Enhanced Ca2+ Current and Decreased Ca2+ Efflux Restore Sarcoplasmic Reticulum Ca2+ Content After Depletion
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.
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
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
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.
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 MΩ 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 MΩ). 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.
In these experiments (Figs 1⇓ and 8⇓), the perforated-patch technique with amphotericin was used.11 Electrodes had resistances of 1 to 3 MΩ 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.
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 (kcont) and (2) with the Na+-Ca2+ exchange inhibited with 10 mmol/L Ni2+ (kNi). Multiplying a measured Na+-Ca2+ exchange flux by kcont/(kcont−kNi) 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⇓).
All values are presented as mean±SEM for n experiments.
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.
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 18×10−18 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+ current25 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.
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.
- © 1997 American Heart Association, Inc.
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