Role of Sarcoplasmic Reticulum in Arterial Contraction: Comparison ...

Role of sarcoplasmic reticulum in arterial contraction: comparison of ryanodines's effect in 

a conduit and a muscular artery. 

T Ashida, J Schaeffer, W F Goldman, J B Wade and M P Blaustein 

Circ Res. 1988;62:854-863 

doi: 10.1161/01.RES.62.4.854 

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854 

Role of Sarcoplasmic Reticulum in Arterial 

Contraction: Comparison of Ryanodine's Effect 

in a Conduit and a Muscular Artery 

Terunao Ashida, Juergen Schaeffer, William F. Goldman, James B. Wade, 

and Mordecai P. Blaustein 

Ryanodine interferes with sarcoplasmic reticulum function in various types of muscle; in vascular 

smooth muscle, it can inhibit contractions that depend on sarcoplasmic reticulum calcium release, 

probably by depleting the sarcoplasmic reticulum calcium store. We tested ryanodine and calcium 

channel blockers (verapamil, diltiazem, and nitrendipine) on small rings of rat thoracic aorta (RA) 

and bovine tail artery (BTA) to determine the relative contributions of sarcoplasmic reticulum calcium 

release and gated calcium entry to contractions induced by norepinephrine, caffeine, and 100 mM K 

depolarization. Ryanodine blocked caffeine contractions in both tissues and attenuated norepinephrine 

responses (by 52% in RA, 14% in BTA) but minimally altered potassium contractions. Calcium 

channel blockers almost completely abolished potassium contractions and reduced norepinephrine 

contractions (by 45% in RA, 82% in BTA) but hardly affected caffeine responses. The blocking effects 

of ryanodine and calcium channel antagonists on the norepinephrine responses were additive. 

Ryanodine had no effect on baseline tension in the standard media; however, when calcium extrusion 

via Na-Ca exchange was inhibited by low external sodium (0-calcium, low-sodium solution), tension 

increased progressively after introduction of ryanodine. This indicates that the sarcoplasmic reticulum 

calcium released by ryanodine then accumulated in the cytosol and activated contraction; restoration 

of external sodium caused prompt relaxation.The smaller effects of caffeine and ryanodine in BTA 

indicate that sarcoplasmic reticulum plays a less important role in calcium control in this tissue, with 

gated calcium entry dominating. These functional findings are correlated with electron-microscopic 

evidence that BTA has about 60% less sarcoplasmic reticulum than does RA. Ryanodine appears to 

be a useful tool for determining the functional relevance of sarcoplasmic reticulum for contraction 

in different arterial smooth muscles. (Circulation Research 1988;62:854-863) 

Contraction of mammalian arterial smooth muscle 

is normally triggered by an increase in the 

cytosolic free calcium concentration,' [Ca 2+ ]|. 

This "trigger calcium" can come either from the 

extracellular fluid or from intracellular stores in the 

sarcoplasmic reticulum (SR). Calcium can enter the 

cells through voltage-gated channels or receptoroperated 

channels that admit calcium 1 ; during depolarization, 

calcium entry may also be mediated by 

voltage-sensitive Na-Ca exchange. 2 Alternatively, or in 

addition, calcium may be released from SR by a 

calcium-induced calcium release mechanism, 3 and/or 

by an inositol trisphosphate-activated mechanism. 4 - 5 

The relative roles of the calcium entry mechanisms and 

SR calcium release are likely to vary with different types 

From the Department of Physiology, University of Maryland 

School of Medicine, Baltimore, Maryland. 

Supported by National Institutes of Health grant AM-32276 and 

a grant from the Muscular Dystrophy Association to M.P.B.; by an 

NSF shared instrument grant DMB-8500564 to J.B.W.; by a Veteran 

Administrations research grant to Dr. Bruce P. Hamilton; and by 

postdoctoral fellowships from the Eli Lilly Company to T.A., from 

the American Heart Association, Maryland Affiliate to W.F.G., and 

from the Deutsche Forschungsgemeinschaft to J.S. 

Address for correspondence: Mordecai P. Blaustein, MD, Department 

of Physiology, University of Maryland School of Medicine, 

655 West Baltimore Street, Baltimore, MD 21201. 

Dr. Ashida's present address: Department of Internal Medicine, 

National Cardiovascular Center, Suita City/Osaka, Japan. 

