J Appl Physiol 92: 1603–1610, 2002;
10.1152/japplphysiol.00756.2001.
Silver ions induce Ca2⫹ release from the SR in vitro
by acting on the Ca2⫹ release channel and the Ca2⫹ pump
R. TUPLING AND H. GREEN
Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
Received 23 July 2001; accepted in final form 21 December 2001
calcium ion cycling; calcium ion release; calcium ion uptake;
silver nitrate
THE CONTRACTION-RELAXATION cycle of skeletal muscle is
regulated by the release and accumulation of Ca2⫹ by
the sarcoplasmic reticulum (SR) (24). T-tubule depolarization causes the rapid release of Ca2⫹ from the SR
store, via the Ca2⫹ release channels (CRC), for the
initiation of contraction. Subsequently, cytosolic Ca2⫹
is rapidly decreased to allow muscle relaxation. The
latter involves reuptake of the released Ca2⫹ via the
SR Ca2⫹-ATPase, which is an energy-dependent process (23).
Loss of SR function, such as occurs during and after
repeated intensive muscular contractions, leads to al-
Address for reprint requests and other correspondence: H. J.
Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON, Canada N2L 3G1 (E-mail: [email protected]).
http://www.jap.org
terations in SR Ca2⫹ cycling, both lower Ca2⫹ release
and Ca2⫹ uptake, and lower force (i.e., muscle fatigue)
(2). Curiously, intrinsic reductions in Ca2⫹ uptake and
Ca2⫹ release, after various fatigue protocols, appear to
be qualitatively and quantitatively similar (20, 42).
After eccentric exercise (20), the time course (in days)
for both the reduction and recovery in SR Ca2⫹ uptake
and Ca2⫹ release is also closely associated. Moreover,
in other models of muscular stress, such as ischemiareperfusion injury (28, 37), a similar trend is observed,
namely an apparent coupling between the reductions
in both SR Ca2⫹ uptake and Ca2⫹ release. Conceivably,
the apparent coupling could be explained by a common
mechanism involving specific sites on the Ca2⫹-ATPase
and the CRC and causing structural modifications to
these proteins. Alternately, the possibility exists that
the coupling is artifact, because of limitations in the
measurement assays. In general, Ca2⫹ release is measured after Ca2⫹ uptake and SR Ca2⫹ loading as part of
the same assay.
Recently, we have also shown that 4 h of ischemia
leads to a reduction in both SR Ca2⫹ uptake and Ca2⫹
release in both homogenates and isolated SR vesicles
prepared from rat skeletal muscle (40). In the ischemic
study, we measured Ca2⫹ release in vitro using silver
nitrate (AgNO3), which produced a biphasic Ca2⫹ release response. There was an initial, rapid rate of
release, which we called phase 1, followed by a more
prolonged and slower rate of release, which we called
phase 2. Surprisingly, we showed that, if the Ca2⫹ATPase was inhibited with cyclopiazonic acid (CPA)
just before Ca2⫹ release was induced with AgNO3, then
the rate of Ca2⫹ release corresponding with phase 2
would be greatly reduced in both ischemic and control
SR. To explain this effect, we hypothesized that SR
Ca2⫹ release and Ca2⫹ uptake processes are tightly
coupled such that Ca2⫹ uptake by the SR during net
SR Ca2⫹ release exerts an influence on Ca2⫹ release
and is important for maintaining normal Ca2⫹ release
function.
Alternatively, AgNO3 might induce Ca2⫹ release by
direct interaction with the Ca2⫹-ATPase. Initial reports have indicated that AgNO3 induces Ca2⫹ release
from the SR by acting on the CRC and not on the
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solely to indicate this fact.
8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society
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Tupling, R., and H. Green. Silver ions induce Ca2⫹
release from the SR in vitro by acting on the Ca2⫹ release
channel and the Ca2⫹ pump. J Appl Physiol 92: 1603–1610,
2002; 10.1152/japplphysiol.00756.2001.—Silver nitrate (AgNO3)
is a sulfhydryl oxidizing agent that induces a biphasic Ca2⫹
release from isolated sarcoplasmic reticulum (SR) vesicles by
presumably oxidizing critical sulfhydryl groups in the Ca2⫹
release channel (CRC), causing the channel to open. To
further examine the effects of AgNO3 on the CRC and the
Ca2⫹-ATPase, Ca2⫹ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1.
