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(Circulation Research. 1997;81:477-484.)
© 1997 American Heart Association, Inc.

Articles

Enhanced Ca²⁺ Current and Decreased Ca²⁺ Efflux Restore Sarcoplasmic Reticulum Ca²⁺ Content After Depletion

A. W. Trafford, M. E. Díaz, N. Negretti, , D. A. Eisner

From the Department of Veterinary Preclinical Sciences, University of Liverpool (UK).

Correspondence to Dr D.A. Eisner, Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 3BX, UK. E-mail eisner{at}liv.ac.uk

	Abstract

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Abstract [Ca²⁺]_i was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca²⁺ content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na⁺-Ca²⁺ exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca²⁺ transient recovered, the integral of the L-type Ca²⁺ current decreased and that of the Na⁺-Ca²⁺ exchange tail current increased. For the early pulses, the gain of Ca²⁺ via the Ca²⁺ current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca²⁺ with each pulse. When these are summed, the calculated gain of cell Ca²⁺ agrees well with the increase of SR Ca²⁺ produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca²⁺ content and the magnitude of the systolic Ca²⁺ transient such that at high SR Ca²⁺ content a given increase of content had a greater effect on the Ca²⁺ transient than did an increase at low SR content. In conclusion, the effects of systolic Ca²⁺ on the Ca²⁺ current and Na⁺-Ca²⁺ exchange current provide a means to regulate SR Ca²⁺ content and thence the systolic Ca²⁺ transient.

Key Words: sarcoplasmic reticulum • Ca²⁺ • Ca²⁺ transient

	Introduction

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In cardiac muscle, the sarcoplasmic reticulum (SR) is the source of the bulk of the systolic rise of [Ca²⁺]_i, which activates contraction. An important factor that controls the amount of Ca²⁺ released from the SR is its Ca²⁺ content. Therefore, if the systolic force of contraction of the heart is to be maintained at a steady level, so too must the SR Ca²⁺ content. Previous work has suggested that the effects of various inotropic maneuvers are due to changes of SR Ca²⁺ content. Similarly, the effects of changes of stimulation frequency or rest duration have been attributed to changes in the balance of entry and efflux of Ca²⁺ during the action potential altering of the SR Ca²⁺ content.^{1 2 3}

A quantitative investigation of this area requires being able to measure both SR Ca²⁺ content and sarcolemmal Ca²⁺ influx and efflux. It is now possible to measure the SR Ca²⁺ content by releasing it with caffeine and measuring the Ca²⁺ pumped out of the cell by the electrogenic Na⁺-Ca²⁺ exchange.^{4 5 6} This can be simultaneously compared with the amount of Ca²⁺ that enters the cell on the Ca²⁺ current during each depolarization as well as that which is pumped out of the cell.⁷

The results show that after the SR has been emptied, subsequent stimulation accelerates the rate of refilling. The gain of SR Ca²⁺ content can be accounted for by the fluxes across the surface membrane as a result of the combination of an increased Ca²⁺ current and a decreased efflux of Ca²⁺.

	Materials and Methods

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Experiments were performed on cardiac myocytes isolated from the rat or ferret. Rats were killed by stunning and cervical dislocation, and cells were prepared using a collagenase and protease digestion technique as previously described.⁸ Ferrets were deeply anesthetized using intraperitoneal sodium pentobarbital (200 mg/kg, Euthatal, Rhône Mérieux). The thoracic cavity was then opened, and the heart was removed, weighed, and retrogradely perfused through the aorta with a nominally Ca²⁺-free solution for 6 minutes at 37°C. (The Ca²⁺-free solution contained [mmol/L] NaCl 134, glucose 11, HEPES 10, KCl 4, MgSO₄ 1.2, and Na₂HPO₄ 1.2, pH 7.2 with NaOH.) Collagenase (type IV, Sigma Chemicals) and protease (Sigma type XIV) were then added to a final concentration of 14.9 and 0.16 U/mL, respectively, and perfused for 9 to 15 minutes until the heart was flaccid. The perfusate was then changed to a taurine-containing solution for 10 minutes (mmol/L: NaCl 108, taurine 50, glucose 11, HEPES 10, KCl 4, MgSO₄ 1.2, and Na₂HPO₄ 1.2, pH 7.2 with NaOH). The left ventricle was then dissected from the heart and cut into small pieces, suspended in the taurine-containing solution, and gently triturated to release single cells. The isolated cells were stored at room temperature in the taurine-containing solution until use.