Received Jury 13, 1987; accepted November 5, 1987. 

of vasoconstrictors (e.g., potassium depolarization promotes 

calcium entry, 6 - 7 while caffeine promotes SR 

calcium release 8 ) and in different arterial tissues. The 

actual amount of SR is likely to be a key determinant in 

the mode of activation of the individual arteries. For 

example, large conduit arteries appear to have a more 

extensive SR than do smaller muscular arteries. 9 Thus, the 

SR may play only a limited role in excitation-contraction 

coupling in small muscular arteries; conversely, the 

movement of calcium across the sarcolemma is likely to 

play a larger role in these vessels. 

The contribution of gated calcium entry to contraction 

can conveniently be studied by application of 

selective organic calcium channel blockers. Functional 

assessment of the role of the SR in contraction is more 

difficult, however, and has involved measurements of 

contractions in calcium-free media 10 " 12 or in the presence 

of caffeine (to deplete the SR calcium stores). 8 

Unfortunately, studies employing caffeine are complicated 

by the fact that it has at least two major 

antagonistic actions: caffeine promotes contraction by 

releasing calcium from the SR 8 and inhibits cyclic 

nucleotide phosphodiesterase, 13 eliciting a rise in 

cyclic nucleotide levels that may be expected to 

promote smooth muscle relaxation." 

Ryanodine is another naturally occurring alkaloid 

that interferes with SR function. 1516 It inhibits evoked 

SR calcium release, in part by promoting a slow 

depletion of the calcium store. 1720 Several investigators 



have shown that ryanodine is effective on vascular 

smooth muscle. 3>2O ~ 22 In contrast to caffeine, which 

causes a rapid release of calcium from SR with a 

transient contraction, ryanodine usually does not itself 

induce a rise in tension. Furthermore, ryanodine does 

not appear to affect the contractile apparatus or the 

sarcolemmal calcium transport mechanisms. 18 Therefore, 

this alkaloid may be especially useful for assessing 

the relative role of the SR in vascular smooth muscle 

contraction. We compared the effects of ryanodine on 

a conduit artery (rat thoracic aorta) and a small 

muscular artery (bovine tail artery). This artery is 

comparable in size to the rat aorta (about 1.5 mm 

diameter) and is particularly interesting because small 

resistance vessels (200-300 (xm diameter), whose cells 

are virtually identical in morphology to those of the 

main tail artery (see "Results"), branch directly from 

the tail artery. These functional studies are correlated 

with morphological evidence that the rat aorta contains 

a much more extensive SR than does the bovine tail 

artery. Preliminary reports of some of these findings 

have been published in abstract form. 23 - 24 

Materials and Methods 

Tissues 

Small rings of artery (approximately 1.5 mm diameter 

and 2-3 mm long) were obtained from bovine tail 

artery (distal end) and rat thoracic aorta. Normal 

Sprague-Dawley rats (200-300 g) were killed by 

decapitation. The thoracic aorta was rapidly excised 

and placed into Krebs solution at 37° C. Tails from 

normal steers were obtained at a local slaughterhouse. 

The distal end of the bovine tail artery was excised 

within 1-1 Vi hours of slaughter and placed into Krebs 

solution. In all tension experiments, the arteries were 

cleaned of surrounding connective tissue and equilibrated 

in the warm Krebs solution until resting tension 

was stabilized at 500 mg (for at least 1 hour) before data 

collection was initiated. 

Solutions 

The tissues were incubated in a modified Krebs 

solution containing (mM) NaCl 138, KC1 4.7, 

NaH2PO4 1.2, CaCl2 1.8, MgSO4 1.2, glucose 10, 

HEPES 10, adjusted to pH 7.4 with Tris and gased with 

100% O2. In some experiments, the potassium concentration 

was increased by replacing some of the 

sodium chloride with equimolar potassium chloride. In 

0-calcium solutions, calcium was replaced with equimolar 

MgCl2. In low-sodium solutions (sodium 1.2 

mM) Af-methyl-glucamine (138 mM) was used as the 

substituting cation and pH was adjusted to 7.4 with 

hydrogen chloride. 

Special Reagents and Drugs 

The following drugs were used: ryanodine (Penick 

Corp, Lyndhurst, New Jersey), dantrolene-Na and 

diltiazem (Tanabe Co, Japan), nitrendipine (Miles 

Pharmaceuticals, Westhaven, Connecticut), verapamil-HCl 

(Knoll Pharmaceuticals, Whippany, New 

Jersey), /-norepinephrine-HCl and caffeine (Sigma 

Ashida et al Ryanodine, Sarcoplasmic Reticulum, and Arterial Contraction 855 

Chemical, St. Louis, Missouri), and prazosin-HCl 

(Pfizer Laboratories, New York). Dantrolene and 

nitrendipine were kept as 10-mM stock solutions in 

poryethylenegh/col (#400) in opaque containers. 