Cyclopiazonic acid (CPA) and ruthenium red (RR) were used
to inhibit the Ca2⫹-ATPase and block the CRC, respectively,
before inducing Ca2⫹ release with both AgNO3 and 4-chlorom-cresol (4-CMC), a releasing agent specific for the CRC.
With AgNO3 and CPA, the early rapid rate of release (phase
1) was increased (P ⬍ 0.05) by 42% (314 ⫾ 5 vs. 446 ⫾ 39
␮mol 䡠 g protein⫺1 䡠 min⫺1), whereas the slower, more prolonged rate of release (phase 2) was decreased (P ⬍ 0.05) by
72% (267 ⫾ 39 vs. 74 ⫾ 7.7 ␮mol 䡠 g protein⫺1 䡠 min⫺1). RR, in
combination with AgNO3, had no effect on phase 1 (P ⬎ 0.05)
(314 ⫾ 51 vs. 334 ⫾ 43 ␮mol 䡠 g protein⫺1 䡠 min⫺1) and decreased phase 2 (P ⬍ 0.05) by 65% (245 ⫾ 34 vs. 105 ⫾ 8.2
␮mol 䡠 g protein⫺1 䡠 min⫺1). With 4-CMC, CPA had no effect
(P ⬎ 0.05) on either phase 1 or 2. With addition of RR, phase
1 was reduced (P ⬍ 0.05) by 59% (2,468 ⫾ 279 vs. 1,004 ⫾ 87
␮mol 䡠 g protein⫺1 䡠 min⫺1), and RR completely blocked phase
2. Both AgNO3 and 4-CMC fully inhibited Ca2⫹-ATPase
activity measured in homogenates. These findings indicate
that AgNO3, but not 4-CMC, induces Ca2⫹ release by acting
on both the CRC and the Ca2⫹-ATPase.
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SARCOPLASMIC RETICULUM FUNCTION AND SILVER IONS
METHODS
Animal description and care. Adult female Sprague-Dawley rats weighing between 225 and 275 g were housed in an
environmentally controlled room (temperature 22–24°C, 40–
60% relative humidity) with reversed light-dark cycles. Animals were fed ad libitum on laboratory chow and water until
the time of the experiment. All experiments (muscle sampling) were initiated at approximately the same time each
day to avoid large diurnal variations in muscle glycogen (6).
Experimental protocols were approved by the Animal Care
Committee of the University of Waterloo.
Sample preparation for SR assessment in vitro. SpragueDawley rats (n ⫽ 9) were weighed and anesthetized using an
intraperitoneal injection of pentobarbital sodium (6 mg/100 g
body wt). After anesthetization, the gastrocnemius muscle
(both red and white portions), along with the entire tibialis
anterior muscle, was excised from each limb and placed in
ice-cold buffer to be used for preparation of muscle homogenates according to Heilmann et al. (16). Mixed gastrocnemius
and tibialis anterior muscles were diluted ⬃1:5 (wt/vol) in
buffer containing (in mM) 5 HEPES (pH 7.5), 250 sucrose,
0.2% sodium azide, and 0.2 phenylmethylsulfonyl fluoride
[no dithiothreitol (DTT)] and mechanically homogenized with
a polytron homogenizer (PT 3100) at 16,500 rpm, for 2 ⫻ 30-s
bursts. Aliquots of muscle homogenate were then rapidly
frozen in liquid nitrogen and stored at ⫺70°C for later analysis of SR function.
J Appl Physiol • VOL
SR Ca2⫹ release measurements. AgNO3-induced Ca2⫹ release was measured in muscle homogenates according to the
methods of Ruell et al. (29), using the Ca2⫹ fluorescent dye
indo 1, with minor modifications as previously described (40).
