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(Circulation Research. 1995;77:354-360.)
© 1995 American Heart Association, Inc.

Articles

Effects of Rest Interval on the Release of Calcium From the Sarcoplasmic Reticulum in Isolated Guinea Pig Ventricular Myocytes

Cesare M. N. Terracciano, Ruby U. Naqvi, Kenneth T. MacLeod

From the Department of Cardiac Medicine, National Heart and Lung Institute, University of London, London, UK.

Correspondence to Kenneth T. MacLeod, Department of Cardiac Medicine, National Heart and Lung Institute, University of London, Dovehouse St, London, SW3 6LY, UK. E-mail 100044.761@compuserve.com k.t.macleod@ucl.ac.uk.

	Abstract

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Abstract Guinea pig cardiac myocytes were loaded with the fluorescent dye indo 1, and cell contraction was measured by a video edge-detection system. Ca²⁺ was released from the sarcoplasmic reticulum (SR) by rapidly cooling the myocytes or by rapid application of 10 mmol/L caffeine. Estimates of the amount of Ca²⁺ released from the SR after different rest intervals (ie, under different loading conditions) were obtained by measuring the current evoked by rapid application of 10 mmol/L caffeine, which we call Na⁺/Ca²⁺ exchange current. This current is completely inhibited by removal of extracellular Na⁺ and Ca²⁺ or by application of 5 mmol/L Ni²⁺. SR Ca²⁺ release after rest intervals of 5 to 120 seconds (assuming cell volume to be 30x10^-12 L) was estimated to be 57.8±5.7 to 25.7±4.5 µmol/L accessible cell volume, respectively, equivalent to 23 to 10 µmol/kg wet wt, respectively. There was an exponential decline in Ca²⁺ released from the SR after rest intervals of 2 to 120 seconds (rate constant, 0.029 s^-1; t_1/2, 24 seconds); thereafter, there remained a portion (56%) of Ca²⁺ releasable to caffeine application. We found a similar exponential decay (rate constant, 0.020 s^-1; t_1/2, 35 seconds) of the size of rapid cooling contractures with increasing rest intervals. The time to peak of the Na⁺/Ca²⁺ exchange current in the presence of caffeine slowed at long rest intervals, ie, at smaller SR loads. A decrease in SR load of 50% increased the time to peak of the exchange current by 213±37% (n=6). The rate of generation of a rapid cooling contracture was faster after short rest intervals and was associated with a faster rise in the corresponding increase in indo 1 fluorescence. A decrease in SR load of 50% decreased the rate of cell shortening to 34±5% and decreased the rate of change in fluorescence to 54±2% (n=5).

Key Words: sarcoplasmic reticulum • cardiac myocyte • Ca²⁺ release • caffeine • Na⁺/Ca²⁺ exchange current

	Introduction

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Calcium-induced calcium release (CICR) from the cardiac sarcoplasmic reticulum (SR)¹ has been shown to be the main mechanism responsible for the generation of contraction in cardiac muscle, although the way this may function in the intact cell is still a matter for considered debate.^{2 3 4} What is presently uncertain is why the release process under some circumstances gives rise to uncoordinated and spontaneous release of Ca²⁺, forming Ca²⁺ waves that may or may not propagate further,^{3 5} and under other circumstances produces coordinated and controlled release in the form of apparently homogeneous Ca²⁺ transients throughout the length of the cell. The "gain" of the CICR process is a term that has been recently introduced to try to explain the above differences in behavior.^{2 4} The gain is generally defined as the amount of Ca²⁺ released from the SR compared with that entering the cell. It has now been demonstrated that the CICR process can have variable gain,⁴ but it is uncertain what causes the variation. It is clear that the gain can be affected by the amount of Ca²⁺ stored within the SR because greatly loaded SR predisposes to Ca²⁺ waves and uncoordinated release.^{6 7} Apart from a theoretical study by Tinker et al,⁸ little is known about the influence of the amount of Ca²⁺ stored within the SR on the rate of release. The rate at which Ca²⁺ is released may have important consequences for activation of neighboring release-channel clusters² and cardiac contraction in general. We have investigated the relationship between SR Ca²⁺ load and indexes of rate of release in intact, undialyzed, isolated cardiac myocytes by using rapid cooling or caffeine application to bring about Ca²⁺ release. The results suggest that the rate with which Ca²⁺ is released from the SR is proportional to the amount of Ca²⁺ stored within it.

	Materials and Methods

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Cell Isolation
The methods used have been described in detail elsewhere.^{9 10} Guinea pig cardiac myocytes were prepared using protease (4 U/mL, Sigma Chemical Co), then collagenase (0.3 mg/mL, Worthington) and hyaluronidase (0.6 mg/mL, Sigma) digestion. After isolation, the cells were stored in DMEM buffered with 25 mmol/L HEPES (GIBCO) at room temperature.