Measurement of [Ca²⁺]_i
Cells were loaded with the [Ca²⁺]_i-sensitive fluorescent indicator indo 1 either as the cell-permeant AM form (2.5 µmol/L for 5 minutes, followed by at least 30 minutes of deesterification) or as the pentapotassium salt (100 µmol/L through the voltage-clamp pipette) and placed in the superfusion chamber on the stage of an inverted microscope adapted for epifluorescence (Nikon Diaphot 300, Nikon). Indo 1 fluorescence was excited at 340 nm and measured at 400 and 500 nm.⁹

Voltage-Clamp Control
All experiments were performed using the switch-clamp facility of the Axoclamp-2B voltage-clamp amplifier (Axon Instruments Inc). The chopping rate of the switch clamp was 3 to 14 kHz. Electrophysiological and fluorescence signals were digitized at 2 kHz and stored on a 586 computer using Axoscope 1.1 software (Axon Instruments Inc) for later analysis using custom software (Visual Basic 4, Microsoft Corp).

Experiments were performed with both conventional whole-cell¹⁰ or perforated-patch techniques.¹¹ The latter technique has the advantage that cell dialysis is minimized but at the expense of having to use the AM rather than the free-acid form of indo 1. In some experiments (see below), as an alternative to the perforated patch, high-resistance patch pipettes were used (again with the AM ester). The details of the solutions are as follows.

Whole Cell
The majority of experiments (Figs 2, 3, 4A, 5, and 7) were performed on ferret myocytes in which the fluorescent indicator indo 1 was loaded into the cell as the free-acid form through the pipette. Electrodes were fabricated from borosilicate glass and had resistances of 3 to 4 M when filled with the following solution (mmol/L): CsCl 120, tetraethylammonium chloride 20, HEPES 10, MgCl₂ 5, Na₂-ATP 5, and K₅-indo 1 0.1, pH 7.2 with CsOH. Some whole-cell experiments (Figs 4B and 6) were also performed on rat myocytes using higher resistance pipettes (10 to 15 M). In these experiments, the cells were loaded with indo 1-AM, and the pipette solution contained (mmol/L) KCl 140, MgCl₂ 5, Na₂-ATP 5, HEPES 3, and K₂-EGTA 0.1, pH 7.2 with KOH.

View larger version (14K): [in this window] [in a new window]   Figure 2. Stimulation accelerates SR refilling (ferret myocyte). A, The effects of a short (20-second) rest after removing caffeine. Top trace shows the time course of the indo 1 ratio. The membrane potential was held at -40 mV, and 100-millisecond pulses were applied to 0 mV at 0.33 Hz. Stimulation was stopped, and 10 mmol/L caffeine was applied for the period indicated by bar i. Caffeine was then removed and reapplied at bar ii. After this removal of caffeine, stimulation was resumed before again stopping stimulation and readding caffeine at bar iii. The caffeine-evoked currents and their cumulative integrals are shown below. B, The effects of a longer (60-second) rest after removing caffeine showing current (top) and cumulative integral (bottom). The left record was obtained after steady state stimulation, and the right record was obtained after 60 seconds of rest after removing caffeine. Panels A and B are from different cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. The effects of refilling the SR on sarcolemmal Ca2+ movements (ferret myocyte). A, Time course of indo 1 ratio. Before the trace shown, the SR had been depleted by exposure to caffeine (10 mmol/L, solid bar). Caffeine was removed as shown, and 100-millisecond depolarizing pulses were applied to 0 mV at 0.33 Hz. Finally, stimulation was stopped, and caffeine was applied. B, Specimen records of membrane current (top) and calculated Ca2+ gain (bottom). The records are taken from the pulses indicated in panel A. The integral trace shows the cumulative integral of the Ca2+ current () as an increasing upward deflection (Ca2+ gain) and the efflux on repolarization () as a downward one. The efflux has been calculated from the integral of the Na+-Ca2+ exchange tail current and corrected for the fact that 33% of the efflux occurs via mechanisms other than Na+-Ca2+ exchange. C, Expanded versions of the current tails on repolarization.

