, the mechanism of activation appears 

to involve a redistribution of Stim1 in the ER when 

Ca dissociates from it, i.e., as the ER depletes. This redistribution 

brings Stim closer to the plasma membrane 

(within 10–25 m) in a punctate pattern on the ER, with 

some workers (541) but not all (148) suggesting that it 

takes up a lipid raft distribution (541). This redistribution 

that precedes I crac by 6–10 s (775) presumably enables 

Stim1 to directly interact with Orai channels, and Ca 

enters in these discrete regions of the plasma membrane 

(416). Evidence for this comes from a variety of biophysical 

and imaging techniques as well as coimmunoprecipitation. 

It may be, however, that there is no direct coupling 

between Orai and Stim and another factor, such as “Cainflux 

factor” (CIF), is stimulated by Stim redistribution 

and CIF (or another messenger) is needed for Orai activation, 

perhaps involving activation of phospholipase A 2 

by CIF displacing calmodulin (63). Another protein identified 

in the Stim family, Stim2, is structurally 60% identical 

to Stim1, but does not appear to play any major role 

in capacitative Ca entry (595), but may contribute to 

regulating basal [Ca] via ER Ca sensing (82). 

Most of the studies mentioned above were performed 

on immune or exocrine cells. How much evidence is there 

for these mechanisms and proteins in smooth muscle? 

Although only a few years from their discovery, the excitement 

around Stim and Orai has already led to studies 

investigating their expression and role in smooth muscle 

tissues. 

3. Orai, Stim, and TRPCs in smooth muscle 

The study of capacitative/store-operated Ca entry in 

smooth muscles is more complicated than it is in nonexcitable 

cells, due to the greater variety of Ca entry mechanisms, 

i.e., voltage-gated and receptor-operated channels, 

in addition to channels that are opened in response


to a depletion of SR Ca. Previous work had investigated 

TRPC channels as the putative Ca entry mechanism 

opened by store Ca depletion, but the growing consensus 

is that they are not responsible for I crac. Therefore, it may 

be argued that TRPCs are unlikely to be responsible for 

SOCE in smooth muscles. This has already been suggested 

by some (153, 722, 738) but contested by others 

(e.g., Ref. 7). TRPC1, the TRP most thought to be involved 

in SOCE and highly expressed in smooth muscles, had no 

effect on vascular function or SOCE when knocked out 

(12). In TRPC6 knockout mice, enhanced DAG and agonist-induced 

current in smooth muscle cells was reported, 

i.e., the opposite result expected if TRPC6 is involved in 

SOCE (188). RNA interference (RNAi)-based high-throughput 

screens also revealed no effect on SOCE activation of 

TRP channel knockdown, in contrast to Stim (400). As 

with studies on nonexcitable cells, caution must be applied 

to studies where genes have been knocked out or 

overexpressed, and in smooth muscle, when cells have 

been cultured, the physiological relevance of such studies 

is even harder to interpret (274). Nevertheless, while it is 

premature to rule TRPCs out of involvement in SOCE in 

smooth muscle cells, especially as much of the Orai work 

has been conducted on nonexcitable cells, it nevertheless 

does not appear profitable to cover in great depth previous 

studies related to a role in SOCE of TRPC, especially 

as this has recently been reviewed (7). A recent report by 

Dehaven et al. (148) demonstrated that TRPC functions 

independently of Orai1 in vascular myocytes (A7r5 and 

A10 cell lines) and suggests that TRPCs are activated by 

PLC and do not involve Stim1 as the Ca sensor and TRCPs 

open nonselective cation channels when stimulated by 

arginine vasopressin. Roles for TRCPs other than in storeoperated 

Ca entry in smooth muscle have been well demonstrated 

for receptor-operated channels, where they 

constitute nonspecific cation channels (39, 457) and 

stretch channels (658), but these are outside the scope of 

this review. 

There is already evidence to support the Stim-Orai 

model for SOCE in some smooth muscles: airway, Stim-1 

(549), Orai (550); vascular, Stim (153, 413, 685), Stim and 

Orai (47, 210); and uterus, Stim and Orai (611). The list of 

smooth muscles expressing Stim and Orai is expected to 

grow rapidly as more studies are performed. To date 

however, there is already evidence that capacitative Ca 

entry/SOCE in some smooth muscles at least occurs via 

the Stim-Orai mechanism. Dietrich et al. (153) inhibited 

SOCE using siRNA against Stim1, whereas TRCP1 knockout 

did not affect it. Further support for a physiological 

function for these proteins in smooth muscles comes 

from data showing that their expression levels are altered 

at different sites and in disease. Thus Orai and Stim were 

upregulated in proliferating arterial myocytes, leading the 

authors to suggest that they play a critical role in the 

altered Ca handling that occurs during vascular growth 


and remodeling (47). Stim1 expression was greater in the 

distal than proximal part of the pulmonary artery, where 

SOCE is also greatest (413). Stim1 and Orai1 were also 

found to be upregulated in aortas from hypertensive rats 

(210). Knockdown of Orai1 reduced thapsigargin-induced 

Ca influx and reduced I crac (210). 

Thus, in summary, Orai and Stim appear to constitute 

the molecular mechanism underlying capacitative Ca entry 

and I crac in nonexcitable cells, and there is mounting 

evidence for this also to be the case in smooth muscle. To 

us, the most urgent questions revolve around whether 

there is only one form of SOCE in smooth muscle and if 

this involves only the Stim-Orai mechanism. The relative 

simplicity of Ca entry in nonexcitable cells falls away in 

studies of smooth muscle, as SOCE is a variable feature 

between myocytes, and several other Ca entry pathways 

exist. In addition, there are only relatively few studies that 

have gone beyond demonstrating a putative SOCE, i.e., 

mechanistic details, measurement of I crac, and biophysical 

and imaging studies are sparse, leading to interpretation 

based on only a few myocyte preparations. The interaction 

of TRPCs and Orai channels, if any, also requires 

resolution. 

4. Evidence for store-operated Ca entry 

in smooth muscle 

As one might anticipate from knowing that the contribution 

of voltage-gated Ca entry varies between smooth 

muscles, being high for example in uterine (771) and 

intestinal (64) and low in many large blood vessels and 

tracheal myocytes (7), so the contribution of Ca entry 

through SOCE and/or other voltage-independent routes 

such as nonspecific receptor-operated cation channels 

(ROCs) shows considerable variation between smooth 

muscles (44). Although ROC activity is a feature of agonist 

binding, e.g., norepinephrine, and is important for 

their physiological effects (39), it does not involve SR Ca 

depletion, and therefore, these channels are not discussed 

in detail further. 

In smooth muscles, SOCE has been associated with a 

second, delayed Ca response to agonist applications; the 

IP 3 formed causes an initial fast and transient release of 

Ca from the SR, followed by the slower process of SOCE 

which produces a more or less sustained Ca signal, often 

of lower amplitude than the initial transient rise. If Ca 

sensitization mechanisms are also stimulated by the agonist, 

then the force responses will not be directly related 

to the Ca signal and force may continue long after [Ca] 

has fallen. 

A sustained increase in [Ca] due to extracellular Ca 

entry following agonist application has been taken as 

evidence for SOCE by many investigators, sometimes augmented 

by data from putative blockers of SOCE such as 

SKF96365 and La 3 . It is generally accepted that there are

no specific blockers of SOCE, so pharmacological approaches 

are helpful but not definitive. Separating SOCE 

from voltage-gated Ca channel and ROC-mediated Ca entry 

must also be accomplished. This is often done by not 

using agonists, depleting the SR of Ca using SERCA inhibitors, 

and preventing L-type Ca entry, and interpretation 

is further simplified by the use of single cells. Such 

conditions make it hard to relate the findings to physiological 

conditions. With the use of these approaches, rises 

in [Ca] considered to be due to SOCE have been reported 

in many smooth muscles, e.g., colonic (370), portal vein 

(538), cultured A10 cells (784), inferior vena cava (120), 

aorta (265), coronary arteries (731), ileal myocytes (526), 

pulmonary (216), esophagus (740), and gallbladder myocytes 

(492). 

There have been few studies in smooth muscle of the 

membrane currents or conductance changes associated 

with SOCE. Recordings of what would be called I crac in 

nonexcitable cells are often denoted as I soc in smooth 

muscle, underlining the uncertainty around whether 

SOCE in smooth muscle uses the same entry mechanism 

as in nonexcitable cells. An added complexity to the study 

of I crac/I soc in smooth muscle is that the plethora of ion 

channels means that any changes in cytoplasmic [Ca] due 

to SOCE could evoke changes in other currents, e.g., 

opening of Ca-activated Cl channels, giving rise to inward 

current and therefore complicating interpretation. Studies 

of I crac should ideally therefore be performed with heavy 

buffering of Ca, e.g., 10 mM EGTA or BAPTA. From 

experiments using whole cell and single-channel recordings, 

evidence for SOCE has been provided in a variety of 

smooth muscles. The first recording of I crac was made in 

mouse anococcygeous myocytes in 1996 (746). Subsequent 

recordings of a conductance elicited by store Ca 

depletion were made in portal vein (5, 403), mesenteric 

(609) and pulmonary arteries (216, 705), and airway (549). 

Thus, as expected, most evidence for significant SOCE 

comes from blood vessels, where reliance on L-type Ca 

entry is less than in many phasic muscles. 

Work particularly from Large’s group (7) has questioned 

whether SOCE is the same process in smooth 

muscle as it is in nonexcitable cells and if different forms 

of SOCE occur in smooth muscle cells. As discussed 

above, there is mounting evidence for Orai and Stim in 

smooth muscles, including vascular and airway, although 

this does not prove that I crac and SOCE are identical to 

those described in nonexcitable cells. In a careful review 

of the biophysical properties and activation mechanism 

for SOCE in a small number of different smooth muscles, 

Albert et al. (7) concluded from measurements of unitary 

conductance (not current) that the Ca permeability of 

SOCs in smooth muscle myocytes was much smaller than 

measured for I crac in nonexcitable cells and that therefore 

in myocytes SOCE is via nonspecific cation channels. 

Such channels will contribute to SR Ca refilling but also to 



depolarization of the myocytes. Albert et al. (7) argue that 

such channels may also have TRPCs associated with them 

and be activated by factors other than Stim; mention has 

been made of Ca-independent phospholipase A 2 (iPLA 2) 

activating SOCE (644), and there is also evidence for a 

role for PKC (6, 609). Finally, store-independent activation 

of SOCE involving PKC has been described by this 

group in portal vein (6), with the suggestion that there is 

a constitutively active driver of SOCE. 

What the relation of these channels is to classical 

SOCE/capacitative Ca entry remains to be established, 

but clearly such channels fall outside the conventional 

definition of SOCE. The use of single-channel recording in 

isolated patches of membrane clearly provides experimental 

clarity, but it is difficult to relate these events to 

physiological activity in intact cells or tissue, where [Ca] 

would not be clamped and Stim-Orai activity would be 

present. 

There are data which indicate that refilling of the 

store occurs directly from the myoplasm (267, 486). It was 

shown that when intracellular [Ca] was clamped around 

resting levels, using Ca chelators, store refilling occurred 

in the absence of external Ca, suggesting that mechanisms 

directly linking Ca influx to store refilling might not 

be necessary (54, 267, 486). Certainly, SR Ca uptake when 

cytoplasmic [Ca] rises (in the absence of external Ca) has 

been directly demonstrated (636). 