Fluorescence measurements were made on a spectrofluorometer (RatioMaster system, Photon Technology International)
equipped with dual-emission monochromators. The measurement of cytoplasmic (buffer) free Ca2⫹ ([Ca2⫹]f) using the
indo 1 procedure is based on the difference in the maximal
emission wavelengths between the Ca2⫹-bound form of indo
1 and the Ca2⫹-free form. The excitation wavelength was 355
nm, and the emission maxima were 485 and 405 nm for
Ca2⫹-free (G) and Ca2⫹-bound (F) indo 1, respectively. The
ratio (R) of F to G is used to calculate [Ca2⫹]f. With the use of
Felix software (Photon Technology International), the ionized Ca2⫹ concentration was calculated by the following
equation (15)
关Ca 2 ⫹ 兴f ⫽ Kd 䡠 共Gmax/Gmin兲 共R ⫺ Rmin兲/共Rmax ⫺ R兲
(1)
where Kd is the equilibrium constant for the interaction
between Ca2⫹ and indo 1, Rmin is the minimum value of R at
the addition of 250 ␮M EGTA, Gmax is the maximum value of
G at the addition of 250 ␮M EGTA, Gmin is the minimum
value of G at the addition of 1 mM CaCl2, and Rmax is the
maximum value of R at the addition of 1 mM CaCl2. The Kd
value for the Ca2⫹-dye complex is 250 nM for muscle homogenates (15).
Photon counts per second were recorded simultaneously
for both emission wavelengths. The Ca2⫹-independent (background) fluorescence was measured in the reaction medium
(without indo 1) at each emission wavelength before the
experiment was started. Background fluorescence was automatically corrected before each assay was started using the
Felix software.
The reaction buffer contained 200 mM KCl, 20 mM
HEPES, 10 mM NaN3, 5 ␮M N,N,N⬘,N⬘-tetrakis(2-pyridylmethyl)ethylenediamine, 5 mM oxalate, and 15 mM MgCl2,
pH 7.0. Before emission spectra were collected, 1.5 ␮M indo 1
were added to a cuvette containing 2 ml of reaction buffer. In
all trials, 2.5 ␮l CaCl2 (10 mM) were also added to the
cuvette, which gave a consistent starting [Ca2⫹]f of ⬃3.2 ␮M.
Immediately after data collection was initiated, 40 ␮l of
homogenate were added to the cuvette. Shortly after the
addition of homogenate, 40 ␮l of ATP were added, to give a
final concentration of 5 mM and to initiate active Ca2⫹
uptake and SR loading. Once the SR was loaded with Ca2⫹
and a steady-state [Ca2⫹]f ⬍100 nM was achieved, 3 ␮l of
AgNO3 were added to give a final concentration of 141 ␮M.
The Ca2⫹ release reaction was then allowed to proceed for ⬃3
min. Ca2⫹ release was also induced by adding 10 ␮l 4-CMC
(final concentration ⫽ 5 mM dissolved in dimethyl sulfoxide)
in a subset of muscle homogenate samples (n ⫽ 7). Dimethyl
sulfoxide had no effect on Ca2⫹ release. In some trials (n ⫽ 5),
100 ␮M of ruthenium red (RR) were added to the buffer at the
start of the assay to check the specificity of both AgNO3 and
4-CMC for the CRC.
Because it was important to determine whether Ca2⫹ATPase inhibition causes a reduction in Ca2⫹ release, which
may be specific to the releasing agent, we used CPA to inhibit
SR Ca2⫹-ATPase activity and Ca2⫹ uptake. A 40 mM stock
solution was prepared by dissolving CPA in chloroform. Chloroform alone had no effect on either Ca2⫹ uptake or Ca2⫹
release under these assay conditions. In the trials with CPA,
2 ␮l of CPA (final concentration ⫽ 40 ␮M) were added after
active loading of the SR with Ca2⫹ and just before the
addition of either AgNO3 or 4-CMC. To assess the specific
impact of varying degrees of SR Ca2⫹-ATPase inhibition on
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Ca2⫹-ATPase (1, 31). On that basis, we, among many
others (12, 20, 29, 41, 42), have used AgNO3 to assay
for Ca2⫹ release in vitro to assess CRC function after
various perturbations. However, there are reports in
the literature suggesting that the Ca2⫹-ATPase may be
the primary pathway for Ca2⫹ release induced by
AgNO3 (14, 36). If this is the case, inhibition of the
Ca2⫹-ATPase by CPA could be responsible for the reduction in Ca2⫹ release observed in phase 2. It is
noteworthy that, in several previous reports utilizing
AgNO3 as the Ca2⫹-releasing agent, a two-phase response is clearly indicated but not used in the analyses
(27, 29, 41).