View larger version (20K): [in this window] [in a new window]   Figure 4. Pulse-by-pulse analysis of changes of sarcolemmal fluxes and SR Ca2+ content. A, Calculated fluxes in a ferret cell: top, sarcolemmal Ca2+ movements (, Ca2+ influx via Ca2+ current [ICa]; , Ca2+ efflux obtained by correcting the Na+-Ca2+ exchange current [INa-Ca]); middle, net gain of Ca2+ per pulse obtained from the difference between Ca2+ entry and efflux on that pulse); and bottom, cumulative Ca2+ gain obtained by adding the gains per pulse. B, Continuous trace of [Ca2+]i (top) and cumulative Ca2+ gain (bottom) in a rat myocyte. C, Representative specimen traces from the points indicated in panel B. In addition to the measured indo 1 ratio (top) and calculated fluxes (bottom), the membrane currents are shown (middle).

View larger version (17K): [in this window] [in a new window]   Figure 5. Mean data showing (from top to bottom) Ca2+ transient magnitude (), Ca2+ efflux () and influx (), calculated Ca2+ gain per pulse (), and cumulative Ca2+ gain (). Data are mean±SEM from 20 refillings on 14 ferret myocytes.

View larger version (11K): [in this window] [in a new window]   Figure 7. The dependence of systolic Ca2+ transient on SR Ca2+ content in ferret cells. The ordinate shows the magnitude of the systolic Ca2+ transient normalized to the maximum in that cell; the abscissa, the SR Ca2+ content similarly normalized. The data are mean±SEM from 20 refillings in 14 cells.

View larger version (20K): [in this window] [in a new window]   Figure 6. External Ca2+ removal postpones the decay of the Ca2+ current (rat myocyte). A, Time course. Two traces are shown. In each case, caffeine had been applied and removed just before the record shown. In the first response, electrical stimulation was begun after removing caffeine; in the second one, the cell was exposed to a Ca2+-free solution for 60 seconds before replacing Ca2+ and recommencing stimulation. B, Specimen records of (from top to bottom) the indo 1 ratio, membrane current, and calculated integral of the Ca2+ current. C, Time course of decay of the integral of the Ca2+ current as a function of the number of pulses applied after removing caffeine. Symbols are as follows: , control; , 60-second interpolated exposure to Ca2+-free solution.

Perforated Patch
In these experiments (Figs 1 and 8), the perforated-patch technique with amphotericin was used.¹¹ Electrodes had resistances of 1 to 3 M when filled with (mmol/L) KCH₃O₃S 125, KCl 20, NaCl 10, HEPES 10, and MgCl₂ 5, titrated to pH 7.2 with KOH and a final amphotericin concentration of 240 µg/mL.

View larger version (20K): [in this window] [in a new window]   Figure 1. The effects of membrane potential on caffeine-evoked changes of [Ca2+]i and current in a ferret ventricular myocyte. A, Original records. In each panel, the traces show (from top to bottom) the indo 1 ratio, membrane current, and the cumulative integral of current converted to equivalent changes of total cell Ca2+ content. The membrane potential is as indicated above the traces. The time of application of caffeine (10 mmol/L) corresponds to the simultaneous increases of [Ca2+]i and inward current. Caffeine was removed after the end of each record, and the cell was stimulated at 0.33 Hz until a steady state was regained (not shown). The membrane potential was then stepped to the desired holding potential for 30 seconds, and caffeine was then applied. The smooth curves through the data are single exponential fits. B, Normalized records of [Ca2+]i (left) and current (right). Traces from the responses at -80 and 0 mV are superimposed.

View larger version (23K): [in this window] [in a new window]   Figure 8. Comparison of the recovery of the Ca2+ transient in current-clamped and voltage-clamped ferret cells. In both parts, the top trace shows the time course of recovery of the Ca2+ transient after removing caffeine. Caffeine was removed 5 seconds before the traces start. Expanded versions of specimen records taken from the times indicated are shown below. Note that i and ii are single transients, whereas iii is the average of four steady state responses. The traces for the specimen records show (from top to bottom) [Ca2+]i, membrane potential (Em), and current. Both panels begin 5 seconds after removing caffeine (10 mmol/L). A, Current-clamp stimulation (2 milliseconds, 4 nA at 0.33-Hz inward current pulses). Note that the current traces have been attenuated. B, Voltage-clamp stimulation (200 millisecond depolarizing pulses from -40 to 0 mV at 0.33 Hz).