If the plasma membrane Ca leak is small and SERCA 

activity large, then many cycles of SR Ca release and 

uptake could occur without the need for Ca entry. In 

those smooth muscles where L-type Ca entry is large and 

agonists produce depolarization, then there is little need 

for a separate SOCE/CCE mechanism. For smooth muscles 

such as the uterus, SOCE may be a mechanism that is 

only important during the strong agonist-driven contractions 

of labor, and that is absent/unused at other times 

(518, 707). The heterogeneous nature of the stores (see 

sect. VII) may also contribute to differences in SOCE 

between smooth muscles. For example, in arterial myocytes, 

the ryanodine/caffeine-sensitive store was not sensitive 

to either thapsigargin or CPA, whereas the IP 3sensitive 

Ca store was depleted by both of them (307). 

From the results obtained on colonic myocytes, which 

have two functionally distinct SR Ca stores, it was suggested 

that the common store (S ) expressing both IP 3R 

and RyR was dependent on an external Ca source for 

replenishment, and the second store containing only RyR 

(S ) refills directly from the myoplasm (185). Also in 

colonic myocytes, the mechanisms that control Ca entry 

into the cytoplasm involve two types of channels, one of 

which is voltage sensitive and the other is voltage insensitive 

(453). It is also suggested that the influx of Ca via 

these two channels produces a high subplasmalemmal 

[Ca] accessed by the common store (453). However, other 

authors have found that the magnitude of SOCE in colonic


myocytes was the same irrespective of whether just IP 3R 

or IP 3R and RyR stores were depleted (370). The refilling 

mechanisms may depend on the location of the SR. Peripherally 

located stores may be functionally more closely 

linked to Ca entry via SOCE channels (215), while those 

located centrally could be refilled from the myoplasm. 

Another consideration is the type of SERCA associated 

with each of the stores. The Ca binding affinity (K m) for 

SERCA2a is 0.31 M and for SERCA2b is 0.17 M (479). 

SERCA2b activity can be modulated by calmodulin (229) 

and phospholamban (162), and again this could affect 

cross-talk between the store and plasma membrane. If 

different isoforms of SERCA are associated with different 

stores, this could explain differences in their ability to 

refill the SR in the presence of different [Ca]. 

6. Summary 

It seems to us, based on the current state of knowledge, 

that SOCE has been demonstrated in some but not 

all smooth muscles and that this will occur through a 

Stim-Orai mechanism when the channel involved gives 

rise to I crac. Variations on this mechanism may occur in 

some smooth muscles, but it remains to be established if 

these are true variations on SOCE, or different channels 

such as ROCs. TRPCs may contribute to variation of 

SOCE in smooth muscle, leading to decreased Ca selectivity, 

but this is far from certain. More studies, both 

molecular and physiological, are clearly called for, and it 

is important to have more information from nonvascular 

myocytes. This remains a controversial and complex area 

(396) but one of some importance. The role of SOCE 

pathways may change with disease states, e.g., hypertension 

and bronchoconstriction, as well as phenotype, e.g., 

changes in arterial myocytes with proliferation, migration, 

or contraction (396), and thus an increased understanding 

could lead to new therapeutic targets. 

There is little doubt that TRP channels are expressed 

in many smooth muscles, particularly, but not exclusively 

tonic ones. As discussed, this expression does not necessarily 

equate to SOCE function. One recent interesting 

paper has provided evidence that TRPC channels become 

converted to SOCE channels as Stim1 leads to their movement 

from non-raft (TRCP) to raft (SOCE) membrane 

locations (10), whereas another recent study had TRCP 

channels in raft domains not Orai1 (148). There is evidence 

from only a limited number of smooth muscles of 

I crac; whether this scarcity is due to the difficulties of 

recording these tiny currents or their absence from most 

smooth muscles is impossible to determine at the moment. 

Studies on cultured cells are limited models for 

native smooth muscle cells, as the downregulation of the 

normal components of E-C coupling and changes in SOCE 

will distort findings (512). The recent discoveries of Orai 

and Stim should help better study store-operated Ca entry 

in all cell types. Firm conclusions must await the ability to 

demonstrate store-operated entry under physiological 

conditions, e.g., agonist depletion, along with direct measures 

of luminal SR Ca content. It is worth noting that in 

cardiac muscle where the SR plays a dominant role in E-C 

coupling, no such SOCE mechanism exists. Perhaps in 

those smooth muscles in which Ca entry is the dominant 

feature of force production, SOCE will also not be important. 

In contrast, in those smooth muscles that are more 

reliant on SR Ca contributions to contraction and have 

significant neurohormonal stimulation, then store-operated 

Ca entry may have an important role. 

VIII. COMMON OR SEPARATE CALCIUM POOLS 



The presence of two types (at least) of Ca release 

channels on the SR, which can interact with each other 

to control local and global Ca signals, adds another 

level of complexity to the regulation of Ca signaling by 

the SR in smooth muscles. The scope for such interactions 

in any particular smooth muscle will depend on 

the morphological arrangement of the store(s), isoform 

expression, receptor distribution, and the cytoplasmic 

and luminal [Ca] (Fig. 5). There is reasonable evidence 

to support the view that the SR has heterogeneous 

compartments of releasable Ca in smooth muscle cells. 

In view of this, it is helpful to frame the discussion that 

follows according to the type of Ca store. These have 

been classified into three subtypes: S (containing both 

IP 3 and RyRs), S (containing only IP 3 receptors) (281, 

285), and S (containing only RyRs) (307). The relative 

distribution of each of these stores can vary considerably, 

not only among different types of smooth muscles 

but also among different species and with development. 

In the studies described below, a couple of general 

points should be borne in mind. First, a variety of 

different agonists, appropriate to particular smooth 

muscles, have been used as a mechanism to generate 

IP 3, and any differences in their other actions are often 

ignored. Second, when conclusions are based on pharmacological 

data, the specificity or otherwise of the 

drugs must not be overlooked. Mention has already 

been made of the problems associated with this approach 

to IP 3 receptor blockade, and similar concerns 

over some agents used to inhibit RyRs have also been 

raised (433). 

B. One Store With One Type of Receptor 

One of the clearest examples of cells having just one 

type of Ca release channel is provided by the ureter. In rat

ureteric myocytes, SR Ca release was shown to be agonist 

sensitive but not caffeine sensitive. In the guinea pig 

ureter, Ca release was caffeine sensitive but agonist insensitive. 

These data suggest that IP 3R plays the key role 

in control of Ca release in rat and RyRs in the guinea pig 

ureteric cells (91, 92). Within the above classification, rat 

has an S store and guinea pig ureter S . 

It was subsequently shown that rat ureteric myocytes 

generated ryanodine-resistant, heparin-sensitive Ca puffs 

and Ca waves when stimulated by IP 3 or ACh (60, 91), 


FIG. 5. Diagram of proposed SR arrangements and possible function in smooth muscle cells. A, a–e: schematic diagram showing proposed 

arrangements of the SR Ca stores reported for different types of smooth muscles. The Ca store in many types of smooth muscle cells can be 

compartmentalized into smaller SR elements that might contain either RyR (green) or IP 3R (red) Ca release channels or a mixture of these channels 

(blue). RyRs can be clustered to form specialized Ca release units located in close proximity to the cell membrane where they generate Ca sparks. 

IP 3Rs in most types of smooth muscle are homogeneously distributed in the cell and are mainly involved in generation of Ca waves and Ca 

oscillations, with or without involvement of the RyR channels. B: schematic diagram illustrating Ca spark/spontaneous transient outward current 

(STOC) coupling mechanism discovered by Nelson et al. (507), which acts as a powerful negative-feedback mechanism controlling excitability and 

electrical activity and has been reported in the majority of smooth muscles. Ca sparks can be activated by Ca entering the cell via L-type Ca channels 

(Ca-induced Ca release; CICR) or an increase in the luminal level of Ca by SERCa pump. BK Ca, Ca-activated K channels; VOC, voltage-operated Ca 

channels. C: possible mechanisms for the generation of global Ca waves in smooth muscle cells. In a, the Ca wave is produced by activation of RyR 

channels via CICR triggered by Ca. In b, the Ca wave is triggered by IP 3 but propagated and amplified by RyR channels (cooperative action between 

two types of Ca release channels). In c, the Ca wave is triggered, amplified, and propagated by IP 3R channels. 


while guinea pig ureteric cells generated ryanodine-sensitive 

Ca sparks enhanced by low concentrations of caffeine 

(76, 87). The functional data obtained on rat ureter 

were also supported by the data obtained using molecular 

biology and immunocytochemistry techniques (60, 493). 

Thus rat myocytes expressed predominantly IP 3Rs (all 

three isoforms) and only a trace of one isoform of RyR 

(RyR3) (60). 

In single myometrial cells from pregnant rats (17), 

oxytocin and ACh evoked an initial peak in [Ca] followed


by a smaller sustained rise. The transient increase in [Ca] 

was abolished by heparin, an inhibitor of IP 3-induced Ca 

release (IICR) and thapsigargin. In contrast, the transient 

[Ca] response induced by oxytocin was unaffected by 

ryanodine. In permeabilized fibers of pregnant rat myometrium, 

caffeine did not produce contraction, whereas 

both IP 3 and the ionophore A23187 evoked contractile 

responses (620). These data show that myometrial cells 

possess an S but not S store. 

C. One Store With Two Types of Receptors 

The majority of smooth muscle cells are both agonist 

sensitive and caffeine sensitive and have one common 

store (S ) shared by both IP 3R and RyR (62, 449, 537, 755). 

In addition, these cells may have additional, i.e., separate, 

agonist- or caffeine-sensitive Ca stores (673, 687). For 

example, in rat mesenteric artery myocytes (32), norepinephrine 

and caffeine produced a transient increase in 

[Ca] in Ca-free solution. In the presence of norepinephrine, 

caffeine or thapsigargin elevated [Ca]. However, if 

thapsigargin or caffeine were added first, the subsequent 

application of norepinephrine did not increase [Ca]. 

These results suggest the existence of two types of Ca 

stores: one store sensitive to both caffeine and agonist 

(S ) and one sensitive to caffeine and thapsigargin but not 

to agonists (S ). Evidence that in some smooth muscles 

there is just one functional store but that it possesses both 

receptor types comes from direct imaging of the store Ca 

(755). One difficulty in demonstrating either one or multiple 

Ca stores comes from the reciprocal actions of both 

the release channels on luminal Ca, i.e., if the Ca store is 

shared, as both IP 3R and RyR are sensitive to cytosolic 

and luminal [Ca], the release of Ca from one population 

will affect the P o of the other. There are many examples 

described below of reciprocal and synergistic effects on 

Ca signaling arising from having both IP 3R and RyRs in 

the same smooth muscle cells. This does not, however, by 

itself prove that they access entirely the same Ca store. 

Identical patterns of store depletion upon exposure to 

caffeine or agonist, consistent with the idea that IP 3Rs 

and RyRs release Ca from the same store, have been 

demonstrated, highly suggestive of a single Ca store (755). 