Monitoring of Intracellular Ca²⁺ and Cell Length
Monitoring of intracellular Ca²⁺ was carried out using the fluorescent dye indo 1 as described previously.^{9 10} The acetoxymethyl ester form of indo 1 (10 µmol/L; indo 1-AM, Molecular Probes) was added to a suspension of cells. After 20 minutes of incubation at room temperature, the supernatant was removed and replaced with fresh DMEM solution. We do not quantify the ratios of indo 1 fluorescence but use them as qualitative indicators of changes in free [Ca²⁺]_i. No background subtraction was carried out. Red light (>630 nm) illuminated the cell under study in a normal bright-field manner and carried the image of the cell to a video edge-detection system for cell length measurements.

Cell Stimulation
When required, cells were field-stimulated at 0.5 Hz with a pair of platinum electrodes placed on either side of the experimental chamber or were impaled with high-resistance (10 to 30 M) borosilicate glass microelectrodes (Clark Electromedical Instruments) filled with a solution containing 2 mol/L KCl, 0.1 mmol/L EGTA, and 5 mmol/L HEPES, pH 7.2, and were stimulated at 0.5 Hz with a 1.0-nA pulse of depolarizing current of 10-millisecond duration. In voltage-clamp experiments, cells were impaled with microelectrodes fabricated as described above and clamped using an Axoclamp 2A (Axon Instruments Inc).

Solutions
The cells were continuously superfused at a rate of 2 to 3 mL/min with a Tyrode's solution containing (in mmol/L) NaCl 140, KCl 6, MgCl₂ 1, CaCl₂ 2, glucose 10, and HEPES 10, pH 7.40±0.01. All experiments were carried out at room temperature (22°C) except during the cooling periods, which bring about the rapid cooling contractures (RCCs) (see below). When Na⁺-free/Ca²⁺-free solution was used, Na⁺ was replaced by 140 mmol/L Li⁺, Ca²⁺ was omitted, and 100 µmol/L EGTA was added, pH 7.40±0.01.

RCCs were generated after varying rest periods after cessation of field stimulation by changing the temperature of the solution superfusing the cells from 22°C to 1°C in less than 1 second by methods already described.^{10 11} During cooling, solutions flowed through the cell chamber at a rate of 12 to 15 mL/min.

Caffeine contractures were produced by application of the normal Tyrode's solution with 10 mmol/L caffeine added in solid form. Tyrode's solution containing 10 mmol/L caffeine flowed at a rate of 12 to 15 mL/min. Using the procedure described by O'Neill et al,¹² we found that [caffeine]_i increased to 1.84±0.26 mmol/L (n=14) in 500 milliseconds,¹⁰ a value certainly consistent with that found by others.^{12 13} The current we measure on rapid application of caffeine is ascribed to Na⁺/Ca²⁺ exchange because it is completely inhibited in Na⁺-free/Ca²⁺-free solution or by application of nickel.^{10 14} The baseline for integration of the transient current was taken at the point where current had settled to a steady state when diastolic [Ca²⁺] had been reached in the presence of caffeine.

Data Acquisition and Statistics
Signals were filtered at 100 Hz and then simultaneously recorded on tape and on computer at a digitization rate of 62 Hz, from which they were analyzed using AXOTAPE 2.0 software (Axon Instruments Inc). Control software was PCLAMP version 5.5 (Axon Instruments Inc). Unless otherwise specified, data are expressed as mean±SEM. Significance between means was calculated using the Student's t test.