These agents were added to the Krebs solution as 

indicated in "Results." 

Contraction Measurements 

Two thin (0.4 mm diameter) stainless steel hooks 

were inserted through the lumen of the artery ring. One 

hook was fixed to the floor of a small jacketed tissue 

chamber (volume 0.75 ml); the other hook was 

connected to a Harvard Model #52-9529 or Model 

#363 force transducer (South Natick, Massachusetts) 

that was mounted immediately above the tissue chamber. 

Isometric tension was continuously monitored and 

recorded on a strip chart recorder. The tissue was 

steadily supervised at a rate of 2 ml/min with welloxygenated 

incubation fluid at 37° C. Most drugs and 

other reagents were added directly to the superfusion 

fluids and allowed to reach a steady concentration in the 

incubation fluid within the tissue chamber. Usually, 

however, norepinephrine (NE) was applied as a small 

(25 |il) bolus injection into the superfusion line; the NE 

was thus diluted 30-fold when it reached the tissue 

chamber. Under these circumstances, the concentration 

of NE in the incubation chamber rose rapidly to a peak 

that lasted 15-25 seconds; the NE concentration then 

declined with a half-time of 15 seconds. Very reproducible 

NE-activated contractions were normally obtained 

under these conditions (see "Results"). 

Electron Microscopy 

Fresh segments of rat aorta and bovine tail artery (three 

animals each) were fixed in 2.5% glutaraldehyde and 

postfixed for 30 minutes with 1% OsO4 in cacodylate 

buffer. Subsequently, the tissue was dehydrated and 

embedded in Epon. Thin sections (60-90 nm) were 

poststained with uranyl acetate and lead citrate and 

examined in a Zeiss 10 CA electron microscope. 

Morphometry 

Grids were coded, and random micrographs were 

obtained without knowledge of the tissue of origin. If, 

at the magnification necessary to visualize SR, the 

entire cell cross-section could not be included, a 

random region of the cell that included the cell surface 

and part of the nucleus was chosen. Electron micrographs 

of four to six arterial smooth muscle cells from 

each animal (three rats and three steers) were taken on 

a random basis and enlarged to a final magnification of 

25,000 x. The micrographs were evaluated morphometricalry 

using the point grid planimetry method. 25 A 

1-cm point grid was used for quantification of nucleusfree 

cell volume. The area of SR (membrane-bound 

cytoplasmiccisternae, tubules, and vesicles) and rough 

endoplasmic reticulum (RER; ribosome-covered membranes) 

was quantitated with a 0.5-cm point grid. These 

data were expressed as volume percent of nucleus-free 

cell volume, giving a proportional estimate of SR and 

RER content in the cytoplasm. 



856 Circulation Research Vol 62, No 4, April 1988 

-A 

Cat. K 

H nU 100 mil 

.200 mg 

Ryanodhw 10 u 

._• V 

Cat. K 

5 mU 100 mM 

FIGURE 1. Effect of ryanodine (10 \LM) on contractions 

induced by 100 mM K and 5 mM caffeine in rat aortic ring. 

Tissue was incubated in ryanodine for 45 minutes before data 

for right-hand side were collected. Each contraction was 

preceded by a resting phase of at least 20-30 minutes. Resting 

tension, 500 mg. 

Results 

Ryanodine Selectively Blocks Calcium Release From 

Arterial Muscle Sarcoplasmic Reticulum 

As illustrated in Figure 1, ryanodine completely 

blocked caffeine-induced contractions in rat aorta but 

had only a minimal effect on potassium-induced 

contractions. This is consistent with evidence that 

ryanodine selectively interferes with SR calcium 

release 3 but not gated calcium entry. In contrast, 

dantrolene, another agent that is reputed to block SR 

calcium release in muscle, 26 did not affect caffeineinduced 

contractions in this tissue (Figure 2). 

Mechanism of Ryanodine Action 

Inhibition of the caffeine contractions by ryanodine 

can be explained either by direct blockade of calcium 

release from the SR or by depletion of the SR calcium 

store as a result of enhanced release and reduced 

resequestration. 17 -"' 20 - 27 To distinguish between these 

two possibilities, the effects of ryanodine were studied 

under conditions in which calcium extrusion was 

inhibited. In modified Krebs solution with normal 

sodium, ryanodine had no effect on tonic tension. 