D. Two Stores, Two Types of Receptors 

Early experiments based on the ability of caffeine 

and IP 3 to release Ca from permeabilized strips of taenia 

caeci, portal vein, and pulmonary artery demonstrated the 

existence of two separate Ca stores, with one gated by 

both RyRs and IP 3Rs (S ) and the other by IP 3Rs alone 

(S ) (281, 282). In taenia caecum and pulmonary artery, 

IP 3 alone or in combination with caffeine-induced Ca 

release, which was about twofold larger than that induced 


by caffeine alone. Inhibition of S store via its depletion 

produced by caffeine and ryanodine did not affect IICR 

from the S store (281). The contribution of the S store 

to total Ca release induced by IP 3 was 60% in taenia 

caecum, 40% in pulmonary artery, and only 6% in portal 

vein (281). In experiments performed on intact pulmonary 

artery myocytes, depletion of Ca stores with ryanodine 

and caffeine eliminated subsequent caffeine-induced intracellular 

Ca transients, but had little or no effect on the 

IP 3-mediated intracellular Ca transient induced by agonists 

(307). Experiments performed on intact terminal 

arterioles also revealed the existence of two separate 

caffeine-sensitive and agonist-sensitive Ca stores. In these 

blood vessels, Ca oscillations induced by agonists were 

resistant to the combined action of caffeine and ryanodine 

(74, 75). Recent work from McCarron and Olson 

(455) has highlighted the difficulty of using functional 

data to deduce information about the existence of two 

stores. They note that the appearance of two stores may 

arise because of partial depletion of SR Ca and luminal Ca 

regulation of the receptors terminating release from one 

receptor only. 

E. Role of Ca Release Channels and Store Type 

in Ca Signaling 

There are two key questions to be addressed here. 

First, can the complete set of Ca signals associated with 

contraction arise in smooth cells using just IP 3Rs or just 

RyRs? Second, how are IP 3-initiated Ca signals influenced 

by RyR Ca release and vice versa? 

There is general agreement that the initiation of Ca 

signaling is a response to agonists caused by the release 

of Ca from the SR via IP 3Rs (212). However, whether Ca 

released from the IP 3R then activates RyR to generate 

further release by CICR to amplify the signal and support 

propagation of Ca waves in a regenerative way, is still a 

controversial issue. As mentioned above, rat ureteric 

myocytes do not express RyR1 or RyR2, the subtypes 

which play the major role in CICR in smooth muscle 

(133). The ability of these myocytes to generate Ca puffs 

and Ca waves in response to agonists or IP 3, which are 

resistant to ryanodine and antibodies to RyR (60), demonstrates 

that the entire Ca release and signaling process 

in smooth muscles can arise from IP 3R activity alone, i.e., 

without RyR involvement (60, 91, 92). Thus the answer to 

the first question posed above is yes; normal agonistinduced 

Ca signals and contractions can be produced by 

IP 3Rs alone. 

There are examples of interactions between IP 3Rs 

and RyRs with the most noted being an amplification of 

IP 3Rs responses by RyRs. With the use of a variety of 

approaches, including blocking of the RyR with ryanodine 

(or ruthenium red, which may have additional actions) or

using antibodies to RyRs (62), effects on IP 3 Ca signals 

have been demonstrated in a wide variety of smooth 

muscles. These effects include abolition of agonist-dependent 

Ca oscillations (333, 535, 638) and inhibition of Ca 

waves (34, 62, 303, 331). There are also many examples of 

IP 3 modulating RyR events, and affecting both local and 

global Ca signals (299). Receptor activation leading to the 

generation of IP 3, increased spark discharge from frequent 

discharge sites (FDS) in vascular (18, 62, 219) and 

visceral (755) smooth muscle cells. It has been postulated 

in portal vein that IP 3 produced by the basal activity of 

PLC activates IP 3Rs to recruit neighboring clusters of 

RyRs within the FDS to generate large Ca sparks (219). 

Inhibition of IP 3R results in a decrease in the frequency of 

Ca sparks, Ca oscillations, and waves in response to 

agonists in visceral and vascular smooth muscles (18, 62, 

299, 333, 535, 638). 

A physiological consequence of interaction between 

IP 3- and RyR-sensitive Ca channels will be positive 

feedback amplifying Ca release. In porcine tracheal 

smooth muscle cells, ryanodine and ruthenium 

red inhibited ACh-induced Ca oscillations (333, 574). 

Similarly, the P2Y receptor agonists UTP and 2-methylthio-ATP 

each increased the occurrence of Ca waves in 

rat cerebral artery and murine colonic myocytes, effects 

which were blocked by ryanodine (34, 303). In 

rabbit cerebral artery (332) and renal resistance arteries 

(307), agonist-induced Ca oscillations were blocked 

by ryanodine, suggesting that RyR-dependent CICR 

contributed to the [Ca] rise. 

Agonist-induced propagating Ca oscillations that 

originate from FDSs have been demonstrated in several 

smooth muscles. For example, ACh-induced Ca oscillations 

in tracheal smooth muscle originate from areas of 

the cell displaying the highest frequency of Ca sparks but 

require SR Ca release through IP 3 receptor channels for 

initiation (333, 535, 638). In gastric myocytes, Ca waves 

evoked by carbachol were initiated from the same sites as 

those evoked by caffeine, and these sites also colocalized 

with spontaneous spark sites. These observations suggest 

that RyRs were involved in both cases, and the carbachol 

responses were inhibited by both IP 3R and RyR blockers 

(755). Inhibition of RyRs with antibodies or with ryanodine 

abolished agonist-dependent Ca transients and 

waves, in both visceral and vascular smooth muscles with 

one functional store shared by both types of receptor 

channels (62, 223, 300, 755, 756). 

These findings suggest that agonists can stimulate Ca 

release in smooth muscle via mechanisms that are both 

IP 3R and RyR dependent and support a model in which 

activation of IP 3Rs acts as a trigger leading to secondary 

activation of RyRs. Does this mean that all IP 3R-activated 

responses are amplified by RyRs when they are expressed? 

Recent observations strongly suggest the scope 

for such interactions varies among different types of 



smooth muscles and depends on receptor distribution 

and isoform expression. 

There is much evidence that in some smooth muscles 

in which IP 3R and RyR share the same store, there is a 

noncooperative relationship between the two types of 

channels (29, 185, 449). For example, adrenergic activation 

decreased the frequency of Ca sparks in intact rat 

resistance arteries; this was suggested to be due to a 

decrease in the SR Ca content (449). Blockade of IP 3Rs in 

smooth muscle cells from both the gastric antrum and vas 

deferens did not decrease, but in fact increased, both 

caffeine-releasable store content and spontaneous spark 

activity (755, 756). However, in rat resistance artery myocytes, 

IP 3R blockade did not affect the temporal or spatial 

characteristics of Ca sparks (380). 

There is, therefore, clear evidence that increases 

and decreases in IP 3R activity can lead to reciprocal 

changes in the activity of RyRs through changes in 

luminal SR [Ca]. However, some inhibition of Ca sparks 

by agonists could be explained by activation of PKC, 

which also decreases Ca spark frequency, by directly 

inhibiting RyRs (70). 

In equine tracheal myocytes, the Ca release evoked by 

the IP 3-generating muscarinic agonist methacholine was unaffected, 

while that evoked by caffeine was blocked by 

ruthenium red (739). In colonic myocytes, ryanodine alone 

did not inhibit IP 3-evoked [Ca] increases, but when applied 

with caffeine allowing ryanodine to lock RyR channels in a 

subconducting state, IP 3-mediated [Ca] increases were 

blocked. These results were attributed to the drug’s indirect 

inhibition of the IP 3-mediated response by store depletion of 

Ca, rather than to involvement of RyR in Ca release (185). 

Similar data have been reported for the smooth muscle cells 

of interpulmonary bronchioles and arterioles, in which under 

normal conditions the RyR was not essential for generating 

agonist-induced Ca oscillations (29, 556, 557). These 

results were consistent with the observation that the release 

of caged Ca was able to initiate CICR via RyR in the smooth 

muscle of the interpulmonary bronchioles only when the 

cells were previously depolarized with KCl to overload the 

Ca stores and sensitize the RyR. 

Acetylcholine, norepinephrine, and nerve stimulation 

each released Ca in porcine coronary, rabbit mesenteric, 

and rat tail arteries, respectively (284, 299, 342, 449). The 

release of Ca by the neurotransmitters was inhibited by 

ryanodine in each study and attributed to the drug’s ability 

to open RyRs and deplete the Ca store. Norepinephrine-evoked 

Ca waves persisted in tail artery segments 

maintained in organ culture for 3 days with ryanodine, 

while Ca sparks and Ca release evoked by caffeine were 

lost, suggesting that RyR does not directly contribute to 

Ca waves in this preparation (154). Ruthenium red and 

tetracaine (each of which abolished the [Ca] increase 

evoked by caffeine without depleting the store) did not 

affect the Ca oscillations induced by agonists in pulmo-


nary arterial myocytes (279). In guinea pig colonic myocytes 

in the absence of RyR activity, repetitive Ca release 

events induced by photolysis of caged IP 3 were not inhibited 

by tetracaine or ryanodine (185). This suggests that 

IP 3 receptor activity alone accounted for the Ca waves 

and loss of the ability of agonists to induce SR Ca release 

after caffeine results from Ca depletion in a common 

store (185). 

F. Summary 

From the above we propose that the functional implications 

of RyRs in neurotransmitter-induced Ca waves 

or oscillations depend on their density relative to IP 3R 

and that they share the same store. In smooth muscles 

displaying a higher density of IP 3Rs than of RyRs (e.g., 

colonic smooth muscles, Ref. 758), the Ca responses to 

neurotransmitters will mainly depend on activation of IP 3 

receptors alone (e.g., Ref. 366). In smooth muscles displaying 

a higher density of RyRs than IP 3Rs (e.g., rat 

portal vein), the Ca waves and oscillations induced by 

neurotransmitters will depend on activation of both IP 3Rs 

and RyRs. However, comparative functional experiments 

correlated with expression and distribution of IP 3R and 

RyR channels in other types of smooth muscles are 

needed to expand this conclusion to all types of smooth 

muscles. 

To summarize these data, we can conclude that the 

functional studies that employed blockers of SERCAs in 

conjunction with activators of IP 3Rs and RyRs shed some 

light on the morphological organization of intracellular Ca 

stores. In some types of smooth muscles, morphological 

studies employing immunofluorescence and molecular biology 

techniques provided information that could explain 

the functional data. However, to fully understand the 

functional role of the SR in smooth muscles, confocal 

microscopy and immuno-EM are needed to examine the 

relationship between IP 3R and RyR isoforms and SERCA, 

to substantiate conclusions drawn from these functional 

studies. 

IX. ELEMENTAL CALCIUM SIGNALS 

FROM SMOOTH MUSCLE 

SARCOPLASMIC RETICULUM 

A. Ca Sparks 


Calcium sparks were first identified in cardiac (122, 

544) and skeletal (354, 708) muscles, where their fundamental 

role in the generation of global Ca signals underlying 

phasic contractions has been well established. In 

smooth muscle cells Ca sparks were first observed in 

cerebral artery and showed biophysical and pharmacological 

characteristics similar to those of cardiac muscle 

cells (507). Since then, similar events have been described 

in a wide variety of smooth muscle types including different 

types of arteries and arterioles (70, 90, 192, 305, 473, 

568, 749, 751), portal vein (219, 221, 474), urinary bladder 

(127, 254–256, 288, 312), gastrointestinal tract (34, 35, 220, 

223, 351, 808, 809, 811), airways (369, 810), gallbladder 

(572), and guinea pig ureter (76, 87). A lack of Ca sparks 

has been reported for smooth muscle cells of neonatal 

cerebral arteries (213), nonpregnant mouse (476), and 

pregnant and nonpregnant rat myometrium (88). 

Calcium sparks in smooth muscle can occur spontaneously 

(76, 87, 220, 507) or after activation by one of the 

following factors: 1) low concentrations (1 mM) of caffeine 

(76, 87, 213, 307, 807), 2) elevation of global [Ca] 

caused by Ca entering through L-type Ca channels (312, 

508), 3) increase in the SR Ca content (87, 811), and 

4) stretch (312). 