	Results

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Na⁺/Ca²⁺ Exchange Current: Measurement of Releasable SR Ca²⁺ Content
Before describing the effects of SR Ca²⁺ content on the rate of release, we first tried to estimate the amount of Ca²⁺ released under various loading conditions. The amount of Ca²⁺ in the SR (SR load) was varied by altering the rest period between the last stimulation pulse and the onset of the Ca²⁺ release. In these series of experiments, we released Ca²⁺ from the SR by the rapid application of caffeine¹⁵ and measured the transient inward current that was produced (Fig 1A). The current can be inhibited by removing extracellular Na⁺ and Ca²⁺ or by application of 5 mmol/L Ni²⁺¹⁰ and can be ascribed to the electrogenic Na⁺/Ca²⁺ exchange.^{10 14 16} Cells were stimulated at 0.5 Hz by passing current to elicit a series of conditioning action potentials. When twitches and Ca²⁺ transients had reached a steady state, the stimulation was stopped, and the cell voltage was clamped at -80 mV. After a rest period of varying duration, caffeine was rapidly applied to the cell producing the transient inward current (Fig 1B). The size of the current can be used to calculate the SR Ca²⁺ release using assumptions and calculations similar to those used by Varro et al.¹⁴ In guinea pig myocytes, there was no evidence of a maintained outward current as in rat myocytes.¹⁴ Transient inward currents measured from seven cells after rest intervals of 5, 10, 30, 60, and 120 seconds gave an average charge transfer of 135±13, 124±11, 94±10, 77±12, and 60±10 pC (n=5), respectively. From previous experiments, the mean membrane capacitance of our cells is about 150 pF; assuming that the specific membrane capacitance is 1 µF/cm² and that the ratio of surface area to volume is 0.5 µm²/µm³,¹⁷ this gives a cell volume of 30x10^-12 L. Thus, the Na⁺ influx and therefore the equivalent Ca²⁺ efflux (assuming the exchange has a stoichiometric ratio of Na⁺ to Ca²⁺ of 3:1) can be calculated to be 46±5, 42±4, 32±3, 26±4, and 21±4 µmol/L cell volume after rest intervals of 5, 10, 30, 60, and 120 seconds, respectively. Previous work^{10 18} suggested that SR Ca²⁺-ATPase and Na⁺/Ca²⁺ exchange produce about 97% of the relaxation in guinea pigs, so we have not made any correction for other mechanisms responsible for Ca²⁺ removal. There is a suggestion that in rabbits the sarcolemmal Ca²⁺-ATPase and mitochondria play larger roles in relaxation if SR Ca²⁺-ATPase is inhibited.¹⁹ However, we do not know whether a similar alteration in fractional roles takes place in guinea pig cardiac cells if the SR cannot act as a store of Ca²⁺. If we make a correction for the fact that mitochondria make up about 25% of cell volume and so total cell volume would not be accessible to Ca²⁺, then our estimates for SR Ca²⁺ release after rest intervals of 5, 10, 30, 60, and 120 seconds are about 57.8±5.7, 53.1±4.6, 40±4.3, 32.7±5.3, and 25.7±4.5 µmol/L, respectively. There is an exponential decline (with a rate constant of 0.029 s^-1 and t_1/2 of 24 seconds) in Ca²⁺ released from the SR after rest intervals of 2 to 120 seconds; thereafter, there remains a portion of caffeine-releasable Ca²⁺ (56%) that leaves the SR more slowly (Fig 1C). We find a similar exponential decay (rate constant, 0.020 s^-1; t_1/2, 35 seconds) in the size of RCCs with increasing rest intervals.

View larger version (13K): [in this window] [in a new window]   Figure 1. A, Recordings show transient inward current (Na+/Ca2+ exchange current) and indo 1 fluorescence changes on rapid application of 10 mmol/L caffeine. Cells were voltage-clamped at -80 mV. The superfusing solution was changed to one containing 10 mmol/L caffeine at the time indicated by the bar. Dotted line indicates zero current level. B, Recordings show the effect of changing the rest interval on the size of the Na+/Ca2+ exchange current. The left trace shows the current elicited after a rest of 5 seconds and the right trace shows, in the same cell, the current elicited after a rest interval of 60 seconds. Dotted line indicates zero current level. C, Graph shows the exponential decline in calculated sarcoplasmic reticulum (SR) Ca2+ content after varying rest intervals. SR Ca2+ content was determined as outlined in "Results" and is expressed in micromoles per liter of accessible cell volume (a.v.), ie, taking into account the volume occupied by the mitochondria in the cell. The line plotted is the best fit and decreases with a rate constant of 0.028 s-1 and t1/2 of 25 seconds). Bars indicate mean±SEM (n=5).

We make the assumption in the following work that caffeine releases all the Ca²⁺ from the SR; therefore, these values of SR Ca²⁺ release are reasonable approximations of Ca²⁺ stored in the SR after the stated rest interval. Furthermore, we assume that rapid cooling also releases all the Ca²⁺ stored in the SR.

We feel these assumptions are justified because rapid cooling immediately after caffeine application does not evoke any further contraction, as can be seen in Fig 2A. This figure shows a period of rapid cooling that followed a caffeine application. Caffeine application evoked a release of Ca²⁺, and both cell length and indo 1 showed appropriate changes. On cooling there was no change in cell length, but we observed a small rise in the baseline of the indo 1 ratio signal. Fig 2A (right) shows a similar experiment on the same cell; caffeine was applied during cooling, then a second cooling period immediately followed caffeine removal. Again, no RCC was produced, but an offset of similar size was observed in the ratio signal. One could argue that these baseline changes in the indo 1 ratio signal represent real increases in Ca²⁺, but we believe these are artifacts caused by movement of the coverslip, which forms the floor of the cell chamber, during cooling (see Reference 11¹¹ ). Fig 2B supports this idea. The left panel of Fig 2B shows the changes in cell length and indo 1 ratio in a cell exposed to two periods of rapid cooling. The cell was then superfused with a solution containing 500 nmol/L thapsigargin for 5 minutes. Under these conditions, we expect the SR Ca²⁺-ATPase to be completely inhibited. Both periods of cooling produced no cell contracture but again a small increase in indo 1 ratio. The size of the ratio change when the SR was depleted was similar in all instances, consistent with the change being artifactual rather than representing a real change in Ca²⁺ within the cell. Thus, we believe our assumption is correct that both caffeine and rapid cooling release the same amount of Ca²⁺ from the SR.