Understanding the specific effects of AgNO3 on SR
Ca2 release and Ca2⫹-ATPase has important implications. On the one hand, if AgNO3 has effects on both
Ca2⫹ cycling processes, the interpretation of the effects
of perturbations such as exercise or ischemia would be
biased. On the other had, if an intrinsic coupling does
exist, this would represent an important advance in
understanding the role of the SR in Ca2⫹ regulation.
In the present study, our objective was to examine
the specific effects of AgNO3 on the CRC and the
Ca2⫹-ATPase to determine whether an intrinsic coupling does, in fact, exist between SR Ca2⫹ uptake and
Ca2⫹ release or whether the effects that we have observed are specific only for AgNO3. To examine this
issue, we used CPA, a specific inhibitor of the SR
Ca2⫹-ATPase and Ca2⫹ uptake (13, 33), to inhibit SR
Ca2⫹ uptake during net Ca2⫹ release, induced by either AgNO3 or 4-chloro-m-cresol (4-CMC), which is
known to directly activate the CRC (17). Our findings
indicate that AgNO3, but not 4-CMC, induces Ca2⫹
release by acting on both the CRC and the Ca2⫹ATPase.
SARCOPLASMIC RETICULUM FUNCTION AND SILVER IONS
ATPase activity was performed on homogenates using procedures developed by Simonides and van Hardeveld (35), with
minor modifications as previously described (38). Total
(Mg2⫹-activated)-ATPase activity was measured in the presence of ⬃6–10 ␮M [Ca2⫹]f to obtain maximal ATPase activity
and in the presence of the Ca2⫹ ionophore A-23187. Basal
activity was measured in the presence of 40 ␮M CPA, which
completely inhibits SR Ca2⫹-ATPase activity (33). The difference between total and basal activities represents the Ca2⫹activated ATPase activity. To assess the effect of both AgNO3
and 4-CMC on SR Ca2⫹-ATPase activity, in 50% of the trials
either 141 ␮M AgNO3 or 5 ␮M 4-CMC were added to the
cuvette after maximal Ca2⫹-ATPase activity was obtained
and before basal activity was obtained. Both Ca2⫹-ATPase
activity and basal activity were expressed in micromoles per
gram protein per minute. On a given analytic day, samples
from all conditions were analyzed in duplicate.
Data analysis. For Ca2⫹ release measurements with and
without 40 ␮M CPA and 100 ␮M RR, a two-way ANOVA was
used to discriminate between differences due to CPA or RR
(with vs. without) and releasing agent (AgNO3 vs. 4-CMC).
Where an overall interaction between assay and group was
found, post hoc analyses (Tukey’s) were performed to determine specific assay and group effects. For AgNO3-induced
Ca2⫹ release measurements made in the presence of varying
CPA concentrations, a one-way ANOVA was used to test for
differences between means. Where significant differences
were found, Tukey’s post hoc tests were used to compare
specific means. For all other measurements, two-tailed
paired t-tests were used to test for differences between
means. For all comparisons, statistical significance was accepted at P ⬍ 0.05. All data are expressed as means ⫾ SE.
RESULTS
Fig. 1. Changes in free calcium ([Ca2⫹]f) levels during Ca2⫹ uptake
and during phase 1 and phase 2 of Ca2⫹ release initiated by silver
nitrate (AgNO3; A) and by 4-chloro-m-cresol (4-CMC; B). The tracing
is from a typical homogenate assay.
J Appl Physiol • VOL
Ca2⫹ release with AgNO3. With the addition of
AgNO3, Ca2⫹ release proceeded in two distinct phases:
an initial, rapid rate of release that was complete
within seconds (phase 1), followed by a slower, more
prolonged rate of release (phase 2) (Fig. 1A), which
lasted 2–3 min until Ca2⫹ release stopped and the
buffer [Ca2⫹]f plateaued at some level (data not
shown). The effects of CPA were opposite on these two
phases of Ca2⫹ release. In all conditions, inhibition of
Ca2⫹-ATPase activity with CPA after loading of SR
with Ca2⫹ increased (P ⬍ 0.05) the early, rapid rate of
release and decreased (P ⬍ 0.05) the slower, more
prolonged rate of release. The same results are obtained with isolated SR vesicles (data not shown). The
percent change in Ca2⫹ release rate was altered with
varying concentrations of CPA, and, therefore, the extent of SR Ca2⫹-ATPase inhibition, for phase 2 but not
for phase 1. A typical response for one animal showing
the effects of different CPA concentrations on Ca2⫹
release is shown in Fig. 2. On average, phase 2 Ca2⫹
release rate was reduced (P ⬍ 0.05) by 44, 57, and 72%
with 2, 5, and 40 ␮M CPA, respectively, compared with
no CPA (Fig. 3). However, the average increase in
phase 1 Ca2⫹ release rate with CPA was similar across
all CPA concentrations used.