Supervision with 0-calcium, 1.2-mM-Na solution, 

however, will inhibit calcium extrusion via Na-Ca 

exchange. 2 Under these conditions, ryanodine caused 

a slow increase in tonic tension that was promptly 

reversed when normal external sodium was restored 

(Figure 3). This effect was also observed in the 

presence of 1 \JM prazdsin and 10 JJLM verapamil (not 

shown) and therefore was not due either to endog- 

Caf 

NE NE SmM 

30 min 

200 mg 

NE NE NE NE 

DantroUne 10 pM 

Caf. 

S mM 

enously released NE or to calcium entry via voltagegated 

channels. These data suggest that ryanodine 

depleted the SR calcium store by slowly releasing 

calcium into the cytosol. 

In the absence of ryanodine, both caffeine and NE 

induced contractions in rat aorta superfused with 

0-calcium, 1.2-mM-Na solution (not shown; see Itoh 

et al 6 for data on rabbit mesenteric artery). Under these 

calcium-free, low-sodium conditions, ryanodine reversibry 

blocked the contractile responses to NE (not 

shown) and caffeine. In the presence of ryanodine, 

caffeine evoked only relaxation (Figure 3); this is 

additional evidence that ryanodine depletes the SR 

calcium stores. 

Estimation of Relative Role of Sarcoplasmic Reticulum 

in Vascular Smooth Muscle Contractions 

NE contractions in rat aorta. Contractions elicited 

by NE are, in part, dependent on calcium entry from 

the extracellular fluid and, in part, on calcium release 

from the SR. 111 As illustrated in Figures 2 and 4-7,10 

(JLM ryanodine reduced substantially the NE contractions 

in rat aorta but did not abolish them. The 

dose-response curve (Figure 4) shows that maximal 

concentrations of ryanodine (10-30 ^M) inhibited the 

NE-induced contractions by about 45%, suggesting 

that about half of the contraction elicited by 2-6 x 10~* 

M NE could be attributed to calcium release from the 

SR. Furthermore, ryanodine (10 ^M) appeared to 

inhibit contractions elicited by lower concentrations of 

NE to a slightly greater degree than contractions 

induced by higher NE doses (Figure 5). This may 

indicate that contractions elicited by low concentrations 

of NE are more dependent on calcium released 

from SR than area contractions produced by higher 

doses. 

When rat aortic rings were superfused with a steady 

concentration of NE, tension rose rapidly and then was 

maintained (Figure 6). At all NE concentrations, 

ryanodine predominantly attenuated the initial rise in 

tension. This is consistent with data from the rabbit ear 

artery 21 in which only the initial rapid component of NE 

contractions was blocked by ryanodine; this effect can 

be explained by depletion of the SR store. The tonic 

phase, on the other hand, appeared to be slightly 

enhanced, possibly due to impairment of SR calcium 

resequestration by ryanodine. 

In contrast to ryanodine, dantrolene had no effect on 

the NE-induced contraction (Figure 2). 

A A & A A A 

NE NE NE NE NE NE NE 

Ryanodine 10 pM 

FIGURE 2. Effects of 10 \xMdantrolene and 10 \iMryanodine on contractile response of rat aortic ring to6x 10'' Mnorepinephrine 

(NE) and 5 mM caffeine (Caf.). Periods of superfusion with dantrolene and ryanodine, respectively, are indicated by bars at top. 

Resting tension, 500 mg. 



100 mg 

Caffeine 

10 mM 


-Ryanodine 10 pf 

-OCa- 

-12 mM Na / 138 mM NMG- 


10 mM 


10 mM 

Additive effects of calcium channel blockers and 

ryanodine on NE-induced contractions in rat aorta. 

Calcium channel blockers, which are known to block 

some receptor-operated channels 7 - 28 as well as voltagegated 

calcium channels 7 ' 29 in vascular smooth muscle, 

almost completely abolished contractions evoked by 

potassium depolarization (e.g., Figure 7 and below). 