View larger version (23K): [in this window] [in a new window]   Figure 2. A, Recordings show the effect of rapid cooling immediately following caffeine application. Caffeine application evokes a release of Ca2+, and both cell length and indo 1 ratio show appropriate changes. The experiment shown in the right panel was carried out on the same cell, but caffeine was applied during cooling; then a second cooling period immediately followed caffeine removal. B, The left panel shows the changes in cell length and indo 1 ratio in a cell exposed to two periods of rapid cooling. The cell was then superfused by a solution containing 500 nmol/L thapsigargin for 5 minutes. The effect of periods of cooling on cell length and indo 1 ratio after this time in thapsigargin is shown in the right panel.

Na⁺/Ca²⁺ Exchange Current: Time to Peak
The time to peak of the exchange current will give an estimate of the time Ca²⁺ takes to reach the exchange after release from the SR. We found that changes in the time to peak of Na⁺/Ca²⁺ exchange current were produced by rapid application of caffeine after changes in rest interval. As the rest interval was increased (ie, as SR load decreased), the time to peak of the current increased (Fig 3, inset). A decrease in SR load of 50% increased the time to peak of the exchange current by 213±37% (n=6) (Fig 3).

View larger version (24K): [in this window] [in a new window]   Figure 3. Graph shows the relationship between the time to peak (TTP) of the Na+/Ca2+ exchange current that accompanies the caffeine application and the releasable sarcoplasmic reticulum (SR) Ca2+ content (expressed as micromoles per liter of accessible cell volume [a.v.]). Cells were voltage-clamped at -80 mV, and Na+/Ca2+ exchange current was elicited after rapid application of 10 mmol/L caffeine as shown in the inset (top). SR Ca2+ content was calculated as described in "Results." Bars indicate mean±SEM (n=5).

Rapid Cooling: Changes in Rate of Cell Shortening and Indo 1 Fluorescence
Fig 4 shows how the rate of cell shortening and the rate of change in indo 1 fluorescence alter with different rest intervals when SR Ca²⁺ is released by rapid cooling. Cells were conditioned by field stimulation until a steady state was reached, then stimulation was stopped and cooling applied after different rest intervals (in the case illustrated in Fig 4, the rest intervals were 2, 30, and 60 seconds). The upper two traces of Fig 4 show the changes in cell length that occurred at the onset of cooling (arrows) and the rate of shortening of cell length ( shortening/t). The associated changes in indo 1 fluorescence and the derivatives are shown in the lower two traces. As the rest interval increased (from 2 to 60 seconds), the rate of increase in indo 1 fluorescence and the rate at which cell shortening developed decreased. Data gathered from four other experiments are shown in Fig 5. In this figure we have used estimates of SR load gathered from experiments like those shown in Figs 1 and 3 and plotted these against the rate of change in indo 1 fluorescence and the rate of change in cell shortening. Over the range of SR loading conditions measured, a decrease in SR load of 50% decreased the rate of cell shortening to 59% and decreased the rate of change in fluorescence to 53% (n=5). Using two separate techniques, we have demonstrated that when Ca²⁺ is released into the cytoplasm, its rate of accumulation is dependent on the amount of Ca²⁺ stored in the SR. The two independent methods used to assess these changes in rate produce similar results. When SR load is decreased by 50%, the time to peak of the Na⁺/Ca²⁺ exchange current increases by about twofold, while the rate of cell shortening and the rate of change in fluorescence decreases by about half.

View larger version (15K): [in this window] [in a new window]   Figure 4. Recordings show the effect of rest-interval length on the rate of change of indo 1 fluorescence (lower traces) and the rate of change of cell shortening (upper traces). Rapid cooling was applied at the time shown by the arrows after 2-, 30-, and 60-second rest intervals. The derivatives of the cell shortening and indo 1 fluorescence are shown.

View larger version (12K): [in this window] [in a new window]   Figure 5. Graphs show the relationship between the releasable sarcoplasmic reticulum (SR) Ca2+ content (expressed as micromoles per liter of accessible cell volume[a.v.]) and the rate of change in the indo 1 ratio (top) and cell shortening (bottom) at the onset of a rapid cooling contracture. Points are mean±SEM (n=5).