Cells were bathed in a control solution of the following composition (mmol/L): in ferret myocytes, NaCl 140, glucose 10, KCl 6, HEPES 5, CaCl₂ 2, and MgCl₂ 1, pH 7.4 with NaOH; in rat myocytes, NaCl 135, glucose 11, HEPES 10, KCl 4, MgCl₂ 1.2, and CaCl₂ 1, pH 7.4 with NaOH. To avoid interference from outward currents, all experiments were performed in the presence of 5 mmol/L 4-aminopyridine and 0.1 mmol/L BaCl₂. Ca²⁺-dependent Cl^- currents¹² were inhibited by the addition of 100 µmol/L DIDS to the superfusate. DIDS is fluorescent, but this produces a constant offset that contributes only to the background fluorescence and is therefore subtracted off and does not interfere with the [Ca²⁺]_i signals. All experiments were performed at 27°C.

Quantification of SR Ca²⁺ Content and Sarcolemmal Ca²⁺ Fluxes
The Ca²⁺ current during depolarization and the Na⁺-Ca²⁺ exchange tail current on repolarization were integrated. The baselines chosen for the integrations were as follows: (1) For the Ca²⁺ current, the baseline was the steady state level of current. During steady state stimulation, the current had relaxed to a steady level at the end of the 100-millisecond pulses used. However, for the first pulse after caffeine, the slower rate of inactivation meant that the steady state was not reached. In this case, the same baseline used during steady state stimulation was chosen. (2) For the tail currents, the baseline used was the mean level of current between 1.5 and 2 seconds after the pulse.

The inward Na⁺-Ca²⁺ exchange currents produced by the application of 10 mmol/L caffeine to a voltage-clamped cell were integrated and converted to total Ca²⁺ fluxes as described previously.⁶ Briefly, one needs to correct for the fact that during a caffeine response, some of the Ca²⁺ is removed from the cytoplasm by mechanisms other than Na⁺-Ca²⁺ exchange and does not, therefore, generate current. The magnitude of this correction was estimated as follows. The rate constant of decay of the caffeine response was measured (1) under control conditions (k_cont) and (2) with the Na⁺-Ca²⁺ exchange inhibited with 10 mmol/L Ni²⁺ (k_Ni). Multiplying a measured Na⁺-Ca²⁺ exchange flux by k_cont/(k_cont-k_Ni) gives the corrected total Ca²⁺ flux. This correction was used for the Na⁺-Ca²⁺ exchange fluxes activated by either caffeine or on repolarization (tail currents). Previous work on rat ventricular myocytes has shown that 33% of Ca²⁺ efflux, during a caffeine response, occurs by mechanisms other than Na⁺-Ca²⁺ exchange.^{6 7} In ferret myocytes, we have found (authors' unpublished data, 1996) that, again, inhibiting Na⁺-Ca²⁺ exchange with 10 mmol/L Ni²⁺ decreases the rate of relaxation of the caffeine-evoked rise of [Ca²⁺]_i to 33% of the control level, and we have used this figure for correcting the data from ferret cells. This is slightly greater than the value of 25% found by others in ferret cells.¹³ Cell volume was calculated from the membrane capacitance assuming a membrane capacitance-to-volume ratio of 6.76 and 5.39 pF/pL for the rat and ferret, respectively.¹⁴ The majority of experiments (and all the mean data presented) were carried out on ferrets. Qualitatively similar results were, however, obtained on rat myocytes, and some of these data are also presented (eg, Figs 4B and 6).

Statistics
All values are presented as mean±SEM for n experiments.