In contrast, even high concentrations (10-30 \iM) of 

§ 100 - • 

c 

>d I 

trol 

Is 

1 O 

• 50 

eplnephi 

o 

z 

(Perce 

n 

- 

- 

• 

V 

8R I V 

Ca Channels 

• i 

0.01 0.1 1 

Ryanodine (pM) 

Oftlizam NMrendfekn Vaf 

10 yM 10 CM 10 CM 

FIGURE 4. Dose-response curve show- ing effect of ryanodine 

on norepinephrine-evoked contraction tension of rat thoracic 

aortic rings (•). In the presence of a maximum concentration 

of ryanodine (10 pM), a number of the preparations were 

exposed to verapamil (a, n = 3), diltiazem (o, n = 5), or 

nitrendipine (A, n=2), as indicated on right-hand side. *, 

mean ± SEM for nine preparations. NE concentrations were 

2-6 x W M. Resting tension, 500 mg. Results suggest that 

norepinephrine contraction can be divided into three components: 

1) a ryanodine-sensitive component, 2) a component due 

to calcium entry from the extracellular fluid via calciumselective 

channels (i.e., it is blocked by three types of calcium 

channel blockers), and 3) an unidentified component (?) that is 

due to neither calcium release from SR nor calcium entry via 

calcium channels. Latter component accounts for 10-20% of 

NE-induced contraction. See text for further details. 


10 mM 

FIGURE 3. Ryanodine-induced tension increase 

in 0-calcium (0-Ca), low-sodium (low- 

Na) solution, rat aortic ring. After stabilization 

and caffeine control contraction in Krebs solution, 

tissue was perfused for 30 minutes with 

a 0-calcium, 1.2-mM-Na, 138-mM N-methylglucamine 

solution, without change in baseline 

tension. Addition of 10 \LM ryanodine led to a 

slow but marked increase in tension that was 

reversed rapidly by restoration of a normal 

sodium gradient in spite of the continuing 

presence of ryanodine. Caffeine contraction 

was completely blocked by ryanodine (only 

relaxation was seen) but was fully restored 

after 2 hours in ryanodine-free Krebs solution. 

There was a 35-minute interval between the first 

and second section of the trace and 110 minutes 

between the second and third section. Resting 

tension, 500 mg. 

the calcium channel blockers reduced NE contractions 

in rat aorta by less than 50%. 2 Combining ryanodine 

with a calcium channel blocker had an additive effect 

and suppressed contractions to a much greater degree 

than did either inhibitor alone (Figures 4, 5, and 7). 

Nevertheless, a small portion of the NE-induced 

contraction was insensitive to high concentrations of 

ryanodine and the calcium channel blockers but was 

dependent on external calcium (not shown). 

Sources of trigger calcium in bovine tail artery. 

Morphological data 910 - 30 indicate that there is large 

variation in SR content between different vascular 

smooth muscles: more peripheral vessels may have 

much less SR than do the large conduit vessels. We 

studied the bovine tail artery, which is similar in 

diameter to the rat aorta, to explore the functional 

correlates of these morphological observations. 

100r 

.-?>• 

,-- < + 10 |1M 

Ryanodln* 

' ••• . ' 

0 pU 

nodln* 

0 pM 

WH.r indlclix 

N o r a p l n a p h r l n a < H ) 

10" 10' 

FIGURE 5. Effect of 10 \iM ryanodine and 10 \iM nitrendipine 

(in presence of ryanodine) on norepinephrine dose-response 

curve of rat aortic rings. 




TABLE 1. Contractile Responses to Various Stimuli in Rat Aorta and Bovine Tail Artery 

A. Maximal contractions in response to potassium-depolarization, norepinephrine, i 

and caffeine 

Maximum contraction amplitude (mg tension + SEM) 

Treatment 

100 mM K solution 

6X 10~ 3 M Norepincphrine 

10 mM Caffeine 

Rat aorta 

1,508 + 206 (6) 

1,090 ±433 (4) 

158±12 (14) 

Bovine tail 

artery 

1,313±237(7) 

678+163(6) 

61 ±17 (5) 


artery/rat aorta 

0.87 

0.62 

0.39 

B. Rate of tension increase in 0-calcium, low-sodium solution 

Rate of tension increase (mg tension/hr + SEM) 



Treatment 

Rat aorta 

artery 

artery/rat aorta 

Control 

None 

None 

10 ^M Ryanodine 

n, number in parentheses. 