One possible factor that might affect these measurements is the presence of SR uptake and Na⁺/Ca²⁺ exchange efflux systems. These may attenuate the rate of rise of Ca²⁺ and cell shortening progressively at the longer rest intervals, ie, at lower SR loads. Thus, it might appear that the release of Ca²⁺ is slowed. Although cooling will tend to inhibit these systems, it will not do so completely. To try to overcome this problem, we carried out the releases in the above experiments in Na⁺-free/Ca²⁺-free solution to inhibit the Na⁺/Ca²⁺ exchange, but this still leaves SR Ca²⁺-ATPase possibly influencing the apparent rate changes. However, we think that the operation of Ca²⁺-ATPase does not influence the rate changes greatly because of results shown in Fig 6. The observations presented are typical of those found in a number of experiments (n=10).

View larger version (17K): [in this window] [in a new window]   Figure 6. Recordings show the effect of 200 nmol/L thapsigargin on rate of change of indo 1 fluorescence and cell shortening. Control changes to cooling after a 30-second rest are shown at left. The cell superfusate was changed to one containing 200 nmol/L thapsigargin, and after 3 minutes in the presence of thapsigargin, stimulation of the cell was stopped. After another rest period of 30 seconds, cooling of the cell was carried out. Note that the speed of relaxation when the cell was rewarmed in the presence of thapsigargin was greatly increased compared with control. Under these conditions, where uptake has been greatly affected, the release is still slowed, as can be seen more clearly in the lowest panel, where the Ca2+ traces have been scaled. The trace on the right is in the presence of thapsigargin.

Fig 6 shows the responses of a cell to cooling and the generation of an RCC in control conditions and in the presence of 200 nmol/L thapsigargin, which inhibits SR Ca²⁺-ATPase (see Reference 20²⁰ ) and at this concentration takes between 5 and 15 minutes to produce complete inhibition. The left side of the figure shows a series of Ca²⁺ transients and corresponding twitches induced by field stimulation. Stimulation was stopped, and after a rest interval of 10 seconds an RCC was induced. The right side of the figure shows the same protocol carried out in the presence of 200 nmol/L thapsigargin. When this dose of thapsigargin was applied for 3 minutes, SR Ca²⁺-ATPase was partially inhibited. This is confirmed by examination of the speed of relaxation when the cell was rewarmed. This was greatly slowed compared with control. Under these conditions, where uptake has been greatly affected, the release was still slowed at lower SR loads, as can be seen more clearly in the lowest panel, where the Ca²⁺ traces have been normalized. In further experiments using RCCs in the presence of thapsigargin and with Na⁺-free/Ca²⁺-free solution (not shown), the release was still slowed at smaller loads.

	Discussion

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The main observations are that (1) measurements of Na⁺/Ca²⁺ exchange current in the presence of caffeine (ie, under conditions that allow measurement of SR release) reveal a slowing of onset of the peak current at long rest intervals, (2) the rate of generation of an RCC is faster after short rest intervals (ie, when the SR is more loaded with Ca²⁺), (3) the faster generation of the RCC is consistent with a faster rise in the corresponding increase in indo 1 fluorescence, and (4) in the presence of submaximal doses of thapsigargin (where SR loading is severely depressed), the rate of rise in Ca²⁺ produced by cooling is greatly slowed.

Quantification and Validation of Results
It is tempting to propose that the experimental approaches used in this work indicate that there are changes in the rate of release of Ca²⁺ from the SR when SR load is altered. However, several important caveats should be noted before any such conclusions are drawn. First, it is well known that indo 1 may distribute into noncytoplasmic compartments,²¹ so there may be contamination of the cytoplasmic Ca²⁺ signal. Second, there is a nonlinear relationship between indo 1 fluorescence and [Ca²⁺]_i that makes it difficult to analyze the relationship between SR load and putative rate of release. Third, since the K_d of indo 1 increases as temperature decreases and indo 1 reaches saturation as free [Ca²⁺] increases past about 10 µmol/L even at 1°C,¹¹ it is difficult to obtain meaningful data while the cells are cooled. For these reasons, we have not attempted to calibrate our ratio signal but have used it in a qualitative approach to assess how a change in SR load may alter the rate of change of Ca²⁺.

From a normal experimental steady-state condition with stimulation at 0.5 Hz, the SR loses approximately half its Ca²⁺ after a rest of 45 seconds. Such a loss of Ca²⁺ decreased the rate of cell shortening to 59%, decreased the rate of change in fluorescence to 53%, and increased the time to peak of the exchange current 2.1-fold. The close correlation between the approximate halving of the cell shortening and fluorescent rate changes and the doubling of the time to peak of the exchange current tends to support the validity of both measurements despite our concerns noted above, particularly because the exchange current measurements were made independently with no indo 1 in the cells.

