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Am J Physiol Regul Integr Comp Physiol 284: R1330-R1339, 2003. First published January 16, 2003; doi:10.1152/ajpregu.00404.2002
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Vol. 284, Issue 5, R1330-R1339, May 2003

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes

Leif Hove-Madsen1, Anna Llach1, Glen F. Tibbits2, and Lluis Tort1

1 Unitat de Fisiologia Animal, Departamento de Biologia Cel.lular, Fisiologia i Immunología, Facultat de Ciencies, Universitat Autonoma de Barcelona, 08193 Cerdanyola, Barcelona, Spain; and 2 Cardiac Membrane Research Laboratory, Department of Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada V5A 156


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell patch clamp and intracellular Ca2+ transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca2+ trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca2+ carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca2+/pF, giving an SR Ca2+ release rate of 279 amol Ca2+ · pF-1 · s-1 or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca2+ release, intracellular Ca2+ transient, and cell shortening at 10 mV. Although 100 µM CdCl2 abolished this local maximum, it had no effect on SR Ca2+ release elicited by a depolarization to 110 or 150 mV, and the SR Ca2+ release was proportional to the membrane potential in the range -50 to 150 mV with 100 µM CdCl2. Increasing the intracellular Na+ concentration ([Na+]) from 10 to 16 mM enhanced SR Ca2+ release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca2+ release was potentiated with 16 mM but not 10 mM pipette [Na+]. Comparison of the total sarcolemmal Ca2+ entry and the Ca2+ released from the SR gave a gain factor of 18.6 ± 7.7. Nifedipine (Nif) at 10 µM inhibited L-type Ca2+ current (ICa) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca2+ release was 16.0 ± 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl2. The gain of the Nif-insensitive but NiCl2-sensitive SR Ca2+ release was 14.8 ± 7.1. Thus both reverse-mode Na+/Ca2+ exchange (NCX) and ICa can elicit Ca2+ release from the SR, but ICa is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca2+ released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca2+ release.

Ca2+ current; Ca2+ transients; caffeine; cardiac; excitation-contraction coupling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INASMUCH AS CA2+ release from the sarcoplasmic reticulum (SR) does not give rise to an easily quantifiable transsarcolemmal ionic current, estimates of the Ca2+ released from the SR during contraction are largely based on measurements of the Ca2+ transient and knowledge of passive Ca2+ buffering in mammalian cardiac myocytes (2, 8, 10, 41). Different experimental designs have been used to examine the contribution of SR Ca2+ release to the activation of contraction under specific conditions (3, 12, 23, 28, 42, 50). With the use of Ca2+-sensitive fluorescent dyes combined with the patch-clamp technique, the efficiency of the L-type Ca2+ channel in releasing Ca2+ from the SR has been described with a gain factor that represents the ratio of Ca2+ released from the SR and Ca2+ entering through the L-type Ca2+ channel (3, 5, 28, 41). In recent years, the relationship between L-type Ca2+ current (ICa) and SR Ca2+ release has to a large extent been examined using fluorescent dyes and confocal microscopy, because this approach allows a determination of the relationship between one or a few Ca2+ channels and a small cluster of SR Ca2+ release channels (7, 8, 33). Indeed, this approach has shown the Ca2+ spark as an elementary event in the Ca2+-induced Ca2+ release (CICR) from the SR (7, 8, 33), and a single L-type Ca2+ channel has been reported sufficient to elicit a Ca2+ spark (7, 9, 14). Furthermore, the amount of Ca2+ released from a cluster of SR Ca2+ channels is modulated by the probability of channel opening (7). It has also been shown that SR Ca2+ release gives rise to Ca2+ sparks that are triggered by L-type Ca2+ channels at physiologically relevant membrane potentials (9, 33).