Ca2⫹ release with 4-CMC. The biphasic Ca2⫹ release
rate response induced by 4-CMC was similar to the
AgNO3-induced Ca2⫹ release rate response (Fig. 1B).
However, the absolute release rate for phase 1 was
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SR Ca2⫹ release with AgNO3, we also used concentrations of
2, 5, and 40 ␮M CPA. Submaximal Ca2⫹ uptake was inhibited by 51, 92, and 98% with 2, 5, and 40 ␮M CPA, respectively (pilot data). CPA and the releasing agent (AgNO3 or
4-CMC) were added in rapid succession to prevent CPAinduced SR Ca2⫹ leakage before Ca2⫹ release was initiated.
We assessed the extent of Ca2⫹ leakage induced by CPA
alone, which averaged 26.8 ⫾ 0.5 ␮mol 䡠 g protein⫺1 䡠 min⫺1.
On addition of both AgNO3 and 4-CMC, Ca2⫹ release
consistently proceeded in two distinct phases. There was an
initial, rapid rate of release (phase 1) followed by a slower,
more prolonged rate of release (phase 2) (see Fig. 1). The
generated curve from Eq. 1 was smoothed over 21 points and
differentiated. The maximal rate of Ca2⫹ release for each
phase was calculated by taking the maximum positive derivative of each phase expressed in ␮moles per gram protein per
minute. In general, phase 1, which began with the initial
increase in [Ca2⫹]f, was complete in the first few seconds. In
contrast, phase 2 extended for 2–3 min. Protein was determined by the method of Lowry, as modified by Schacterle and
Pollock (32). On a given day, all samples for a given variable
were analyzed in duplicate.
SR Ca2⫹-ATPase activity measurements. Spectrophotometric (Schimadzu UV 160U) measurement of SR Ca2⫹-
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SARCOPLASMIC RETICULUM FUNCTION AND SILVER IONS
approximately sixfold higher (P ⬍ 0.05) with 4-CMC
compared with AgNO3 (1,961 ⫾ 112 vs. 314 ⫾ 51
␮mol 䡠 g protein⫺1 䡠 min⫺1). There were no differences
(P ⬎ 0.05) in the absolute rate of release for phase 2
between 4-CMC and AgNO3 (281 ⫾ 17.1 vs. 267 ⫾ 39
␮mol 䡠 g protein⫺1 䡠 min⫺1, respectively). Unlike AgNO3,
40 ␮M CPA after Ca2⫹ loading of the SR had no effect
on the 4-CMC-induced rate of release corresponding
with both phase 1 and phase 2 (Fig. 4).
Ca2⫹ release with RR. A typical response showing
the effects of RR for both AgNO3- and 4-CMC-induced
Ca2⫹ release rate is shown in Fig. 5. With AgNO3, RR
had no effect on phase 1 rate of release (P ⬎ 0.05)
(342 ⫾ 68 vs. 323 ⫾ 51 ␮mol 䡠 g protein⫺1 䡠 min⫺1) and
Fig. 4. Phase-dependent effect of CPA on 4-CMC-induced Ca2⫹ release kinetics in homogenates. Values are means ⫾ SE (n ⫽ 7).
decreased phase 2 release rate (P ⬍ 0.05) by 65%
(245 ⫾ 34 vs. 105 ⫾ 8.2 ␮mol 䡠 g protein⫺1 䡠 min⫺1) (Fig.
5A). Unlike AgNO3, RR reduced phase 1 rate (P ⬍ 0.05)
by 59% (2,468 ⫾ 279 vs. 1,004 ⫾ 87 ␮mol 䡠 g
protein⫺1 䡠 min⫺1) and completely blocked phase 2 (see
Fig. 5B) when 4-CMC was used to induce Ca2⫹ release.