The SR Ca²⁺ content calculations also assume that caffeine and rapid cooling release similar amounts of Ca²⁺ and that these are good indicators of SR Ca²⁺ content. The experiments of the type shown in Fig 2 suggest that if either method of Ca²⁺ release is applied first, subsequent application of either method is ineffective at producing release. However, we cannot determine whether these methods cause the entire release of all Ca²⁺ that may be used in contraction. Paired caffeine applications have been used by several groups as indicators of complete SR Ca²⁺ depletion,^{22 23} but whether there is complete release of SR Ca²⁺ or just of caffeine-releasable SR Ca²⁺ is uncertain. Weber and Herz¹⁵ noted that 8 to 10 mmol/L caffeine produced up to 50% release of accumulated SR Ca²⁺ in frogs and produced 35 to 48 µmol/kg muscle Ca²⁺ release from rabbits, a quantity similar to that measured by us.

SR Load at Different Rest Intervals
It is possible to draw some quantitative conclusions from our measurements of Na⁺/Ca²⁺ exchange current in the presence of caffeine. We used the same technique as Varro et al,¹⁴ which supposes that the SR cannot function as a store of Ca²⁺ in the presence of caffeine, so the Na⁺/Ca²⁺ exchange expels the Ca²⁺ that is released from the SR on application of caffeine minus a portion that is removed from the cytoplasm by exchange-independent mechanism(s). The value of SR Ca²⁺ content derived in this way depends greatly on estimates of cell volume and the percentage of contribution of exchange-independent mechanisms. We have not made any correction for processes other than Na⁺/Ca²⁺ exchange that may remove Ca²⁺ from the cytoplasm because previous results^{10 18} show that such processes account for about 3% of the decline in cytoplasmic Ca²⁺ during a twitch in guinea pigs. Our calculations may, therefore, be slight underestimates. The greatest source of error is likely to be in estimating cell volume and accessible cell volume. The size of our caffeine-activated inward current (135±13 pC after 5 seconds of rest) is similar to that found by others (113 pC¹⁶ and 94 pC¹⁴ ); however, our measure of total Ca²⁺ released is 61 µmol/L, which is about 50% of the value derived by Varro et al (1993), largely because of the difference in cell size. If we assume the cell volume to be 30x10^-12 L (as in "Results") and 25% of this is taken up by mitochondria, then by extrapolation, at 0.5 Hz stimulation, 61 µmol/L of Ca²⁺ can be released from the SR. This is the equivalent of 24 µmol/kg wet wt, assuming a conversion factor of 2.5.²⁴ If half of the Ca²⁺ in the SR is released per beat²² and the passive buffering of the cell is near that described by Hove-Madsen and Bers,²⁵ then some 15 to 27 µmol/kg wet wt must be supplied from other sources to change free [Ca²⁺] from 0.1 to 500 nmol/L or 1.0 µmol/L, respectively. We anticipate that Ca²⁺ flux via Ca²⁺ current will not be large immediately after the initial depolarization phase of the action potential because the membrane potential will be about +50 mV. Ca²⁺ influx after the peak of the transient is reached does not play a role in activation of contraction, so a period from 4 to 5 milliseconds into the action potential until the peak of the Ca²⁺ transient is reached (eg, up to 30 milliseconds) seems an appropriate period over which to integrate the Ca²⁺ current.

Using the formulations of Hilgemann and Noble,²⁶ DiFrancesco and Noble,²⁷ and Noble et al²⁸ in the simulation program HEART version 3.4 (Oxsoft), we can calculate approximate limits for Ca²⁺ influx via the Ca²⁺ current in guinea pig cardiac myocytes. The calculations are for Ca²⁺ flowing via the L-type Ca²⁺ channel during the action potential, not under voltage clamp. After 15, 20, and 30 milliseconds, Ca²⁺ influx is 8.5, 15.7, and 41.0 µmol/kg wet wt, respectively. In this particular simulation, very little Ca²⁺(1.0 µmol/kg wet wt) is supplied by Na⁺/Ca²⁺ exchange. Enough Ca²⁺ could be supplied by the Ca²⁺ current to produce contraction. However, in our calculations of SR Ca²⁺ release we did not take account of the volume of the cell taken up by the myofilaments. This is approximately 45% in guinea pig ventricular muscle cells.²⁹ If this is done, our estimates of Ca²⁺ release are much higher, so much less Ca²⁺ would be required to enter the cell.