2. Frequent discharge sites 


Irrespective of the mechanism of activation, Ca 

sparks repeatedly arise from a few specialized regions 

adjacent to the superficially located SR within the myocytes; 

these have been termed FDS (65, 87, 220, 221, 288, 

312, 508). These areas are enriched with clusters of RyR2 

channels (133, 213, 525). It should, however, also be noted 

that frequent discharge events away from the cell membrane 

have also been reported in smooth muscle cells 

from rat portal vein (18, 65) and gastrointestinal tract 

(220, 807). The number of FDS varies not only among 

different types of smooth muscle cells (65, 87, 219, 220, 

807), but also among different populations of the same 

type of smooth muscle (65). The number of FDS is increased 

in the presence of low concentrations of caffeine 

(76, 307, 807) or after increasing the SR Ca content (87, 

811). Such a non-uniform distribution of FDS in smooth 

muscle may arise from either the irregular clustering of 

RyR Ca release channels, or nonuniform distribution of 

the SERCA pump on the SR. Clustering of RyR2 in peripheral 

zones of SR was an absolute requirement for the 

formation of sites generating spontaneous Ca sparks coupled 

to STOCs in arteriolar myocytes (213). 

The properties of Ca sparks in isolated cells appear 

to be relatively similar in different types of smooth 

muscle cells (for details, see reviews in Refs. 536, 750) 

and do not differ from those observed in intact preparations 

(70, 87, 305, 507, 568). Thus, although variations 

in their biophysical parameters have been noted, Ca 

sparks appear to be more stereotypic in different types 

of smooth muscles than the global Ca signaling controlling 

contractility.

3. Functional role of Ca sparks 

The physiological role of Ca sparks has been a subject 

of intense investigation in a variety of smooth muscles. 

Calcium sparks are the fundamental units of SR Ca 

release during E-C coupling in cardiac muscle, synchronized 

in time by the action potential. Cardiac sparks occur 

through the CICR process, and the physical and functional 

coupling between Ca influx and RyR channels is tight and 

intimate (50, 122, 127). RyR channels are positioned in junctional 

SR elements within short distances (20 nm) of voltage-dependent 

Ca channels in the transverse tubules (50, 

759). Pharmacologically CICR in cardiac muscle can be 

easily tested using ryanodine, or a low concentration of 

caffeine. In cardiac muscle, ryanodine produces a strong 

negative inotropic effect associated with marked inhibition 

of the global Ca transient, but with no effect on the action 

potential. Caffeine produces a transient positive inotropic 

effect associated with an increase in systolic [Ca] with no 

change of the Ca current. 

Cardiac myocytes express RyR2. The majority of 

smooth muscles also express RyR2 and 1C L-type Ca 

channels (511), which colocalize with them (490, 525). It 

is perhaps not surprising then that a cardiac-like mechanism 

of CICR was expected to be present, acting as an 

amplifying system for the global Ca transient, especially 

in phasic smooth muscle cells. However, it should be 

recalled that the highly organized junctional arrangement 

between plasma membrane and SR seen in striated muscles 

does not occur in smooth muscles, and junctional 

proteins such as junctin and triadin may be absent (see 

sect. IV). 

Even more importantly, there are significant differences 

in the activation by Ca between cardiac and smooth 

muscles. In cardiac muscle, global Ca is the determinant 

of contraction under normal physiological conditions. 

However, in smooth muscle cells, myosin light chain 

phosphorylation catalyzed by MLCK and activated by the 

Ca-calmodulin complex is a key factor in the control of 

mechanical activity. Data obtained in some types of 

smooth muscle cells show that MLC phosphorylation can 

achieve its maximal level at submaximal levels of Ca 

(654). Thus, unlike cardiac muscle where high cytoplasmic 

[Ca] is required to achieve a high level of force, in 

smooth muscle cells a high level of force can be achieved 

at moderate levels of Ca, especially during agonist-induced 

stimulation, when sensitizing pathways involving 

PKC or Rho-associated kinase inhibition of myosin lightchain 

phosphatase (MLCP) can also be activated (625, 

692). 

Finally, Ca-activated ion channels are conspicuously 

expressed in smooth muscle cells but are largely absent 

from the heart. Thus, as will be described below, a role for 

Ca sparks in affecting excitability, as well as modulating 



Ca signals, has emerged as their prime function in many 

smooth muscles. 

The first work in which smooth muscle Ca sparks 

were observed (507), in myocytes of cerebral arteries, 

showed that sparks did not contribute to contraction. 

Rather, the authors demonstrated that sparks were involved 

in relaxation of tone via activation of Ca-activated 

large-conductance K channels (BK). The discovery of Ca 

sparks provided the missing link in explaining the origin 

and functional role of STOCs, which had been observed 

earlier in a number of smooth muscle cells (41). The role 

of a Ca sparks/STOCs coupling mechanism and its functional 

role in tonic smooth muscles has now been thoroughly 

investigated (507), and well reviewed (304, 750). 

In contrast, the role of Ca sparks in phasic smooth 

muscles, particularly their possible role in amplification 

of the global Ca signal via CICR, is still under investigation, 

and there is no consensus on this issue. It appears to 

us that different types of smooth muscles utilize Ca 

sparks in a variety of ways to match their physiological 

function. Unlike cardiac muscle, smooth muscle cells express 

Ca-activated K and Cl channels, which can be targeted 

by Ca sparks, and in this way control excitability. 

4. Ca sparks in phasic smooth muscles 

In cardiac myocytes, each action potential results in 

a contraction that derives from RyR-mediated calcium 

release, triggered by an increase in [Ca] due to influx via 

L-type Ca channels. The signal gain is quite high, since 

each channel opening results in a Ca spark (activation of 

several RyRs), the duration of which is longer than the 

L-type Ca channel opening (104). Phasic smooth muscles 

are also activated by Ca transients controlled by the action 

potential. However, information on the Ca signal in 

the cytosol following the action potential is limited, even 

though it is crucial to the understanding of the coupling of 

membrane excitation to Ca-sensitive cellular functions. 

Thus, despite the broad expression of L-type Ca channels 

and RyR in many phasic smooth muscles, the existence 

and nature of CICR in these nonsarcomeric cells, in which 

the distribution of L-type Ca channels and RyR differs 

substantially from an orderly dyadic pattern in sarcomeric 

muscles, is not well established. 

Simultaneous recording of electrical activity and intracellular 

[Ca] have been performed in some intact phasic 

smooth muscles, e.g., guinea pig ureter (87), pregnant 

rat myometrium (88), and urinary bladder (288) and isolated 

cells from guinea pig ileum (247, 248, 254), guinea 

pig vas deferens (361), and mouse urinary bladder (494). 

In urinary bladder, vas deferens, uterus, and ileum, the 

action potential usually appears as brief (20–40 ms) 

spikes, while in ureter it consists of a spike followed by a 

long-lasting (400–800 ms) plateau component. In all these 

four smooth muscles, the Ca transient is triggered by the


action potential, and both are inhibited by L-type Ca 

channel blockers, indicating that Ca influx is a key factor 

in the generation of the global Ca transients. Is CICR 

contributing to the global Ca transients associated with 

the action potential in smooth muscles? 

In the guinea pig ileum where Ca sparks have been 

observed (220), ryanodine and thapsigargin produced 

stimulant rather than inhibitory effects on the Ca transients 

associated with the action potential by increasing 

the amplitude and duration of the spike (203, 288, 312, 

494). Voltage-clamp experiments performed on ileum 

(361), cerebral arteries (330), portal vein (133), vas deferens 

and bladder (288), and pregnant myometrium (630) 

demonstrated that Ca entry via L-type Ca channels can 

trigger some Ca release via RyRs. At the same time, other 

workers have failed to detect CICR in voltage-clamped 

smooth muscle cells isolated from other or even the same 

type of smooth muscles. For example, CICR was not 

detected in voltage-clamped smooth muscle cells of portal 

vein (329), gastrointestinal tract (79, 781), and airways 

(178). 

A discussion of the physiological role of CICR in 

urinary bladder smooth muscle cells illustrates the complexity 

in this area and the data which are hard to unify. 

Elegant work performed on voltage-clamped guinea pig 

urinary bladder myocytes by Ganitkevich and Isenberg 

(203) showed that Ca transients induced by long depolarizing 

voltage steps were inhibited 70% by ryanodine. 

This suggested that a relatively high gain “cardiac type” of 

CICR operates in these myocytes, at least under these 

experimental conditions. The importance of a functional 

contribution of CICR to E-C coupling and contraction in 

urinary bladder smooth muscles has been assessed by 

several research groups, using different species and experimental 

models (127, 247, 254, 490, 494, 525). Results 

from these studies addressing the susceptibility to contraction 

in bladder myocytes treated with ryanodine are 

not in agreement. For example, Hashitani and Brading 

(247) showed the intracellular [Ca] rise induced by spontaneous 

action potentials in the guinea pig was reduced to 

60% by ryanodine. In a later publication (494), the stimulant 

action of ryanodine on the mechanical activity of the 

guinea pig bladder was reported. Reduction of spontaneous 

or electrically evoked contractions by ryanodine have 

been reported for mouse bladder myocytes (494). However, 

ryanodine has no significant effect on the [Ca] and 

spontaneous contractions in rat bladder myocytes (252). 

These data suggest that the contribution of RyRs and 

CICR to E-C coupling in the bladder differs between 

species and experimental conditions. 

Imaizumi et al. (288) in guinea pig urinary bladder 

myocytes using brief (50 ms) depolarizing voltage steps, 

obtained the first direct evidence that Ca “hot spots” 

(equivalent to Ca sparks) could be transiently activated in 

cells depolarized to 20 mV. These events lasted for 0.5 

s and spread as Ca waves when depolarization was to 0 

mV. The increase in global [Ca] elicited by 50 ms depolarization 

to 0 mV reached a peak 60–100 ms after the 

start of depolarization, indicating that the spread of Ca 

hot spots continued even after most voltage-dependent 

Ca channels had closed. Recently, Kotlikoff and co-workers 

(127) have also investigated the temporal relationship 

between the Ca current and local and global Ca signaling 

in rabbit urinary bladder myocytes, under voltage- and 

current-clamp conditions with physiologically relevant 

protocols of stimulation. Localized photolysis of caged Ca 

initiated CICR, but multiple excitation pulses were 

needed and IP 3R channels were also activated (313). Calcium 

channels were shown to activate RyRs to produce 

CICR in the form of Ca sparks and propagated Ca waves. 

Both the initial Ca spark and the subsequent Ca waves 

occurred through the opening of RyRs, since both were 

eliminated by ryanodine, and neither was affected by 

dialysis with heparin (127). They concluded that 1) L-type 

Ca channels could open without triggering Ca sparks, 

2) triggered Ca sparks could be observed after channel 

closure, 3) the trigger stimulus for the Ca release process 

is not a local but a global rise in intracellular [Ca], and 

4) this results in a functional uncoupling of a single action 

potential from Ca release. 