	Results

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The approach in the present study depends on the fact that the SR Ca²⁺ content can be estimated from the integral of the caffeine-evoked current and that this, in turn, relies on the assumption that at least at a fixed holding potential, the only significant current that is affected by [Ca²⁺]_i is the Na⁺-Ca²⁺ exchange current. This has been validated in rat myocytes.^{5 6} However, in some species, Ca²⁺-dependent Cl^- currents are also present, and this would invalidate the method. For this reason, the experiments described in the present study were performed in the presence of the Cl^- channel inhibitor DIDS.¹² The data presented in Fig 1 support the idea that under these conditions, in the ferret, Na⁺-Ca²⁺ exchange is the major Ca²⁺-activated current. In this experiment, caffeine was applied at different holding potentials. At each voltage (from -80 to 0 mV), caffeine produces an inward current. The peak of the caffeine-evoked increase of [Ca²⁺]_i and the integral of the current both increase with depolarization, presumably as the cell and therefore the SR Ca²⁺ load is increased. It should be noted that any contribution of a Ca²⁺-dependent Cl^- current will result in an outward current at voltages positive to the reversal potential (here calculated to be -34 mV). Fig 1 also shows that the rate constant of decay of the caffeine-evoked current decreases with depolarization¹⁵ since the Na⁺-Ca²⁺ exchange is less active.¹⁶ The mean calculated SR Ca²⁺ content in the ferret myocytes (after steady state stimulation at 0.33 Hz) was 80±6 µmol/L (n=17; holding potential, -40 mV). These Ca²⁺ contents were derived from the integrated caffeine-evoked currents using the corrections described in "Materials and Methods." The integral of the caffeine-evoked current increased with depolarization (in Fig 1, from 149 at -80 mV to 177 at -40 mV and 189 µmol/L at 0 mV). This is presumably due to the increase of resting [Ca²⁺]_i produced by depolarization (Fig 1A) leading to increased SR Ca²⁺ loading.

The experiment illustrated in Fig 2 shows that refilling of the SR is accelerated by stimulation. In Fig 2A, the cell was initially electrically stimulated with depolarizing voltage-clamp pulses. The application of caffeine (Fig 2, bar i) produced an inward current. Caffeine was then removed and reapplied after an interval of 20 seconds without stimulation. This subsequent application of caffeine (Fig 2, bar ii) produced a small increase of [Ca²⁺]_i and no detectable inward current. After this response, five stimuli were applied. This was sufficient to return the SR Ca²⁺ content to a level similar to the control as shown by the effects of the final caffeine application (Fig 2, bar iii). In Fig 2B, the left record shows a caffeine response obtained after steady state stimulation. The right record was obtained 60 seconds after removing caffeine in the absence of stimulation. Here the calculated SR Ca²⁺ was 36 µmol/L compared with 91 µmol/L after stimulation. In other words, the longer unstimulated recovery period produces a greater recovery of SR Ca²⁺ content than did the shorter one, but the recovery is still less than that produced by an equivalent period of stimulation. We have found (not shown) that in the absence of stimulation, there is a linear gain of SR Ca²⁺ content over the first 70 seconds. A regression through the data gave a rate of gain of 0.55±0.17 µmol · L^-1 · s^-1. Similarly, Terracciano and MacLeod¹⁷ found, using cooling contractions in the presence of caffeine, that the rate of subsequent SR reaccumulation was reasonably linear over the first 1 to 2 minutes. The acceleration of SR refilling by stimulation is consistent with observations of the recovery of the electrically stimulated twitch after removal of caffeine.¹⁸

How Does Stimulation Fill the SR?
The experiments shown above have demonstrated that stimulation increases the SR Ca²⁺ content. In principle, this could result from effects on Ca²⁺ entry into and efflux from the cell. Distinguishing between these possibilities requires examining the individual fluxes. Specifically, we have measured the entry of Ca²⁺ into the cell on the L-type Ca²⁺ current and the efflux from the cell via the Na⁺-Ca²⁺ exchange. The latter was assessed from the "tail" currents on repolarization after short (100-millisecond) depolarizing pulses. Fig 3A shows the recovery of the Ca²⁺ transient after an exposure to caffeine. The current records from the first and the steady state (ninth) pulses are reproduced in Fig 3B. It is clear that the magnitude of the Ca²⁺ current is larger on the first than the ninth pulse and that its rate of inactivation is slowed. On repolarization after the capacity current, the current returns immediately to the baseline on pulse 1, whereas there is a slower tail in pulse 9. The differences in the tail current are more obvious in the expanded record of Fig 3C. The lower traces in Fig 3B show the cumulative net Ca²⁺ gain as calculated from the current records. Here, the Ca²⁺ current and Na⁺-Ca²⁺ exchange current have been integrated and used to calculate the changes of total cell Ca²⁺ content. To do this, we have corrected the Na⁺-Ca²⁺ exchange flux to allow for the fact that a fraction (33%) of Ca²⁺ is removed from the cytoplasm by mechanisms other than Na⁺-Ca²⁺ exchange. The record from the first pulse shows a calculated gain of 9.8 µmol/L with almost no change on repolarization (a loss of 0.5 µmol/L). In contrast, for the ninth pulse, the gain during the depolarizing pulse is less (3.9 µmol/L), and the loss on repolarization is increased (4.0 µmol/L), such that there is very little change of total cell Ca²⁺.