Rate of Ca²⁺ Release
The finding that the rate of cell shortening and the rate of change in indo 1 fluorescence are altered by SR load is, to our knowledge, the first full demonstration of this phenomenon in the intact cardiac myocyte, although it is apparent in earlier studies^{11 30 31} but was not investigated extensively. The finding suggests that SR load may influence not only the amount but also the rate of SR Ca²⁺ release. This feature has to date only been recognized in the isolated SR Ca²⁺ release system⁸ and not tested in the intact single cell.

The increased rate of release at large SR loading could be due to a larger instantaneous conductance, the recruitment of more channels, or an increased open probability of channels during the release period. At present, there is no conclusive evidence for either mechanism being the more important, but given the conditions under which the experiments were carried out, the results point to altered conductance of the SR Ca²⁺ release channel being responsible for the effect more than alterations to gating. The reasons are as follows. Low temperature and caffeine application greatly increase the open probability of the isolated channel studied in an artificial bilayer.^{32 33} Although extrapolations regarding the behavior of the isolated channel in the bilayer to its behavior in the cell should be made with caution, it seems reasonable to conclude that cooling or caffeine application will alter the gating so markedly that any effect of SR Ca²⁺ content will be mediated through a change in conductance rather than a further alteration to gating. However, we cannot rule out an additional effect of stored Ca²⁺ on gating: a recent report suggests that luminal Ca²⁺ may indeed regulate release channel gating, but this action depends on the way the channel is activated from the cytoplasmic side.³⁴

Given the caveats we introduced above, we cannot determine the true relationship between rate of release and SR load. All we can state at present is that when release is produced by caffeine or cooling there is an apparent increase in rate of release as SR Ca²⁺ content increases. The simplest explanation is that because there is more Ca²⁺ in the SR it is released more rapidly. This interpretation assumes that there is homogeneous Ca²⁺ release from the SR throughout the different loading conditions used. If there are inhomogeneities of release at different SR loads, this may result in apparent differences in the rate of release.

	Acknowledgments

 
We would like to thank the British Heart Foundation and Medical Research Council for financial support and Dr Alan Williams for helpful discussion and comments on an early version of the manuscript.

Received October 24, 1994; accepted April 14, 1995.

	References

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1. Fabiato A. Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol. 1985;85:291-320. [Abstract/Free Full Text]

2. Stern MD. Theory of excitation-contraction coupling in cardiac muscle. Biophys J. 1992;63:497-517. [Medline] [Order article via Infotrieve]

3. Lipp P, Niggli E. Microscopic spiral waves reveal positive feedback in subcellular calcium signaling. Biophys J. 1993;65:2272-2276. [Medline] [Order article via Infotrieve]

4. Wier WG, Egan TM, López-López JR, Balke CW. Local control of excitation-contraction coupling in rat heart cells. J Physiol (Lond). 1994;474:463-471. [Abstract/Free Full Text]

5. Valdeolmillos M, O'Neill SC, Smith GL, Eisner DA. Calcium-induced calcium release activates contraction in intact cardiac cells. Pflugers Arch. 1989;413:676-678. [Medline] [Order article via Infotrieve]

6. Lakatta EG, Capogrossi MC, Kort AA, Stern MD. Spontaneous myocardial calcium oscillations: overview with emphasis on ryanodine and caffeine. Fed Proc. 1985;44:2977-2983. [Medline] [Order article via Infotrieve]

7. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993;262:740-744. [Abstract/Free Full Text]

8. Tinker A, Lindsay ARG, Williams AJ. Cation conduction in the calcium release channel of the cardiac sarcoplasmic reticulum under physiological and pathophysiological conditions. Cardiovasc Res. 1993;27:1820-1825. [Abstract/Free Full Text]

9. Naqvi RU, del Monte F, O'Gara P, Harding SE, MacLeod KT. Characteristics of myocytes isolated from the hearts of renovascular hypertensive guinea pigs. Am J Physiol. 1994;266:H1886-H1895. [Abstract/Free Full Text]

10. Terracciano CMN, MacLeod KT. The effect of acidosis on Na⁺/Ca⁺⁺ exchange and consequences for relaxation in isolated cardiac myocytes from guinea pig. Am J Physiol. 1994;267:H477-H487. [Abstract/Free Full Text]

11. Bers DM, Bridge JHB, Spitzer KW. Intracellular Ca²⁺ transients during rapid cooling contractures in guinea-pig ventricular myocytes. J Physiol (Lond). 1989;417:537-553. [Abstract/Free Full Text]

12. O'Neill SC, Donoso P, Eisner DA. The role of [Ca²⁺]_i and [Ca²⁺] sensitization in the caffeine contracture of rat myocytes: measurement of [Ca²⁺]_i and [caffeine]_i. J Physiol (Lond). 1990;425:55-70. [Abstract/Free Full Text]

