Sarcoplasmic Reticulum Function in Smooth Muscle - Physiological ...

136 SUSAN WRAY AND THEODOR BURDYGA
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
A. Introduction
Physiol Rev VOL 90 JANUARY 2010 www.prv.org
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