The data described above compare net Ca²⁺ fluxes on the first pulse with those in the steady state. It is possible to make similar measurements for all the pulses after removal of caffeine. Fig 4A (top) shows that the integral of the Ca²⁺ current decreases and that the integral of the Na⁺-Ca²⁺ exchange tail current increases with successive pulses. As a result of this, the net Ca²⁺ entry per pulse decreases from 14.3 µmol/L on the first pulse to zero in the steady state. The bottom curve shows the calculated gain of SR Ca²⁺ content calculated by cumulatively summing the net gain on all the pulses. In this case, the total gain of Ca²⁺ amounts to 75 µmol/L.

The actual time course of changes of calculated cell Ca²⁺ are plotted in Fig 4B (rat myocyte). These were calculated in the same way as shown for Fig 3. It is clear that the response changes gradually from a large Ca²⁺ influx followed by a small loss to equal and opposite movements. Mean data from 20 refillings from 14 cells are presented in Fig 5. On average, there is a net gain of 8.1 µmol/L on the first pulse. In the steady state (from pulse 9 onward), there is no significant difference between the Ca²⁺ influx and efflux (P>.05).

The work presented above shows that the gain of Ca²⁺ on the initial pulses is due to both increased Ca²⁺ current and decreased Ca²⁺ efflux. The latter is presumably due to the smaller Ca²⁺ transient producing a decreased activation of Na⁺-Ca²⁺ exchange. However, it is less clear what the cause of the increased Ca²⁺ current is. It does not appear to result simply from slow recovery from inactivation of the Ca²⁺ current. This is because if after the removal of caffeine the cell was left unstimulated for several minutes such that there was time for the SR to refill with Ca²⁺, then the first stimulus was accompanied by both Ca²⁺ transients and currents of control size (not shown). Two other possibilities were considered: (1) it could result from reduced Ca²⁺-induced inactivation as a consequence of the decreased systolic calcium, or (2) it could be a consequence of the previous exposure to caffeine, perhaps because caffeine, as a phosphodiesterase inhibitor,¹⁹ increases the concentration of cAMP. The experiment illustrated in Fig 6 was designed to distinguish between these possibilities by adding a period in Ca²⁺-free solution after the removal of caffeine in order that any caffeine-dependent effect could be removed without SR refilling. If the recovery reflects a caffeine removal effect, then one would expect the Ca²⁺ current to have recovered fully by the first stimulus following the Ca²⁺-free period. Two recoveries from caffeine are shown. After the first exposure to caffeine, stimulation was recommenced 10 seconds after removing caffeine. This resulted in a Ca²⁺ entry of 6.3 µmol/L on the first pulse. After a further 60 seconds of stimulation, the Ca²⁺ entry had decreased to 3.6 µmol/L. After the second exposure to caffeine, the cell was exposed to a Ca²⁺-free solution for 60 seconds. This will inhibit SR refilling while still allowing any effects of inhibition of phosphodiesterases to decay. When stimulation was recommenced, the Ca²⁺ entry on the first pulse was 6.6 µmol/L and then decreased to 3.5 µmol/L in the steady state. The data show, therefore, that the first pulse has a large Ca²⁺ current and small Ca²⁺ transient. Indeed the recovery of Ca²⁺ entry on the Ca²⁺ current is the same whether or not the Ca²⁺-free period is interposed. Therefore, we conclude that the magnitude of the Ca²⁺ entry through the Ca²⁺ current depends not on the time following caffeine removal but on the time following the commencement of stimulation and therefore on the SR Ca²⁺ content.