5. Loose coupling 


The small number of Ca spark sites evoked by the Ca 

current, the delay between Ca channel and RyR channel 

openings, as well as the inhibitory effect of BAPTA on Ca 

sparks in urinary bladder smooth muscle cells suggested 

that compared with cardiac myocytes, a fundamentally 

different coupling process operates in smooth muscle, 

which was termed “loose coupling” (127, 368). Features of 

the loose coupling system are low gain (multiple Ca channels 

must open to produce sparks), discriminated responses 

(release takes the form of local Ca sparks or 

globally propagated Ca waves), and a marked lengthening 

of signal duration (Ca waves last far longer than the 

action potential). The delayed activation of CICR and the 

subsequent propagation of a Ca wave, which could last up 

to 1 s, suggests that CICR in contrast to cardiac muscle 

(122, 127) cannot be controlled by a brief (40–60 ms) 

single action potential, and its functional role is yet to be 

established (368). Thus, in urinary bladder myocytes paradoxically, 

global rises of [Ca] are required to activate Ca 

sparks. The concept of loose coupling can also be explained 

by an increase in the SR [Ca] and activation of 

[Ca] sparks. An increase in the luminal [Ca] produces 

profound effects on both the frequency and the amplitude 

of Ca sparks in smooth muscle (811). 

Both cytosolic and SR Ca levels would be expected to 

increase with enhanced Ca entry. The proximity of the SR 

to the plasma membrane and the existence of the uptake

mechanisms by the SERCA pump provide the structure 

for what has been termed by van Breemen the “superficial 

buffer barrier” (714), discussed also in section II. Since 

inhibition of SERCA has little effect on the global rise of 

[Ca] induced by depolarizing voltage steps (e.g., Refs. 77, 

507), it can be suggested that only a small portion of the 

SR is involved in active ATP-dependent accumulation of 

Ca, which does not affect the global rise of [Ca]. Similarly, 

many myocytes have FDS which, when releasing Ca in the 

form of Ca sparks, have little effect on the global rise of 

[Ca] (330). However, this suggestion should be experimentally 

tested by studying the spatial distribution of 

SERCA pumps in smooth muscle cells. Since the effects 

of ryanodine on the guinea pig urinary bladder were mimicked 

by CPA, the BK channel inhibitor iberiotoxin, and 

BAPTA (247), it was suggested that CICR targets BK 

channels via Ca sparks and in this manner controls excitability 

of this smooth muscle. 

Another well-studied smooth muscle with respect to 

CICR and the role of Ca sparks is the guinea pig ureter. In 

contrast to bladder smooth muscles, Ca transients and 

phasic contraction in the guinea pig ureter are controlled 

by a single long-lasting (400–800 ms) plateau-type action 

potential of myogenic origin, which gives rise to a longlasting 

Ca transient (76, 87). Parameters of the Ca transients 

are tightly coupled to the parameters of the action 

potentials (76, 87, 93) and thus meet the criteria of both 

tight and loose coupling. Early contractile studies in intact 

guinea pig ureteric smooth muscle preparations identified 

ryanodine-sensitive calcium release by caffeine (93, 

287) but not agonists (89, 91, 92), and thus these myocytes 

serve as an excellent experimental model to investigate 

the role of CICR in phasic smooth muscles. The STOCS, 

associated with spontaneous transient hyperpolarizations 

and sensitive to caffeine, were recorded in myocytes under 

voltage- and current-clamp conditions (91, 92). We 

observed Ca sparks arising from several FDS in isolated 

cells and intact preparations (87). In guinea pig ureteric 

smooth muscle cells, low caffeine concentration increased 

the frequency of Ca sparks and inhibited the 

action potential, Ca transients, and phasic contractions. 

These components of E-C coupling were restored by ryanodine, 

CPA or TEA, and iberiotoxin (blockers of BK 

channels) (76). Under voltage-clamp conditions, caffeine 

at low concentrations had no inhibitory effect on the Ca 

current and slightly potentiated global rises of [Ca], suggesting 

some contribution of CICR to the generation of 

depolarization-evoked increases in global [Ca]. However, 

caffeine markedly increased the frequency and amplitude 

of STOCs and increased the overlap between Ca and BK 

channel currents when voltage-clamped ureteric myocytes 

were depolarized to 0 mV (76). Caffeine had no 

effect on the action potential and Ca transients in the 

presence of TEA, again suggesting that CICR had little 

contribution to global rises of [Ca] controlled by the 



action potential. The lack of effect of ryanodine and CPA 

on the Ca transients and the reversal of the inhibitory 

effects of caffeine by these agents suggest that the contribution 

of CICR to global Ca signaling is minimal, and 

the major source of Ca is action potential-stimulated Ca 

entry. 

In the guinea pig ureter, the duration of the plateau 

component of the action potential is affected by inward 

Ca current (I Ca) and outward BK (I BK) currents (287, 382). 

Normally there is a substantial delay between I Ca and 

STOCs in ureteric myocytes (287, 382), which enables the 

generation of the long-lasting plateau. However, if this 

delay is shortened or eliminated, as is the case with 

caffeine, the action potential is blocked or markedly reduced 

in duration leading to the inhibition or reduction in 

the duration of the Ca transient. Thus these data fit the 

hypothesis of loose coupling between I Ca and CICR. This 

mechanism is an important factor in controlling the duration 

of the plateau component of the action potential, 

which in turn plays a key role in controlling the amplitude 

of phasic contractions (93). A decrease or elimination of 

the delay between I Ca and STOCs, and a slight increase in 

I Ca-independent amplitude of the global Ca transient occur 

under voltage-clamp conditions in the presence of 

caffeine, suggesting that I Ca can be tightly coupled to Ca 

sparks. The mechanism of loose coupling between I Ca and 

STOCs (Ca sparks) in guinea pig ureteric cells can be 

explained by a prominent role of luminal Ca in the activation 

of Ca sparks/STOCs coupling mechanism. Indeed, 

we and others have recently shown that the Ca content of 

the SR following a global Ca signal is substantially increased 

(77, 633), and this was well correlated with a 

long-lasting discharge of Ca sparks after L-type Ca channels 

were closed (87, 93). 

The Ca content of the SR was also shown to play a 

critical role in the generation of spontaneous Ca sparks 

and STOCs in smooth muscle of the gastrointestinal tract 

(811). We have established that the transient eruption of 

Ca sparks plays a key role in the control of the longlasting 

period of refractoriness, which serves as an important 

pacing and protecting mechanism in the ureter 

(87). In the guinea pig bladder, the repolarizing phase of 

the action potential, as well as the frequency of spike 

discharge in one burst, are also potentiated by inhibitors 

of the SR or BK channels (247, 248, 254, 256). These data 

would also fit a loose coupling between I Ca and STOCs, 

acting as an important mechanism controlling the parameters 

of the action potential and excitability of these types 

of smooth muscles, as observed by Collier et al. (127). 

To summarize these data, we conclude that the large 

contribution of BK channels activated by superficial CICR 

to the negative feedback of membrane excitability and/or 

the regulation of action potential shape, is well-established 

and a characteristic of many types of smooth muscles. 

The question of a possible role of CICR as a contrib-


utor to the global Ca signal is still a matter for future 

experiments. That inhibition of SR function by SERCA 

inhibitors results in 70% inhibition of the [Ca] rises in 

the area of discharge sites in urinary bladder and vas 

deferens strongly suggests that CICR is present in these 

tissues and can produce a significant contribution to the 

cytoplasmic [Ca] rise. However, as under the same conditions, 

that global rises of [Ca] were not inhibited, but in 

fact were slightly potentiated, as also reported for a number 

of intact phasic smooth muscles, suggests that CICR 

is confined to microdomains. Studies using other types of 

smooth muscles and different species should be carried 

out. High temporal and spatial resolution and analyses of 

the Ca release from the SR should be correlated with the 

isoform expression and spatial distribution of different 

types of RyRs. 

6. Ca sparks and myogenic tone 

Many resistance arteries possess an intrinsic “myogenic” 

tone, activated in response to elevation of intravascular 

pressure by a graded membrane depolarization, 

opening Ca channels, and leading to elevation in [Ca] and 

constriction (507). It has been proposed that Ca sparks, 

by targeting BK channels, generate STOCs, which cause a 

tonic hyperpolarizing influence on the membrane potential 

of small pressurized arteries with myogenic tone 

(507). Inhibition of Ca sparks or BK channels causes 

membrane depolarization, leading to further elevation of 

[Ca], and additional increases in muscle tone in pressurized 

cerebral arteries (330, 356, 507). Thus, in resistance 

arteries, a Ca sparks/STOCs coupling mechanism can act 

as a negative-feedback mechanism to oppose myogenic 

Ca entry via Ca channels and promote relaxation (507). 

However, under voltage-clamp conditions, global rises of 

[Ca] induced by depolarizing voltage step in myocytes 

isolated from these arteries were significantly inhibited by 

ryanodine. This suggested that CICR is also present in 

these cells and can act as an amplifying mechanism (507). 

However, under normal physiological conditions, this 

mechanism may contribute little, as blocking Ca sparks in 

the presence of blockers of BK channels has little effect 

on global [Ca] and arterial tone (330). Myogenic tone is 

associated with moderate depolarization (356, 357, 507), 

and it was suggested that L-type Ca channels operate in a 

“low-activity mode,” i.e., the myocytes never depolarize 

sufficiently to achieve a high channel P o. 

We conclude that the Ca sparks/STOCs coupling 

mechanism acts as a powerful vasodilator in vascular 

beds where membrane potential depolarization acts as an 

activating mechanism to generate tone. If muscle tone is 

activated via stimulation of Ca entry by SOC or ROC 

channels, this mechanism will be ineffective, as it will be 

in controlling tone produced by the Ca oscillations associated 

with activation of IP 3Rs. However, this mechanism 

can play an important role in disabling the mechanisms of 

generation of the action potential, which is a feature of 

most of the arteriolar smooth muscle cells and enables 

them to be regulated by local factors. 

B. Ca Puffs 


The term Ca puffs refers to the small local increases 

in [Ca] that occur when IP 3Rs open spontaneously or in 

response to agonists. Unlike sparks, Ca puffs have not 

been detected in vascular myocytes despite the obvious 

presence of IP 3Rs. So far, Ca puffs have been detected in 

only two smooth muscles: rat ureter (60) in which agonist-induced 

Ca release plays a dominant role in modulating 

contraction (34, 86) and murine colonic myocytes. In 

rat ureteric myocytes, Ca puffs were observed during 

release of low concentrations of IP 3 from a caged precursor, 

or by low concentrations of ACh. They were also 

observed spontaneously in Ca-overloaded myocytes (91). 

Spontaneous Ca puffs were observed in intact rat ureteric 

preparations (60). 

Calcium puffs in tissues appear to have a wide 

range of amplitudes, time courses, and spatial spread, 

suggesting that the IP 3Rs exist in clusters of variable 

numbers of channels and that within these clusters a 

variable number of channels can be recruited (86). 

Calcium puffs in the ureteric myocytes were blocked 

selectively by intracellular applications of heparin and 

an antibody to IP 3R, but were unaffected by ryanodine 

and intracellular application of an antibody to RyR (60). 

When stimulated by agonists, propagated, ryanodineresistant 

Ca waves appeared to result from the spatial 

recruitment of Ca-release sites by diffusion. This is 

consistent with data from multicellular preparations, 

where ryanodine-resistant agonist-induced Ca release 

was reported (34). Both Ca signals provide an integrated 

mechanism to regulate contractility in smooth 

muscle cells, where RyRs are absent, or poorly expressed. 

The possible role of Ca puffs in the control of excitability of 

rat ureteric smooth muscle cells has not been investigated, 

although we have observed spontaneous transient outward/ 

inward currents (STOICs) (Burdyga and Wray, unpublished 

data), which could result from activation of the BK and Cl Ca 

channels (91, 92). 