205 ±12 (3) 

38+14 (3) 

0.19 

The contractile responses of bovine tail artery to 

potassium-rich solutions, to brief pulses of NE, and 

to caffeine are illustrated in Figures 8 and 9; the data 

are compared with rat aorta in summarized form 

in Table 1A. Potassium-induced contractions in bovine 

tail artery were comparable to those in rat 

aorta: they were almost completely abolished 

by 10 u,M verapamil (Figure 8) but were unaffected 

by ryanodine (Figure 9). Further, a small external 

calcium-dependent fraction of the response persisted 

in the presence of verapamil. In contrast, the 

effects of verapamil on NE-induced contractions 

were different in the two vessels. On average, 

NE-induced bovine tail artery contractions were 

inhibited by 82 ± 8% (SEM, n = 6) as compared with 

45 ±2% (n = 4) in rat aorta. Conversely, ryanodine 

inhibited NE-induced contractions by only about 

14 ± 3% (n = 6) in bovine tail artery (Figure 9) but by 

about 52 ±2% (n = 8) in rat aorta (see Figure 4). In 

bovine tail artery, caffeine elicited brief, small contractions 

(Figure 9 and Table 1A), followed by 

sustained relaxation (Figure 9). As in rat aorta, 

ryanodine blocked the initial caffeine-induced contraction 

but did not affect the subsequent relaxation 

200 mg 

NE 

10" 6 M 

FIGURE 6. Effect of 10 \iM ryanodine on the norepinephrine 

(NE) response. In this experiment, unlike others with the rat 

aortic rings (see text), tissue was superfused with 10 "* M NE 

continuously for periods indicated by bars under contraction 

records. Tissue was exposed to 10 p.M ryanodine for 60 minutes 

before record on right-hand side was obtained. Resting tension, 

500 mg. 

(Figure 9). Note that in bovine tail artery, the average 

amplitude of the caffeine-induced contractions was 

less than 5% of that induced by high potassium but was 

about 10% of the average potassium-induced contraction 

in rat aorta. 

When sodium-dependent calcium extrusion was 

suppressed by superfusing 0-calcium, 1.2-mMsodium 

solution, bovine tail artery exhibited qualitatively 

the same response to ryanodine as did rat aorta 

(Figure 3): a progressive increase in tension, with 

rapid relaxation when the standard (139.2 mM) 

sodium medium was reintroduced. However, the rate 

at which this tension developed was much slower in 

the bovine tail artery than in rat aorta (Table IB). This 

observation, as well as the smaller contractile response 

to caffeine and the smaller ryanodine-sensitive 

component of the NE contraction, indicates that the 

bovine tail artery has a relatively small SR store of 

calcium as compared with rat aorta. These data imply 

that most of the "activator calcium" in bovine tail 

artery comes from the extracellular fluid. 

Morphology and Morphometry of Rat Aortic and 

Bovine Tail Artery Cells 

The 15 arterial myocytes from each species that 

were analyzed morphometricalry were a representative 

sample of the population. These cells appeared 

relaxed and did not exhibit swollen vacuoles or other 

artifacts. 

In overall structure, rat aortic cells (Figure 10) 

differed markedly from those of the bovine tail artery 

(Figures 11 and 12). As described by others, 31 the rat 

cells were roughly cuboidal and ranged from about 

3 x 7 to 10 x 15 ^m in size (Figure 10). SR and rough 

endoplasmic reticulum (distinguished by the presence 

of ribosomes on the surfaces of the latter) as well as 

mitochondria were abundant and were distributed 

throughout the cytoplasm. 

Bovine tail artery cells were spindle shaped and 

much larger: 10-15 jim in diameter and usually more 

than 100 \im in length (Figure 12, light microscopy 

indicated that most cells were about 10—15 x 125-150 



100 mg 


Ryanodine 10 |iM 

NE K 

100 mM 

Verapamil 10 

H,m). The cytoplasm of these cells (Figures 11 and 12) 

was characterized by numerous fibrils and dense bodies 

that were not prominent in the rat aortic cells. The SR 

was very sparse and was concentrated in the region just 

under the plasma membrane; some SR, as well as rough 

endoplasmic reticulum, was also located in the region 

around the nucleus. The few mitochondria observed 

were generally confined to the region around the 

nucleus and to the periphery, just under the plasma 

membrane. 

Morphometric analysis (Table 2) indicated that rat 

aortic myocytes contain substantially more SR and 

rough endoplasmic reticulum than do bovine tail artery 

myocytes. The SR accounts for about 5.4% of the 

nucleus-free cytoplasmic volume in the rat aortic cells 

but for only about 2.3% in the bovine tail artery cells. 

Furthermore, the latter value may be an overestimate. 

Because much of the SR in these cells was located 

adjacent to the plasma membrane, it was often difficult 

to distinguish surface invaginations ("caveolae") from 

SR. Thus, some surface invaginations, which did not 

exhibit clear openings to the extracellular space, were 

included in the SR counts. 

Discussion 

Mechanism of Action of Ryanodine 

Our results demonstrate that in arterial smooth 

muscle, ryanodine selectively blocks evoked SR calcium 

release but has little effect on gated calcium entry. 