Ca2⫹-ATPase activity. Maximal Ca2⫹-ATPase activity was inhibited by 95.4 ⫾ 1.9% in the presence of 141
␮M AgNO3 (Fig. 6). Ca2⫹-ATPase activity in the presence of AgNO3 was not different (P ⬎ 0.05) from basal
activity. Even concentrations as low as 5 ␮M AgNO3
appeared to partially inhibit Ca2⫹-ATPase activity
(data not shown). Surprisingly, we also found that
4-CMC inhibited Ca2⫹-ATPase activity by 97 ⫾ 1.4%.
DISCUSSION
Fig. 3. Concentration-dependent effects of CPA in muscle homogenates on AgNO3-induced Ca2⫹ release kinetics for overall group in
phase 1 and phase 2. Values are means ⫾ SE (n ⫽ 9). Significantly
different from * No CPA, ** No and 2 ␮M CPA, and *** No, 2, and 5
␮M CPA, P ⬍ 0.05.
J Appl Physiol • VOL
The purpose of this study was to determine whether
SR Ca2⫹ uptake and Ca2⫹ release are intrinsically
coupled or whether the apparent coupling that has
been observed is specific to the Ca2⫹-releasing agent
employed. We have demonstrated that AgNO3 induces
a biphasic Ca2⫹ release rate response measured in
homogenates in vitro, as we have shown previously
(40). We also confirmed our earlier finding, namely
that Ca2⫹-ATPase inhibition with CPA administered
after SR Ca2⫹ loading and before inducing Ca2⫹ release causes a reduction in Ca2⫹ release rate (phase 2).
However, this effect is only specific for AgNO3. If Ca2⫹
release is induced with 4-CMC, inhibition of the Ca2⫹ATPase with CPA after Ca2⫹ loading has no effect on
either phase of Ca2⫹ release. Moreover, blocking the
CRC with RR had no effect on phase 1 and inhibited
phase 2 by 65% when AgNO3 was used. When 4-CMC
was employed as the releasing agent in conjunction
with RR, phase 1 was reduced by 59%, and phase 2 was
completely blocked. These results suggest that AgNO3,
in contrast to 4-CMC, has a unique effect on Ca2⫹-
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Fig. 2. Concentration-dependent effects of cyclopiazonic acid (CPA)
in muscle homogenates on Ca2⫹ release kinetics during a typical
assay in phase 1 and phase 2.
SARCOPLASMIC RETICULUM FUNCTION AND SILVER IONS
ATPase activity, resulting in reversal of the pump and
extrusion of Ca2⫹ into the cytosol.
Recently, Ikemoto and colleagues (18, 30) proposed a
tight coupling between SR Ca2⫹ uptake and Ca2⫹ release and suggested that the early kinetic changes in
SR luminal Ca2⫹ during Ca2⫹ release provided the
signal for a coordinated response from the Ca2⫹ATPase and subsequent increased rate of Ca2⫹ uptake.
Theoretically, the rate of Ca2⫹ uptake during net Ca2⫹
release could also affect SR luminal Ca2⫹ and thus the
rate of Ca2⫹ release. A tempting hypothesis was that
this could explain both the effects of CPA on SR Ca2⫹
release that we observed previously (38) and the observation from the literature that reductions in Ca2⫹
uptake and Ca2⫹ release after exercise (20, 42) and
ischemia-reperfusion are closely matched (28, 37).
However, in this experiment, we wanted to examine
whether such a relationship exists intrinsically after
the SR was loaded with Ca2⫹ or whether the relationship is specific to AgNO3.
To investigate this problem, it was first necessary to
demonstrate that the reduction in AgNO3-induced
Ca2⫹ release (phase 2) due to inhibition of the Ca2⫹J Appl Physiol • VOL
ATPase with CPA is a general phenomenon and is not
just specific for AgNO3. In fact, we found that CPA had
no effect on the rate of Ca2⫹ release for either phase
when 4-CMC was employed as the Ca2⫹ releasing
agent. Although this does not contradict the findings
by Ikemoto and Yamamoto (18) or the possibility that
SR Ca2⫹ release and Ca2⫹ uptake are coupled, it does
suggest that the mechanism by which AgNO3 and
4-CMC act to cause Ca2⫹ release from SR vesicles is
different. These results with 4-CMC also suggest that,
if the SR is preloaded with Ca2⫹, inhibition of Ca2⫹ATPase activity and Ca2⫹ uptake per se do not impair
Ca2⫹ release, in either phase 1 or phase 2, at least
under the conditions of our assay.