Localized Ca transients and puffs, resistant to ryanodine 

and inhibited by xestospongin, U-73122 (an inhibitor 

of PLC), occurred spontaneously or during P2Y receptor 

stimulation in murine colonic myocytes (34). The 

puffs were coupled to the activation of both BK and 

small-conductance Ca-activated K (SK) channels. Thus 

the release of Ca by G protein-mediated activation of PLC 

can be linked to inhibitory responses via Ca puffs targeting 

SK channels. This is in marked contrast to the usual 

finding that IP 3-dependent mechanisms are used by excitatory 

agonists in smooth muscles.

A role for IP 3Rs in the generation of Ca oscillations 

and waves seems to be a common feature of smooth 

muscles. The absence of local Ca signals, i.e., Ca puffs, is 

therefore surprising. The paucity of Ca puffs might be 

explained by there being little or no clustering of the 

receptors in most smooth muscles. It may also be that Ca 

puffs have not been sought. Whether puffs are present in 

other smooth muscles and whether they can contribute to 

the control of excitability seems to us to be an important 

question that should be urgently addressed. 

X. SARCOPLASMIC RETICULUM AND 

PLASMALEMMAL ION CHANNELS 

A. Overview of Ca-Activated Ion Channels 

In contrast to skeletal and cardiac muscle, smooth 

muscle cells express several types of Ca activated ion 

channels on the plasmalemma that can be differentially 

activated by SR Ca release events, such as Ca sparks, Ca 

puffs, and Ca waves. There are two major populations of 

Ca-activated channels that can be activated by SR Ca 

release in different types of smooth muscles: Ca-activated 

K (K Ca) and Ca-activated Cl (Cl Ca) channels. The K Ca 

channels are composed of at least three different members, 

which are separate molecular entities. Their different 

K conductances have led to their being known as big 

(BK), intermediate (IK), or small (SK) K channels. These 

channels also exhibit different inhibition profiles to various 

toxins. (The BK channel is also referred to as the 

maxi K Ca.) Coupling between SR-mediated Ca events and 

these Ca activated ion channels can lead to changes in 

membrane potential and/or the action potential and thus 

serve as powerful negative- or positive-feedback mechanisms, 

controlling excitability and contractility of smooth 

muscles. Ca sparks can activate either or both of these 

Ca-activated channels producing STOCs (507), spontaneous 

transient inward currents (STICs), or STOICs (811). 

Spontaneous transient membrane hyperpolarizations 

have also been reported in coronary artery (202) and 

guinea pig ureter (87) and spontaneous transient membrane 

depolarizations in urethral (249, 287) smooth muscles, 

confirming that STOCs and STICs can affect the 

membrane potential of smooth muscles. A Ca spark/ 

STOCs coupling mechanism can also directly affect the 

parameters of the action potential by increasing the repolarizing 

current and thus decreasing the amplitude and 

duration of the action potential, as was shown for urinary 

bladder (247, 248, 254, 256, 288) and ureter (474). Calcium 

waves may also indirectly influence excitability and contractility 

of smooth muscles through activation of Cadependent 

ion channels. In rat portal vein myocytes, Ca 

sparks were demonstrated to activate STOCs, while Ca 

waves selectively activated Cl Ca channels (474). Interest- 


ingly, STICs but not STOCs were selectively blocked by 

inhibition of IP 3Rs (623), suggesting that the variability in 

Ca release events can also modulate the cell membrane 

potential through differential targeting of K Ca and Cl Ca 

channels. 

B. BK Channels 


Transient outward K currents were first described in 

smooth muscle cells by Benham and Bolton (1986), who 

used the term STOC to describe these events, which were 

later identified in a wide variety of smooth muscle cells 

(41, 70, 87, 150, 264, 277, 287, 308, 332, 382, 495, 507, 516, 

528, 568, 604, 617, 806, 810, 811). The native BK channels 

consist of four identical subunits that create a functional 

BK channel and four modulatory subunits which 

combine with the channel forming subunits and affect 

their sensitivity to Ca (83, 93, 459, 514, 712). Immunocytochemical 

observations using antibodies against the BK 

channel subunit, Ca channel 1C subunit and RyR, 

showed that the BK and RyR but not the Ca channel form 

clusters in myocytes (525). The BK channels have an 

estimated single-channel conductance of 80 pS at 40 

mV with a physiological K concentration gradient and 

occur at a density of 1–4 channels/m 2 (41, 641, 689). 

Direct evidence for the causal relationship between Ca 

sparks and transient BK currents has been supplied by 

studies combining whole cell patch clamping with simultaneous 

confocal imaging of Ca in voltage-clamped myocytes 

(83, 150, 302, 352, 474, 555, 749, 810). These experiments 

revealed good correlations between the temporal 

characteristics of Ca sparks and STOCs, although BK 

currents showed a faster decay relative to the Ca spark. 

These data are in good agreement with immunohistochemical 

observations that showed the close proximity of 

Ca spark-generating sites and BK channels (525). 

A Ca spark/STOC coupling mechanism is present in 

many visceral and vascular smooth muscles and acts as a 

powerful negative-feedback mechanism, protecting smooth 

muscle cells from overexcitability. Inhibition of Ca sparks 

with ryanodine or SERCA pump inhibitors or their target BK 

channels (by low concentration of TEA or iberiotoxin) results 

in augmentation of vascular tone (507) and an increase 

in the excitability and termination of the refractory period in 

phasic smooth muscles (87, 247). The importance of BK 

channels has been demonstrated with transgenic mice. Targeted 

deletion of regulatory 1-subunits of BK channels 

resulted in animals that developed an increase in vascular 

tone and hypertension (618) as well as other pathologies 

such as ataxia (619), urinary incontinence (465), and erectile 

dysfunction (752). BK channels have a relatively low 

affinity for Ca. For example, at a physiological membrane 

potential of 40 mV, the K D of BK channels for Ca is 20 

M (554). Calcium reaches high levels (10–100 M) close


to the SR release site (505, 555), and the plasma membrane 

is in close proximity; thus BK channels are activated 

despite their low affinity for Ca. A single Ca spark 

can produce 20 mV hyperpolarization in an isolated 

myocyte through activation of BK channels (202, 507). 

Vascular myocytes have a relatively high density of BK 

channels and do not require clustering of BK channels 

above a spark site. However, in visceral smooth muscle 

cells, where the density of BK channels is low, clustering 

of BK channels in the patches of the plasma membrane at 

sites frequently discharging sparks is required (525, 809). 

C. SK and IK Channels 

There are four different isoforms of the SK channel, 

SK1-4 (480), with SK4 also known as IK1. The cloning of 

SK channels revealed the existence of at least three members 

of a family (SK1-3) (362), which were first identified 

in cultured rat skeletal muscle (55). These channels are 

highly sensitive to Ca (EC 50 0.5–0.7 M) and have a 

small single-channel conductance (4–14 pS). In addition, 

they are selectively blocked by the bee venom toxin 

apamin (135, 190, 247, 250, 254, 256). Of the three cloned 

SK channel subtypes (SK1-3), SK1 and SK3 channels are 

least sensitive to apamin (nanomolar apamin was required 

for their inhibition), and SK2 channels were more 

sensitive and inhibited in the subnanomolar range (67, 

362, 626, 727). 

Since their initial discovery, SK channels have been 

described in a number of cell types, including rat pituitary 

cells (381), T lymphocytes (227), and adrenal chromaffin 

cells (543). They have also been identified in some types 

of smooth muscles, e.g., urinary bladder (698), stomach 

(673), colon (364), uterus (360, 450), renal artery (206), 

portal vein (293), and the pancreatic arterioles (666). 

Their distribution and functional importance are currently 

being established (247, 248, 256, 663), but there is 

evidence that SK3 is particularly relevant to contractility 

and excitability in smooth muscle. Functional studies performed 

on the intact bladder revealed SK channels contribute 

to the afterhyperpolarization (AHP) following 

trains of action potentials (255), as apamin blocked the 

AHP and increased excitability and contractility without 

affecting other parameters of the action potential. 

There is evidence that both SR Ca release and plasmalemmal 

Ca entry can activate SK channels. Hererra and 

Nelson (256) reported that SK channels in the bladder are 

activated by the rise in [Ca] which occurs upon Ca entry 

through L-type Ca channels during the action potential 

but not by Ca sparks (256). The inability of Ca sparks to 

activate SK channels was explained by the small density 

of the SK channels near the discharging sites. Indeed, the 

density of SK channels in bladder myocytes was estimated 

to be 200 times lower than that of BK or L-type 


Ca channels (257). The activation of SK channels occurs 

through the binding of Ca to calmodulin, which is constitutively 

bound to the COOH terminus of the channel 

(503). Thus SK channels are well suited to respond to 

changes in global [Ca] (0.1–1 M), since calmodulin is 

saturated by [Ca] below 1 M. However, Kong et al. (368) 

showed that IP 3-induced Ca release in colonic myocytes 

activated SK. Their activation has been suggested to underlie 

the hyperpolarization and relaxation associated 

with inhibitory purinergic input on these cells. The SK 

channels are also responsible for the hyperpolarization in 

response to release of ATP from enteric inhibitory motorneurons 

(360). More studies are required before it is 

known if this is common to other smooth muscles. 

When mice were produced in which SK3 was absent, 

no aberrant phenotype was observed (68). However, overexpression 

of SK3 unexpectedly led to difficulties in 

breathing and parturition. Labor was prolonged and described 

as dystocic. These data suggest SK channel downregulation 

may be required for parturition and labor. Modzelewska 

et al. (485) subsequently showed that apamin 

inhibits NO-induced relaxation of the myometrium, but 

did not affect spontaneous contractions. In bladder, inhibition 

of SK3 channels has been associated with instability 

(257). 

The IK channel, also known as KCNN4, K3.1, and 

formerly SK4 (527, 747), is the product of different genes 

from SK1-3 and exhibits only 40% amino acid sequence 

identity (40). The IK have conductances of 50–70 pS 

(718). These channels are blocked by TRAM-34 and clotrimazole, 

are sensitive to charybdotoxin but insensitive 

to apamin, and are not time or voltage dependent (506). It 

is now considered that IK channels are the Gardos channels 

of erythrocytes, responsible for their high K permeability 

(269). 

In the endothelium, IK channels contribute to endothelial 

derived hyperpolarizing factor (EDHF)-mediated 

responses. Deletion of IK channels attenuates ACh-induced 

hyperpolarization of endothelial cells and vascular 

myocytes (637; see also Ref. 179). Blood pressure was 

elevated in the IK knockout mice in agreement with a role 

for EDHF in its regulation (81). 

The IK channel may be associated with proliferating 

arterial and airway smooth muscle (510, 628). The latter 

authors showed IK expression in human airway and suggest 

that their upregulation plays a role in disease. The 

mRNA for IK channels has also been found in human 

trachea (628). It is likely that IK channels will contribute 

to setting the membrane potential relatively negative in 

the cells that express them. As mentioned already, IK 

channels in endothelial cells can lead to hyperpolarization 

of vascular myocytes. In human pulmonary artery, IK 

expression and current have been shown, but these are 

likely to come from endothelial cells not myoctyes (301, 

564). Thus, although there is a physiological importance

of IK channels to smooth muscle, most of this appears to 

be due to indirect effects. 