The mechanism of action of ryanodine, however, is still 

unresolved. There is evidence that one effect is to 

maintain SR calcium release channels in an open 

state, 17 "" producing a functional blockade of release as 

a result of depletion of the SR calcium store. Nevertheless, 

the effect of ryanodine seems to depend on the 

20 min 

|1 Verapamil 10 

k — 

NE K 

100 mM 

K 

50 mM 

FIGURE 7. Comparison of effects of ryanodine and 

verapamil on the norepinephrine (NE)- and 100 mM 

K-evoked contractions in rat aortic ring. After the 

introduction of 10 \iM ryanodine, the contraction 

response to 6 x 10'' M NE was much more markedly 

attenuated (by 55%) than was the response to 100 mM 

K (by 10%). Responses to NE (in the presence of 

ryanodine) and 100 mM K were both greatly reduced 

by 10 \iM verapamil. Resting tension, 500 mg. 

experimental conditions' 8 - 32 " 34 ; ryanodine blocks caffeine 

contractions under some circumstances, 33 but not 

under others. 34 A possible explanation for these divergent 

observations is that ryanodine locks the SR 

calcium release channels in a subconducting or "leaky" 

state 27 - 35 but does not prevent SR calcium uptake. Thus, 

with a large calcium influx, or with a low concentration 

or brief exposure to ryanodine, the SR calcium store 

may not be totally depleted. 

In tracer flux studies, 20 ryanodine promoted calcium 

efflux from rabbit aorta, presumably because it 

enhanced passive SR calcium release. The subsequent 

suppression of NE- and caffeine-induced contractions 

could then be explained by the depletion of the SR 

calcium store. Our observations are consistent with 

the view that ryanodine causes slow release of calcium 

from the SR into the myoplasmic space. The data also 

suggest that ryanodine, like caffeine, 2 - 8 may be very 

useful for separating sarcolemmal calcium transport 

mechanisms from SR calcium sequestration mechanisms 

because ryanodine does not appear to affect 

calcium movement across the sarcolemma. 18 

Comparison of Contractions in Rat Aorta and Bovine 

Tail Artery: Structure-Function Relations 

Rat aorta exhibits a substantial caffeine contraction 

that is ryanodine-sensitive. The implication is that rat 

aortic smooth muscle has a relatively large SR store 

of calcium. This view is supported by the morphologic 

observations that indicate that the SR accounts for a 

much larger fraction of the nonnuclear cytoplasmic 

volume in this tissue than in bovine tail artery. 

Moreover, evoked SR calcium release may play an 

important role in excitation-contraction coupling in 

rat aorta because nearly half of the NE-induced 


http://circres.ahajournals.org/ by guest on April 6, 2013 

FIGURE 8. Effect of 10 \iM verapamil on contractile 

responses of ring of bovine tail artery to 50 mM K and 

to 2 x 10'" M norepinephrine (NE). Periods during 

which 50 mM K and verapamil were present are 

indicated by appropriately labeled bars. NE was 

introduced (by bolus injection) at times indicated by 

arrowheads. Resting tension, 500 mg.


K 

100 mM 


10 mM 

-Ryanodino 1 0 yM- 

K 

100 mM 

contractions can be attributed to ryanodine-sensitive 

calcium release from SR; the remaining half appears 

to be due to calcium entry from the extracellular 

fluid. Thus, it should be possible to determine 

the relative contributions of calcium entry and SR 

calcium release with other agonists by comparing 

contractions evoked in the absence and presence of 

ryanodine. 

These findings on rat aorta, a large conduit artery, 

were compared and contrasted with observations from 

a similar-diameter peripheral muscular artery, the 

bovine tail artery. In the latter artery, the ryanodinesensitive 

fractions of both NE- and caffeine-induced 

contractions were significantly smaller than in rat aorta. 

This suggests that there is a smaller SR store of calcium 

and that it plays a less important role in excitationcontraction 

coupling in bovine tail artery than in rat 

aorta. The morphologic data, which show that the 

bovine tail artery has about 60% less SR than rat aorta, 

are consistent with the physiological findings. We also 

observed a pronounced difference in rough endoplasmic 

reticulum content between the two arteries. Rough 


10 mM 

t 

NE 

10" 4 M 

FIGURE 9. Contractile responses 

of bovine tail artery to 100 mM K, 10 

mM caffeine, and maximal dose 

(1.2 x 10" M) norepinephrine (NE) 

before and (as indicated by bar) 

during incubation with 10 \LM ryanodine. 