It has been known for some time that heavy metals,
such as Cu2⫹, Hg2⫹, and Ag⫹, can induce a rapid Ca2⫹
release from Ca2⫹-loaded SR vesicles, which is related
to their binding affinity for sulfhydryl groups (1). In the
case of Ag⫹, it has been reported that the rate of Ca2⫹
release is five to six times higher in heavy SR vesicles
compared with light SR vesicles and that Ca2⫹ release
could be blocked by procaine, tetracaine, and RR, now
well-known inhibitors of the CRC (31). Based on these
results, it was argued that heavy metals and specifically Ag⫹ (AgNO3) induce Ca2⫹ release from SR vesicles by interacting with the CRC and not the Ca2⫹ATPase (31). In addition, single-channel activity
measurements demonstrated the ability of Ag⫹ to directly activate the CRC (25).
However, in the study by Salama and Abramson
(31), the light SR vesicles, in which the Ca2⫹-ATPase
comprises 90% of the total protein (19), released 90–
100% of the accumulated Ca2⫹ in the presence of
AgNO3. Moreover, the Ca2⫹-ATPase comprises 55–
65% of total heavy SR vesicle protein (19). It is also
well known that the SR Ca2⫹-ATPase contains 24
cysteine residues (3) and is highly susceptible to sulf-
Fig. 6. Effects of AgNO3 and 4-CMC on maximal Ca2⫹-ATPase
activity. Values are means ⫾ SE (n ⫽ 9). * Significantly different
(P ⬍ 0.05) from basal activity and net AgNO3 and 4-CMC activity.
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Fig. 5. Effects of ruthenium red (RR; 100 ␮M) in muscle homogenates on Ca2⫹ release kinetics initiated by AgNO3 (A) and by 4-CMC
(B). The tracing is from a typical homogenate assay.
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SARCOPLASMIC RETICULUM FUNCTION AND SILVER IONS
J Appl Physiol • VOL
72% and, importantly, had no effect on 4-CMC-induced
Ca2⫹ release. In the presence of lower CPA concentrations, the extent of inhibition of Ca2⫹ release for phase
2 was also lower. Because CPA can inhibit the Ca2⫹ATPase in both directions (forward and reverse) (10,
11) but cannot reduce the leak of calcium ions through
the Ca2⫹-ATPase passive channel (10), it would appear
that AgNO3 triggers the reversal of the Ca2⫹-ATPase
and thus Ca2⫹ release through this pathway. Interestingly, 4-CMC also inhibited Ca2⫹-ATPase activity.
However, because Ca2⫹ release (phase 2) was completely blocked when 4-CMC was combined with RR,
inhibition of Ca2⫹-ATPase would appear to be specific
for the forward direction only.
Gould et al. (14) proposed a similar mechanism in
reconstituted vesicles, suggesting that the Ca2⫹ binding sites on the Ca2⫹-ATPase could remain in a lowaffinity state and cycle between the luminal and cytoplasmic side of the SR membrane, thus releasing Ca2⫹
into the cytoplasm (14). Other studies have also shown
that the Ca2⫹-ATPase can be reversed under various
conditions and can synthesize ATP from ADP and Pi
(7–9).
The nature of the Ca2⫹ release response for phase 1
induced by AgNO3 is unclear from our data. We have
found that various concentrations of CPA increased the
rate of release by a similar extent. If phase 1 reflects
Ca2⫹ release solely from the CRC, this finding would be
expected, providing the rate of Ca2⫹ release simply
reflects a balance between the rate of release and
reuptake of Ca2⫹ during release. On the other hand,
because AgNO3 completely inhibits Ca2⫹-ATPase activity, the increase in phase 1 Ca2⫹ release in the
presence of CPA is not due to reduced Ca2⫹ uptake
during release. Moreover, RR had no effect on phase 1
Ca2⫹ release induced by AgNO3, whereas phase 1 Ca2⫹
release induced by 4-CMC was inhibited by 59% with
RR. Thus the CPA and RR results do not support a role
for either the Ca2⫹-ATPase or the CRC for the early,
rapid rate of Ca2⫹ release induced by AgNO3.