The above data show that both the systolic Ca²⁺ transient and the SR content increase on stimulation. The relationship between these two parameters is shown in Fig 7. It can be seen that the magnitude of the Ca²⁺ transient increases proportionately more than the SR Ca²⁺ content. This supralinear relationship is similar to that previously described by Bassani et al.²⁰

In all the data presented above, the cells were stimulated with step voltage-clamp depolarizing pulses. We have also investigated the effects of SR Ca²⁺ depletion on the action potential. In the experiment illustrated in Fig 8, the cell was initially stimulated with brief depolarizing current pulses to produce action potentials (panel A). After removal of caffeine, the action potential duration was increased as expected from the enhanced Ca²⁺ current mentioned above. The first Ca²⁺ transient shows an initial rapid increase, presumably due to Ca²⁺ entry via the Ca²⁺ current, which is then followed by a slowly developing rise due to Ca²⁺ entry on Na⁺-Ca²⁺ exchange during the long action potential. The magnitude of the systolic Ca²⁺ transient shows a marked overshoot, which may result from the extra Ca²⁺ entry/decreased Ca²⁺ efflux during the long action potentials. The same cell was then stimulated with depolarizing pulses (panel B), and the recovery did not show such an overshoot.

	Discussion

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The present study shows that after depletion of the Ca²⁺ content of the SR, the refilling can be quantitatively accounted for by (1) enhanced inward Ca²⁺ current and (2) decreased Ca²⁺ removal on Na⁺-Ca²⁺ exchange. In addition, during the refilling process, the relationship between SR Ca²⁺ content can be studied and is shown to be supralinear.

We have measured both the Ca²⁺ current during depolarization and the efflux of Ca²⁺ on Na⁺-Ca²⁺ exchange on repolarization. Strictly speaking, a full characterization of Ca²⁺ fluxes should also allow for Ca²⁺ entry and efflux via Na⁺-Ca²⁺ exchange during depolarization. During the short pulses used in the present study, the Ca²⁺ efflux via the exchange is likely to be very small.⁷ We have no measurement of Ca²⁺ entry via "reverse-mode" Na⁺-Ca²⁺ exchange during depolarization or of Ca²⁺ fluxes beyond the first few hundred milliseconds of repolarization. However, the fact that during steady state stimulation the cell is calculated to be in Ca²⁺ flux balance suggests that we are not missing significant Ca²⁺ fluxes.

The Mechanism of SR Refilling
The experiments show that the rate of refilling of the SR is increased by stimulation. At rest, the calculated rate of Ca²⁺ gain by the SR is 0.55 µmol · L^-1 · s^-1. At this rate, it would take almost 1 minute to half refill the SR in the absence of stimulation. In contrast, four pulses (12 seconds at the rate of stimulation used here) are adequate to produce a similar refilling. We do not know the mechanism by which Ca²⁺ enters a quiescent cell. Given the average cell volume (32 pL), the above flux corresponds to a rate of Ca²⁺ entry of 18x10^-18 mol/s per cell, which corresponds to a current of 1.7 pA. Therefore, if the Ca²⁺ entry is through some kind of leak channel, then the expected current would be very hard to measure.

The observation that stimulation increases SR refilling is in agreement with previous measurements of SR Ca²⁺ content or contraction.^{1 18 21 22 23} This observation suggests that when the SR is depleted, there is a net gain of Ca²⁺ with each pulse. This net refilling results from a decrease of Ca²⁺ efflux and an increase of the influx. Quantitatively, the change of influx has a larger effect: the Ca²⁺ entry decreases, on average, by 5.7 µmol/L (from 10.4 to 4.7 µmol/L), whereas the efflux increases by 1.9 µmol/L (from 2.0 to 3.9 µmol/L). The effect on the net Ca²⁺ movement is even more dramatic as it increases from a large net gain on the first pulse to no significant net flux after steady state stimulation. It has previously been suggested that at least 50% of the Ca²⁺ entering the cell during the first pulse is sequestered in the SR.²⁴ In the present study, we find that, on average, the Ca²⁺ entry on the first pulse is 10.4 µmol/L and that 82% of this remains in the cell.

The effect of stimulation on the Ca²⁺ efflux presumably reflects only the fact that since the Ca²⁺ transient magnitude is decreased, the Na⁺-Ca²⁺ exchange is less activated. The increase of the Ca²⁺ current when the SR is depleted may be due to the removal of Ca²⁺-dependent inactivation due to SR Ca²⁺ release. Previous work has shown that SR Ca²⁺ release produced either by caffeine or depolarization can accelerate inactivation of the Ca²⁺ current^{25 26} and thereby decrease Ca²⁺ entry into the cell. In the present study, caffeine was used to empty the SR, and one must consider whether the potentiation of the Ca²⁺ current is due to an increase of cAMP concentration as a result of phosphodiesterase inhibition. That this is not the explanation is shown by the observation (Fig 6) that the interpolation of a period in Ca²⁺-free solution postpones the negative Ca²⁺ current staircase until stimulation is subsequently resumed in a Ca²⁺-containing solution.