13. Bassani RA, Bassani JWM, Bers DM. Mitochondrial and sarcolemmal Ca²⁺ transport reduce [Ca²⁺]_i during caffeine contractures in rabbit cardiac myocytes. J Physiol (Lond). 1992;453:591-608. [Abstract/Free Full Text]

14. Varro A, Negretti N, Hester SB, Eisner DA. An estimate of the calcium content of the sarcoplasmic reticulum in rat ventricular myocytes. Pflugers Arch. 1993;423:158-160. [Medline] [Order article via Infotrieve]

15. Weber A, Herz R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol. 1968;52:750-759. [Abstract/Free Full Text]

16. Callewaert G, Cleeman L, Morad M. Caffeine-induced Ca²⁺ release activates Ca²⁺ extrusion via Na⁺-Ca²⁺ exchanger in cardiac myocytes. Am J Physiol. 1989;257:C147-C152. [Abstract/Free Full Text]

17. Page E. Quantitative ultrastructural analysis in cardiac membrane physiology. Am J Physiol. 1978;235:C147-C158. [Abstract/Free Full Text]

18. Terracciano CMN, Naqvi RU, MacLeod KT. Effects of hypoxia on sodium/calcium exchange in isolated guinea-pig cardiac myocytes. Biophys J. 1993;64:A41. Abstract.

19. Bassani JWM, Bassani RA, Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol (Lond). 1994;476:279-293. [Abstract/Free Full Text]

20. Kirby MS, Sagara Y, Gaa S, Inesi G, Lederer WJ, Rogers TB. Thapsigargin inhibits contraction and Ca²⁺ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca²⁺ pump. J Biol Chem. 1992;267:12545-12551. [Abstract/Free Full Text]

21. Miyata H, Silverman HS, Sollott SJ, Lakatta EG, Stern MD, Hansford RG. Measurement of mitochondrial free Ca²⁺ concentration in living single rat cardiac myocytes. Am J Physiol. 1991;261:H1123-H1134. [Abstract/Free Full Text]

22. Bassani JWM, Bassani RA, Bers DM. Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes. Am J Physiol. 1993;265:C533-C540. [Abstract/Free Full Text]

23. Janczewski AM, Lakatta EG. Buffering of calcium influx by sarcoplasmic reticulum during the action potential in guinea-pig ventricular myocytes. J Physiol (Lond). 1993;471:343-363. [Abstract/Free Full Text]

24. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983;245:1-14.

25. Hove-Madsen L, Bers DM. Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes. Am J Physiol. 1993;264:C677-C686. [Abstract/Free Full Text]

26. Hilgemann DW, Noble D. Excitation-contraction coupling and extracellular calcium transients in rabbit atrium: reconstruction of basic cellular mechanisms. Proc R Soc Lond Biol Sci. 1987;230:163-205. [Medline] [Order article via Infotrieve]

27. DiFrancesco D, Noble D. A model of cardiac electrical activity incorporating ionic pumps and concentration changes. Philos Trans R Soc Lond B Biol Sci. 1985;307:353-398. [Abstract/Free Full Text]

28. Noble D, Noble SJ, Bett GCL, Earm YE, Ho WK, So IK. The role of sodium-calcium exchange during the cardiac action potential. Ann N Y Acad Sci. 1991;639:334-353. [Medline] [Order article via Infotrieve]

29. Forbes MS, Van Niel EE. Membrane systems of guinea pig myocardium: ultrastructure and morphometric studies. Anat Rec. 1988;222:362-379. [Medline] [Order article via Infotrieve]

30. Lewartowski B, Hansford RG, Langer GA, Lakatta EG. Contraction and sarcoplasmic reticulum Ca²⁺ content in single myocytes of guinea pig heart: effect of ryanodine. Am J Physiol. 1990;259:H1222-H1229. [Abstract/Free Full Text]

31. Sipido KR, Wier WG. Flux of Ca²⁺ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. J Physiol (Lond). 1991;435:605-630. [Abstract/Free Full Text]

32. Sitsapesan R, Montgomery RAP, MacLeod KT, Williams AJ. Sheep cardiac sarcoplasmic reticulum calcium-release channels: modification of conductance and gating by temperature. J Physiol (Lond). 1991;434:469-487. [Abstract/Free Full Text]

33. Sitsapesan R, Williams AJ. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum. J Physiol (Lond). 1990;423:425-439. [Abstract/Free Full Text]

34. Sitsapesan R, Williams AJ. Regulation of the gating of the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by luminal Ca²⁺. J Membr Biol. 1994;137:215-226.[Medline] [Order article via Infotrieve]

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