Tissue was incubated in ryanodine 

for 45 minutes before data 

for right-hand side were obtained. 

Each contraction was preceded by 

resting phase of at least 20-30 minutes. 

Small caffeine contraction was 

suppressed by ryanodine, NE response 

was only minimally reduced, 

and potassium contraction was unaffected. 

Caffeine-induced relaxation 

was not altered by ryanodine. 

Resting tension, 500 mg. 

endoplasmic reticulum has been shown to participate 

in the metabolism of [Ca 2+ ], in other tissues. 36 

Because our contraction data indicate that rat 

aorta has a much larger internal calcium store than 

does bovine tail artery, these morphological considerations 

raise the possibility that rough endoplasmic 

reticulum also plays a role in calcium regulation and 

excitation-contraction coupling in vascular smooth 

muscle. 

There have been no systematic studies of SR density 

in various types of arteries. Nevertheless, published 

data'' 10 * 30 suggest that, in general, peripheral, muscular 

arteries have substantially less SR than do central, 

conduit vessels. It is likely that the peripheral blood 

vessels, especially, are tonicalry stimulated by neural 

and humoral factors and are thus tonicalry contracted. 

Consequently, [Ca 2 ^ must be constantly maintained 

above contraction threshold. This situation thus differs 

from that in skeletal muscle, 37 where the SR plays a key 

role in the initiation and termination of contraction by 

phasic release and resequestration of calcium. In 

peripheral artery smooth muscle, the primary control 

TABLE 2. Morphometric Quantitatlon of Sarcoplasmic Reticulum and Rough Endoplasmic Reticulum in Cytoplasm 

of Bovine Tail Artery and Rat Aortic Cells 

Specimen 

Bovine tail artery 

a 

b 

c 

Mean 

Rat aortic cells 

d 

e 

f 

Nucleus-free area 

(cm 2 ±SEM) 

297 ± 24 

3O4±25 

207 ± 36 

269 + 20 

99±17 

162 ±29 

106±31 

Percentage of nucleus-free volume ±SEM 

SR 

RER 

SR + RER 

2.53 ±0.39 

2.30±0.31 

2.12±0.28 

2.32±0.19* 

5.71 ±0.75 

5.40±0.53 

5.27 ±0.67 

0.86±0.13 

1.56±0.09 

1.07±0.11 

1.16±0.10* 

4.65±0.61 

5.92 + 0.67 

4.65 + 0.55 

3.39±0.37 

3.86±0.27 

3.19±0.22 

3.48±0.17* 

10.36 + 0.99 

11.36 ±0.76 

9.92±0.75 

Number 

of cells 

Mean 

126±17 5.44±0.36* 5.16 + 0.38* 10.61 ±0.49* (15) 

*Data combined for all bovine tail artery cells and all rat aortic cells, respectively. Values for rat aortic cells were 

significantly larger than those for bovine tail artery, with p


FIGURE 10. Rat aortic smooth muscle cell, 24,500 x. SR, sarcoplasmic reticulum; RER, rough endoplasmic reticulum. Bar, 1 \un. 

of [Ca 2 *], must reside in the sarcolemmal calcium 

transport systems to maintain and regulate tone. 38 

The relatively sparse SR probably functions primarily 

as a modulator or buffer to help dampen large 

changes in [Ca 2 ^. Our observations confirm that 

Na-Ca exchange plays a role in Ca 2+ transport 

FIGURE 11. Bovine tail artery smooth muscle, 4,500 x. SR, sarcoplasmic reticulum. Bar, 10 

across the plasma membrane in these arterial smooth 

muscle cells. 2 The sarcolemmal sodium gradient most 

likely modulates tonic [Ca 2+ ], and the size of the 

SR calcium store (and, thus, contractility) in these 

arterial smooth muscle cells 38 just as it does in cardiac 

muscle. 39 




FIGURE 12. Bovine tail artery smooth muscle cell, section, 24,750x. SR, sarcoplasmic reticulum; RER, rough endoplasmic 

reticulum; DB, dense body. Bar, 1 \un. 

Acknowledgments 

We thank Drs. W.J. Lederer and W.G. Wier for the 

samples of ryanodine and nitrendipine and Dr. Wier for 

valuable comments on the manuscript. 

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KEY WORDS • ryanodine • calcium • sarcoplasmic reticulum 

arterial smooth muscle

Role of Sarcoplasmic Reticulum in Arterial Contraction: Comparison ...

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