We have previously examined the effects of suddenly reducing the duration of a depolarizing pulse.⁷ This results in a gradual decrease of systolic Ca²⁺ transient due to increased Ca²⁺ efflux on Na⁺-Ca²⁺ exchange and, therefore, decreased SR Ca²⁺ content. Interestingly this was accompanied by little or no effect on the Ca²⁺ current. It is possible that in the previous work the long pulses (800-millisecond duration) had initially increased the SR Ca²⁺ content to a level above that in the present study and that there is a maximum degree of inhibition of the Ca²⁺ current.

The data (Fig 5) show that the calculated gain of SR Ca²⁺ content, which can be accounted for by changes of Ca²⁺ entry and efflux after 1 minute of stimulation, is 53 µmol/L. Over the same period, the expected entry without stimulation would be 33 µmol/L (given the calculated resting Ca²⁺ entry of 0.55 µmol/L per second). If we assume that the stimulation-independent component is unaffected by stimulation, then the total calculated influx is 86 µmol/L. This can be compared with the total SR Ca²⁺ content (calculated from the integral of the caffeine-evoked current) of 80±6 µmol/L. The agreement of this value with that calculated from the refilling shows that the various pathways for SR refilling have been accounted for.

It has recently been suggested that the depression of the Ca²⁺ current by increased SR Ca²⁺ release provides a mechanism for stabilizing the SR Ca²⁺ content and thence the magnitude of the systolic Ca²⁺ transient.²⁷ Our work shows that changes in the Na⁺-Ca²⁺ exchange–mediated Ca²⁺ efflux also regulate SR Ca²⁺ content. Most important, we also quantify the contributions of these two processes to SR refilling.

The Ca²⁺ content of the SR is generally thought to be an important factor controlling the magnitude of the systolic Ca²⁺ transient.²⁸ The present data allows us to quantify this relationship. As shown in Fig 7, the slope of the dependence of the Ca²⁺ transient on SR Ca²⁺ content increases with increasing content. It should be noted that the curvature in this relation cannot be due to the nonlinear relationship between the indo 1 fluorescence ratio and [Ca²⁺]_i. This nonlinearity would tend to decrease the slope at higher systolic [Ca²⁺]_i, and the observed relationship must therefore be an underestimate of the curvature. A similar relationship has been obtained in a previous study.²⁰ In that study, the SR Ca²⁺ content was measured from the change of free Ca²⁺ by making use of the previously determined relationship between free and total Ca²⁺ concentration. There are several possible explanations for the nonlinear relationship: (1) Work on single SR Ca²⁺ release channels has shown that their opening probability is increased by intra-SR or luminal Ca²⁺.²⁹ (2) Another possibility suggested previously²⁰ depends on the fact that total SR Ca²⁺ content will be a saturating function of the free SR Ca²⁺ concentration, and it is possible that the initial rate of release of SR Ca²⁺ depends on the free rather than total SR Ca²⁺. In contrast, Janczewski et al³⁰ found a linear relationship between the magnitude of the systolic Ca²⁺ transient and the rise of [Ca²⁺]_i produced by brief (200- to 300-millisecond) pulses of caffeine. This latter result may not be inconsistent with that in the present study. It is likely that as [Ca²⁺]_i increases, some of the cytoplasmic buffer sites will become more saturated with Ca²⁺ ions. Therefore, at higher [Ca²⁺]_i, a given increase of [Ca²⁺]_i during the caffeine response may require a smaller additional release of total Ca²⁺ from the SR. A linear correlation between the systolic Ca²⁺ transient and that produced by caffeine need not, therefore, be taken as showing a linear relationship between systolic Ca²⁺ and SR Ca²⁺ content.

	Acknowledgments

 
This study was supported by grants from the British Heart Foundation, Wellcome Trust, and the European Community. M.E. Díaz was supported by Consejo Nacional de Investigaciones Cientificas y Tecnologicas (Venezuela) and an Overseas Research Student Award.

Received February 3, 1997; accepted July 2, 1997.

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