In summary, although there is now good evidence for 

SK channels playing an important role in control of excitability 

and contraction in some types of smooth muscles, 

the functional role of these channels in other types of 

smooth muscles is still not clear. The Ca signals that 

activate SK and IK channels in different types of smooth 

muscles are not well established. The situation is even 

more complex for blood vessels, which contain both endothelial 

and smooth muscle, both of which can express 

different sets of BK, SK, and IK channels. However, one 

can speculate that SK channels are expressed and functional 

in smooth muscle cells that are controlled by a 

burst of action potentials. Data obtained on intact urinary 

bladder myocytes suggest that SK channels are involved 

in control of the AHP which sets a “mini refractory period” 

between action potentials. During this period, voltage 

inactivation of Ca channels is prevented; thus the 

channels are available for generation of the next action 

potential. SK channels are well suited for this function, 

i.e., controlling AHP as they are more sensitive to [Ca] 

than BK and thus can be activated by a relatively small 

rise in cytoplasmic [Ca] produced by a brief action potential. 

To achieve a graded rise of force in bladder, uterus, 

and intestinal muscles, a burst of action potentials is 

required which produces Ca oscillations. This in turn will 

result in a gradual summation of force and the generation 

of the large synchronized contractions needed for voiding 

or parturition. This mechanism of generation of muscle 

“tone” in phasic action potential-generating smooth muscles 

has many advantages over muscle tone produced by 

sustained membrane potential depolarization. One can 

also speculate that Ca oscillations are well suited to the 

generation of gradually rising force induced by changing 

the frequency of action potentials. Smooth muscles controlled 

by single action potentials, such as ureter, appear 

not to express SK channels. Instead, a Ca sparks/STOCs 

coupling mechanism operates, setting the refractory period 

to ensure that there is no summation of the action 

potential, which in the case of the ureter would impair the 

flow of urine and lead to kidney damage. 

D. Ca-Activated Cl Channels 

Chloride channels sensitive to Ca are a second population 

of Ca-sensitive plasma membrane ion channels 

that respond to both local (Ca sparks or puffs) or global 

(Ca spikes, waves, and oscillations) SR Ca release events. 

They are encoded by a family of genes and found in many 

smooth muscles (84, 163, 226). In the majority of smooth 

muscle cells, electrochemical driving force for Cl (E Cl) is 

in the range of 20 to 30 mV, i.e., more positive than the 

resting membrane potential, which is in the range of 50 



to 60 mV. Thus the E Cl is outwardly directed, and activation 

of Cl currents will result in diffusion of Cl out of 

the cell, causing membrane depolarization, i.e., an increase 

in the excitability of smooth muscle cells (321). 

Ca-activated Cl channel currents activated by agonistinduced 

global Ca signals from IP 3-sensitive Ca stores 

have been identified in a number of smooth muscles 

including coronary artery (355), anococcygeus muscle 

(97, 98), portal vein (539), pulmonary artery (737), esophagus 

(4), trachea (310), intestine (717), myometrium (17), 

and rat ureter (625). In some types of smooth muscle, Ca 

sparks were shown to activate Cl Ca to promote membrane 

depolarization and contraction (369, 810). STICs (810) 

were first observed in myocytes from trachea (309) and 

portal vein (386, 736) and have subsequently been reported 

in mesenteric vein (716), trachea (251, 308, 810), 

and pulmonary artery (270, 369), suggesting that a Ca 

sparks/STIC coupling mechanism serves a specific 

functional role in these types of smooth muscles. The 

properties of STICs are consistent with Ca sparks causing 

the transient activation of Cl Ca in the plasma membrane, 

as they are abolished by chloride channel blockers 

and are inhibited by the depletion of SR Ca stores 

(251, 386, 736, 810). 

The coexistence of Cl Ca and BK channels in the sarcolemmal 

membrane adjacent to Ca spark sites suggests 

another level of fine control of membrane excitability. In 

tracheal smooth muscle cells, STICs have been demonstrated 

to be activated by Ca sparks in conjunction with 

STOCs, leading to generation of biphasic STOICs (369, 

810). There are some notable differences in the properties 

of STICs and STOCs. The decay of STICs (t 1/2 65–182 

ms, Ref. 810) is similar to the decay of Ca sparks (810) and 

is much slower than the decay of STOCs (t 1/2 9–43 ms, 

Ref. 810). The amplitudes of STICs and STOCs are similar 

[with similar driving forces for Cl and K even though the 

unitary conductance of the BK channel (80 pS) is 30-fold 

greater than the Cl Ca channel (2.6 pS) (369)]. Therefore, a 

STIC appears to represent the activation by a Ca spark of 

at least 600 clustered Cl Ca channels. The activation characteristics 

and Ca sensitivities of BK and Cl Ca channels 

are also different. BK channels are both Ca and voltage 

sensitive, and depolarization increases their sensitivity to 

[Ca] (107, 134, 136, 438, 508). The Cl Ca channels appear to 

have a higher affinity for Ca (half-maximal activation at 

370 nM, Ref. 538) than BK channels and are less voltage 

sensitive. 

The precise effect on membrane potential of the 

simultaneous activation of Cl Ca and BK channels by a Ca 

spark will depend on a number of factors. These include 

the poor voltage dependence of Cl Ca and high voltage 

dependence of BK channels whose P o changes about 

e-fold for a 12- to 14-mV change in potential (42, 383) and 

their conductances in the cell membrane. Thus, at negative 

potentials (70 to 60 mV), the predominant effect


of Ca spark discharge will be to trigger STICs (810), since 

at these potentials the electrochemical driving force for Cl 

will be greater than that for K. The discharge of STICs will 

cause membrane depolarization and thus serve as a positive-feedback 

mechanism to trigger the activation of voltage-gated 

Ca channels and excitation of the myocytes. As 

depolarization increases the frequency of Ca sparks and 

STOCs, sensitivity of BK channels to Ca and the electrochemical 

driving force for K will also be increased. This 

will make BK more active and result in the generation of 

high-amplitude STOCs and hyperpolarization. This would 

lead to closure of voltage-gated Ca channels and relaxation 

of smooth muscle, as was reported for pressurized 

cerebral arteries (507). High sensitivity of Cl Ca to [Ca] and 

low sensitivity to voltage make them ideal ion channels to 

be targeted by agonists to elicit excitatory effects on 

smooth muscle cells. Unlike STICs, the whole cell Cl Ca 

currents decay much faster than global Ca transients as 

the inactivation of Cl Ca current is determined not by 

intracellular [Ca], but by phosphorylation of the channel 

by CAM kinase II (739). Therefore, the decay of a STIC 

appears to be determined by the decline of Ca sparks, 

whereas the decay of the whole cell Cl Ca currents is 

controlled by CAM kinase II activity. 

To summarize these data, we conclude that the expression 

of a range of different ion channels enables 

muscles to perform diverse functions in the human body. 

Recent data clearly indicate a role of the SR in the Ca 

signaling that controls smooth muscle function, via coupling 

Ca release from the SR to activation of ion channels. 

Discovery of Ca sparks, waves, and oscillations that can 

target several Ca-regulated channels (BK, SK, and IK) can 

produce positive or negative feedback controlling contractility. 

For example, some tonic contractions, which 

can be achieved via sustained membrane depolarization 

or discharge of bursts of action potentials, can be effectively 

decreased via activation of a Ca sparks/STOCs coupling 

mechanism. BK channels coupled to Ca sparks may 

produce long refractory periods, whereas SK channels 

targeted by Ca entry may be associated with short refractory 

periods, as discussed above. Still other mechanisms 

may be present in different smooth muscles, and a better 

understanding of them would aid understanding of the 

control of their contractility under normal and pathological 

conditions. 

XI. SARCOPLASMIC RETICULUM AND 

DEVELOPMENT AND AGING 

In the nervous system and heart there is a relatively 

large amount of literature on the role of Ca homeostasis 

and the SR, and a growing understanding of their importance 

to aging, development, and pathology. Less is 

known for smooth muscle, but there are some data spe- 

cific to fetal and/or neonatal smooth muscles and aging 

tissues and their SR, and this will be briefly presented. 

Perhaps given the diversity of expression in Ca release 

channels between different adult smooth muscles, it 

should not be expected that a uniform thesis can be 

presented. As some papers have examined both development 

and aging, these data will be discussed together as 

appropriate. As changes in [Ca] are considered to be 

important for gene expression and pathological conditions 

in very many tissues, their role in development and 

aging in smooth muscles would seem to be worthy of 

study. It should also be acknowledged that changes 

involving the SR may be direct, e.g., SERCA expression, 

or indirect, e.g., due to changes in Ca buffering or to 

protein-coupled receptor sensitivity and IP 3 production. 

Other changes with development, e.g., development 

of force (502, 613), or Ca sensitivity (682), are not 

considered here. 

Although a complete data set is not available for any 

smooth muscle, it would seem reasonable to conclude 

that a change in the role of the SR occurs with the 

transition from fetal and neonatal life to adulthood. There 

is evidence for this at both structural and biochemical 

levels, as well as from physiological studies. Care has to 

be taken in the interpretation of these data as it is not 

always possible to distinguish SR from ER, and thus to 

establish if the greater ER protein production in the neonates 

contributes to these differences. In addition, not all 

studies have been able to report directly on either 

intracellular or luminal [Ca]. Consequently, although 

changes in SR Ca uptake and release and Ca pool size 

can explain the data, effects on processes such as Ca 

sensitization or excitability cannot always be excluded. 

Nevertheless, a greater contribution of SR to smooth 

muscle function in the neonate has been identified in a 

number of instances, including uterus, bladder, and 

some blood vessels. 

Following examples in other tissues, where aging has 

been linked to dysfunctions in Ca homeostasis and the SR 

(409, 570, 616), studies in smooth muscles have also 

pointed to alterations in Ca signaling (448, 593, 779). From 

this review of the data, it is apparent that cytoplasmic 

[Ca] can rise with aging in smooth muscles. This in turn 

may be attributed to a decline in SERCA activity, although 

more studies on this point are required. This rise in resting 

[Ca] will produce overcontraction and contribute to 

dysfunctions associated with aging such as hypertension 

and incontinence urge. 

XII. GENDER 


The effects of female hormones on the vasculature 

and other smooth muscles are well recognized. While a 

discussion of these interesting differences is beyond the

scope of this review, a little can be said about the SR and 

gender. 

The earliest and still best documented effects of 

gender on SR function come from studies on cardiac 

muscle, including human myocardium (119, 144, 365, 

388, 547). SERCA levels are depressed in failing hearts, 

as is phospholamban phosphorylation. The latter characteristic, 

however, was only found in male hearts. Men 

have a greater incidence of coronary heart disease and 

have augmented myocardial ischemic reperfusion injury, 

which has been related to increased SR Ca load 

(“overload”) (119). In smooth muscle, the few studies 

we have located have been on vascular smooth muscle. 

The findings of lower blood pressure in women than 

men, and the demonstration of male rats in hypertensive 

strains developing a greater elevation of blood 

pressure and with earlier onset than females, can be 

partly attributed to estrogen. There is an estrogendependent, 

protective enhanced release of NO in female 

endothelial cells (112). The cardiac studies mentioned 

above would also point to mechanisms directly 

present in the vascular myocytes and involving the SR, 

as well as other aspects of Ca homeostasis. In addition, 

female hypertensive rats still exhibit an enhanced vasodilatory 

response in the absence of endothelium, 

compared with males (328). 

Aortic cells isolated from male hypertensive rats 

proliferated more rapidly than those from female rats 

(28). There were no differences in resting [Ca] between 

the cells, but a more pronounced increase in [Ca] to 

angiotensin II occurred in males (411). As the authors 

noted, this could indicate a greater ability of agonist to 

mobilize Ca from the SR or release threshold in males. 

In a follow up study, intracellular [Ca] was measured 

and the augmented Ca response to angiotensin II in 

males confirmed. However, SERCA inhibition with 

thapsigargin produced a much larger [Ca] rise in females. 