The fact that 4-CMC-induced release corresponding
to phase 1 was not completely blocked by RR is consistent with the known mechanism for RR blockade of the
CRC. RR blocks the channel conduction pore of an open
CRC but does not inactivate the CRC or prevent the
CRC from opening (4, 22). Therefore, despite RR being
present at the start of each assay, addition of 4-CMC
would activate the channel and induce Ca2⫹ release
until RR moved into the channel conduction pore to
block further release of Ca2⫹. The question might be
asked as to whether the effects of AgNO3 might be
different with whole muscle homogenates as used in
this experiment and enriched SR fractions that contain
primarily only the Ca2⫹-ATPase. Measurement of the
Ca2⫹-ATPase in whole homogenates is highly specific
as a result of the selective inhibition of other cellular
ATPases (29, 35). Moreover, we have shown that Ca2⫹
release induced from enriched SR preparations demonstrates a similar biphasic release as whole homogenates and is affected by CPA in a similar manner
(unpublished observations). These observations would
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hydryl oxidation (34, 43). Given these considerations, it
is difficult to rule out the involvement of the Ca2⫹ATPase in the AgNO3-induced Ca2⫹ release response
in SR vesicles.
The most compelling evidence to date that Ag⫹ can
induce Ca2⫹ release by interaction with the Ca2⫹ATPase comes from a study by Gould et al. (14) in
which they showed a rapid Ca2⫹ efflux induced by Ag⫹
from reconstituted vesicles that contained the purified
Ca2⫹-ATPase as the only protein. As we have shown for
homogenates in the present study, they also showed
that Ag⫹ greatly inhibited Ca2⫹-ATPase activity in
both reconstituted vesicles and SR vesicles. Evidence
from other cell types, such as liver (44) and HL-60 (36),
suggests that heavy metals (including Ag⫹) induce
Ca2⫹ release from intracellular stores mainly due to
inhibition of the Ca2⫹-ATPase in conjunction with a
direct effect on the CRC. In liver cells, both RR and
tetracaine have no effect on Ca2⫹ efflux induced by
heavy metals (44).
Our results strongly support a dual action of AgNO3
for inducing Ca2⫹ release, at least from muscle homogenates prepared from rat skeletal muscle. Similar to
HL-60 cells, it appears that AgNO3 induces a rapid
Ca2⫹ efflux from muscle homogenates by simultaneously interacting with both the CRC and the Ca2⫹ATPase of the SR. The effects of RR on the AgNO3induced Ca2⫹ release response provides evidence that
AgNO3 directly activates the CRC. However, only
phase 2 was inhibited with RR, and the extent of
inhibition was only 65% compared with 100% for
4-CMC-induced Ca2⫹ release. This suggests that only
part of the AgNO3-induced Ca2⫹ release response can
be explained by activation of the CRC. Nonetheless,
the CRC does contribute to AgNO3-induced Ca2⫹ release as expected, and recent work using single-channel analysis confirms that AgNO3 directly activates
both the skeletal muscle CRC isoform (ryanodine receptor 1) and the cardiac isoform (ryanodine receptor 2)
(G. G. Du, personal communication).
In previous studies, it was shown that 4 mM DTT
completely blocked AgNO3-induced Ca2⫹ release from
skeletal muscle samples (29, 41). In these studies,
DTT, as opposed to RR, was used to validate the Ca2⫹
release assay using AgNO3 and to demonstrate the
specificity of AgNO3 for the CRC. However, DTT is a
sulfhydryl-reducing agent that is not specific for any
particular protein (5), and we have shown that DTT
can alter the effects of skeletal muscle ischemia on
measurements of SR Ca2⫹-ATPase activity (39).
In HL-60 cells, it was concluded that the apparent
Ca2⫹ release induced by AgNO3 was mainly due to
inhibition of the Ca2⫹ pump with increased permeability for Ca2⫹ (10). This mechanism cannot explain the
rapid AgNO3-induced Ca2⫹ release response observed
in the present study. Even though AgNO3 completely
inhibited Ca2⫹-ATPase activity, the rate of Ca2⫹ leak
due to Ca2⫹-ATPase inhibition alone that we observed
only represented ⬃12% of the AgNO3-induced rate of
release in phase 2. Moreover, CPA reduced the AgNO3induced rate of release corresponding to phase 2 by
SARCOPLASMIC RETICULUM FUNCTION AND SILVER IONS
This study was supported from a grant received from the National
Sciences and Engineering Research Council.
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