Thus a role for increased Ca entry rather than SR 

Ca release, in males, would appear more likely (412). 

Gender differences in Ca entry pathways have previously 

been demonstrated (496). This study also found 

no evidence for gender-based differences in SR Ca 

release. Effects of estrogens on SERCA expression in 

smooth muscles have been suggested from a few studies 

(see sect. IIIE). 

It seems to us that there is little direct evidence thus 

far for effects of gender on Ca handling and SR function 

in smooth muscle cells. It is, however, premature to decide 

whether this is due to a lack of significant effects or 

the small amount of data currently available. Given the 

high incidence and morbidity of cardiovascular disease, it 

would be fruitful to have more information from smooth 

muscles on these issues. 


XIII. pH, METABOLISM, AND SARCOPLASMIC 

RETICULUM FUNCTION 


That changes in pH can affect smooth muscle function 

has long been known (645). These changes can be 

intracellular (pH i), occurring for example with changes of 

metabolism and activity, or extracellular (pH o), consequent 

to changes in perfusion or systemic pH disturbance, 

for example (768). The mechanisms by which a change in 

pH i or pH o can affect E-C coupling in smooth muscle are 

numerous. Our focus is on SR Ca release processes and 

SERCA activity. For details on pH and smooth muscles, 

see the following reviews (22, 732, 772). Changes in pH 

are closely associated with metabolic activity and accompanied 

by changes in ATP and P i concentrations, which as 

we discuss will also influence SR function. 

B. pH 


Release through RyR has been shown to be pH sensitive, 

with alkalinization increasing and acidification decreasing 

the sensitivity of the release channels to Ca (151, 

513, 763). In a skinned portal vein preparation (286), 

reduction in pH increased CICR, suggesting in this simplified 

system, where bathing [Ca] is maintained constant, 

that the RyR are either open for longer or have an increased 

P o as pH falls. In intact preparations, this effect 

may be masked, as other processes involved in Ca homeostasis 

are affected. In a recent study in intact rat 

aortic preparations, it was concluded that acidic pH induces 

Ca release from RyRs and contributes to the rate of 

rise of contraction (594). Studies on cardiac SR microsomes 

and lipid bilayers have shown that a fall in pH 

lowers the P o of the RyR (e.g., Refs. 602, 782). In isolated 

cells, Ca sparks are reduced, which is also consistent with 

a fall in RyR P o (30). In pressurized cerebral arteries, 

Heppner et al. (253) showed that a small alkalinization, 

from pH 7.4 to 7.5, increased the frequency of Ca sparks 

and, upon raising it further to pH 7.6, caused a shift from 

sparks to Ca waves in the cells and vasoconstriction. 

These effects were blocked by ryanodine, but not inhibitors 

of IP 3R. The IP 3R, however, is also pH sensitive (71, 

146, 232, 709); alkalinization increases the sensitivity of 

IICR. These effects may be due to changes of pH affecting 

IP 3 binding to the receptor (497), or IP 3 production (8). 

Alkalinization will also increase the P o of IP 3R (324, 767). 

Changes of pH were also reported to be more potent 

when acting at IP 3R3 rather than IP 3R1 (146). Further data 

are required to confirm isoform pH sensitivity. 

As noted in section III, protons are translocated during 

SERCA activity from lumen to cytoplasm (395, 785) 

and may contribute to stabilization of SERCA when its Ca


binding sites are vacant (700). The proton countertransport 

has been reported to stop around luminal pH 8.0 

(551). Thus it may be anticipated that pH will affect 

SERCA Ca pumping. Grover’s group have studied the 

effects of pH alteration on SERCA activity in a variety of 

smooth muscle preparations and concluded that alkalinization 

decreased its activity and acidification increased it 

(165, 166, 228). As a decrease in pH inhibits kinase activity, 

then phosphorylation of phospholamban will be reduced, 

which in turn will produce a decrease in SERCA 

activity (606). Stimulation of SOCE, by SR Ca depletion, 

has also been reported to be pH sensitive and contribute 

to the effects of pH o on vascular tone (748). 

Thus, although sometimes overlooked as investigators 

focus on ion channels and changes in myofilament Ca 

sensitivity, effects of pH alterations on Ca release and 

SERCA activity in smooth muscles will be significant 

factors in functional changes. 

C. Metabolism 

Here we address how metabolic changes such as 

ATP, phosphocreatine (PCr), and P i levels affect SERCA 

activity. Most methods of determining SERCA activity 

couple its cycling to ATP hydrolysis via measurements of 

NADH, with the rate of NADH disappearance being proportional 

to the rate of ATP hydrolysis by SERCA (414). 

Other approaches target the production of P i by SERCA 

activity in colorimetric-based assays (e.g., Ref. 109). In a 

recent paper, Williams et al. (762) described an HPLCbased 

method that also measured ATP, ADP, and PCr. 

The SERCA activity is calculated from the changes in ATP 

and ADP. The authors were also able to confirm that the 

PCr is used locally via creatine kinase to synthesize local 

ATP for SERCA (244, 366). Thus functional coupling and 

localization between PCr, creatine kinase, and SERCA 

occurred. This specific binding of creatine kinase to 

SERCA had previously been reported (e.g., Refs. 367, 

598). As PCr levels are much lower in smooth compared 

with striated muscles (385, 659, 773), this positioning of 

creatine kinase by the SERCA membrane may be particularly 

useful in smooth muscles. Local metabolic 

control of SERCA may be advantageous during conditions 

of metabolic stress, in maintaining SERCA function 

and preventing cytoplasmic [Ca] from becoming 

too high. 

Investigators have suggested, based on the location 

of glycolytic enzymes, that there is also a coupling between 

glycolysis and SERCA activity (168, 780). In smooth 

muscle, a large body of work, particularly from Lorenz 

and Paul (410), has led to the theory of metabolic compartmentation. 

Although beyond the scope of this review, 

there is good evidence that ATP, provided by oxidative 

phosphorylation, supports contraction, whereas that from 


glycolysis supports membrane pumps in smooth muscle 

(246, 295, 420, 421). There is also a much greater lactate 

production and use of glycolysis under normoxic conditions 

in smooth muscle compared with many other cell 

types (422, 770). During hypoxia or ischemia, effects of 

pH and changes in metabolites on SERCA activity will be 

expected to contribute to contractile dysfunction (683). 

XIV. MATHEMATICAL MODELING 

Given the accepted complexities of SR Ca uptake and 

release events, and their reciprocal effect on cytoplasmic 

[Ca], plasmalemmal ion channels, and other organelles 

such as the mitochondria, there is merit in developing 

models of E-C coupling in smooth muscle cells. One 

considerable obstacle to the use of such models is the 

lack of quantitative data for many of the essential parameters, 

e.g., SR [Ca]. Even setting aside experimental differences 

such as temperature, species, and stimulation 

mode, for some smooth muscles, e.g., urethra and anococcygeus, 

many necessary measurements are simply not 

available; others such as vascular and uterine smooth 

muscles have been better studied. 

There are several mathematical models of various 

aspects of E-C coupling in smooth muscle now available 

[e.g., airway, (376), mesenteric artery (335), cerebral artery 

(786)]. Other models have addressed more general 

questions, such as superficial buffer barrier (37), slow 

waves (289), protein interactions (171), and IP 3 changes 

(176). The use of such models could provide significant 

insight into the workings of the SR by taking an integrated 

approach. As such models become more detailed and SR 

focussed, and the “missing” parameters are experimentally 

determined, then a better overall description and 

understanding of the SR in different smooth muscles 

should emerge. 

XV. CONCLUSIONS AND FUTURE DIRECTIONS 

The SR is vital to the modulation and regulation of 

force in smooth muscles. Pathophysiologies are associated 

with perturbation of SERCA, Ca release mechanisms, 

and regulatory proteins, but our appreciation of 

this lags behind studies on striated muscles. This can 

largely be attributed to the lack of clarity around the 

regulation of normal SR function in smooth muscle, 

which in turn is affected by genuine differences between 

tissues. It is uncommon for investigators to address more 

than one smooth muscle in their studies, leading to pockets 

of detailed information in specific areas, but a lack of 

breadth. There are areas where real progress has been 

made, such as unraveling the Ca spark-STOC mechanism 

of regulating excitability and function. Other areas remain 

controversial, such as the role of TRPCs in store-operated

Ca entry. A key question remaining to be answered is 

whether in smooth muscle SR Ca depletion leads to I crac 

only through the Stim-Orai pathway. Another key question 

is how important SOCE is to those smooth muscles 

whose main source of Ca for contraction is L-type Ca 

entry; need such mechanisms be required if SERCA can 

refill from the plasmalemmal Ca entry? More measurements 

of SR luminal Ca are also required, as they could 

provide important mechanistic insight. 

Another area of unfinished business is the role of the 

SR in shaping Ca signals in smooth muscles. With Caactivated 

ion channels that can increase or decrease 

membrane excitability being expressed in the same myocyte, 

elucidation of the role of local SR Ca release to 

global Ca signals is difficult. We have suggested that 

consideration of the pattern of excitation, i.e., single 

short-lasting or plateau-type action potential or trains of 

action potentials, may dictate the involvement or otherwise 

of the SR with BK, IK, and SK and Cl Ca channels 

playing a role. Imaging and molecular biology techniques 

have supported the views expressed over 30 years ago 

that there will be microdomains of Ca and signaling moieties 

within the myocyte. The SR Ca release channels and 

Ca-activated ion channels form part of the underlying 

mechanism, with both the plasma membrane and SR 

membrane having signaling microdomains. 

The existence of different SR Ca stores, which may 

or may not completely intercommunicate, also leads to 

specialization within smooth muscles. Figure 5 illustrates 

some features of the store. This complexity of Ca signaling 

in smooth muscle (and other tissues) is added to by 

the growing evidence that other organelles, including mitochondria, 

lysosomes, Golgi, and nucleus, interact with 

the SR (ER). Furthermore, along with Ca and IP 3 as Ca 

release agents, newer messenger/modulators, including 

NAADP, cADPR, and possibly the L-type Ca channel, need 

to be brought into the mix. Presumably the right mix of 

messenger(s) and Ca store(s), along with the right place 

and time, are how the myocyte decodes the Ca stimulus. 

With time, the SR will give up more of its secrets. 

This requires laboratories applying a range of techniques, 

including functional, electrophysiological, pharmacological, 

and molecular biological studies. We are encouraged 

that the increased use of intact tissues rather than cultured 

cells for molecular biology, along with the success 

of nonviral transfection methods, will result in much new 

data which are physiologically relevant and not dogged by 

concerns about the phenotypic changes occurring with 

culturing. Ultimately, an improved understanding of the 

SR of smooth muscles will contribute to the development 

of new therapeutic approaches to tackle diseases. Given 

the pivotal role of smooth muscle to all the major systems 

of the body, this is a worthwhile goal. 


ACKNOWLEDGMENTS 

We are extremely grateful to M. Nelson for secretarial 

support and help and to Amanda Heath, Sarah Arrowsmith, 

Karen Noble, and Lydmylla Borysova for reading drafts. We 

acknowledge and thank all our colleagues and group members 

who have contributed to the work in this review and engaged in 

discussions with us over the years. 

Address for reprint requests and other correspondence: S. 

Wray, Dept. of Physiology, Univ. of Liverpool, Crown Street, 

Liverpool, Merseyside L69 3BX, UK (e-mail: s.wray@liv.ac.uk). 

GRANTS 

We thank the British Heart Foundation, Action Medical 

Research, WellBeing of Women, the Mothercare Trust, and the 

Wellcome Trust for supporting our research programs. 

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