Whereas the importance of the Na+/Ca2+ exchanger (NCX) in the regulation of contraction and SR Ca content is well documented (2, 34, 43), a direct triggering of SR Ca2+ release by reverse-mode NCX remains controversial (6, 31, 32, 35, 37, 40). Bulk Ca2+ release from the SR may, however, be modulated by a number of factors that directly or indirectly affect triggering of Ca2+ release from the SR by the L-type Ca2+ channel. This includes the colocalization of NCX and ryanodine receptors (35), the synchronization of individual SR Ca2+ release events (42), or factors that affect the increase in the cytosolic Ca2+ concentration ([Ca2+]) required to trigger SR Ca2+ release (15, 32). Furthermore, the NCX plays a more prominent role in the regulation of cytosolic Ca2+ in the lower vertebrate heart (13, 24, 29, 48, 51). It, therefore, remains possible that reverse-mode NCX can activate Ca2+ release from the SR and contraction under physiologically relevant conditions in these hearts.

Whereas the physiological importance of the SR is generally not well characterized in lower vertebrate species (11, 44), SR Ca2+ release appears to occur in the trout and toad heart (23, 29). In this respect, short stimulation pulses (2-20 ms) give rise to a tail current on repolarization that can be ascribed to Ca2+ release from the SR in rainbow trout atrial myocytes (23). Integration of the caffeine (Caf)-sensitive part of this tail current, therefore, provides a measure of the amount of Ca2+ released from the SR (23). This approach should therefore allow an examination of mechanisms that may modulate the bulk Ca2+ release from the SR. In the present study, it has been used to examine the ability and efficiency of different Ca2+ trigger sources to elicit or modulate SR Ca2+ release, intracellular Ca2+ transients, and contraction.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation

Trout atrial myocytes were obtained by enzymatic digestion of the heart as described previously (25). Briefly, a cannula was inserted into the ventricle, and the heart was rinsed 5-10 min with nominally Ca2+-free Tyrode containing (in mM) 125 NaCl, 1.8 MgCl2, 4 NaHCO3, 0.8 NaH2PO4, 10 HEPES, 5 glucose, and 5 pyruvate. pH was adjusted to 7.4 with NaOH. The heart was then perfused for 40 min at 20°C with a nominally Ca2+-free Tyrode containing 50 µM EGTA and 37.5 µM Ca2+, 0.1 mg/ml collagenase (YAKULT), 0.1 mg/ml trypsin (Sigma), and 0.5 mg/ml BSA. After the first 25 min, the collagenase-containing solution was replaced with a fresh solution. At the end of the digestion, the atrium was cut off the ventricle and transferred to a nominally Ca2+-free Tyrode containing vitamins, amino acids, penicillin, and 1 mg/ml BSA. The tissue was gently agitated, and the supernatant was filtered through a nylon filter (100 µm). Ca2+ was gradually increased to 750 µM, and the cells were stored at 6°C. The cell isolation procedure and the experimental protocols were approved by the local ethical committee (DARP #1017) and follows the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Electrophysiological Measurements

Ionic currents were measured using a software-driven patch-clamp amplifier (EPC-9, HEKA, Germany). After seal formation, the cell was placed in front of one of eight capillaries containing the desired extracellular solution. Standard internal and external solutions were used to eliminate Na+ and K+ currents. The pipette solution contained (in mM) 100 CsCl, 3.1 Na2ATP (3.1 MgATP), 4 MgCl2 (1 MgCl2), 5 Na2-phosphocreatine, 0.42 Li2GTP, 0.025 EGTA, 10 HEPES, and 20 tetraethylammonium (TEA). pH was adjusted to 7.2 with CsOH. The external medium contained (in mM) 107 NaCl, 20 CsCl, 1.8 MgCl2, 4 NaHCO3, 0.8 NaH2PO4, 1.8 CaCl2, 10 HEPES, 5 glucose, and 5 pyruvate. pH was adjusted to 7.4 with NaOH. Na+ current in trout atrial cells could be eliminated with 1 µM TTX. Ionic currents were measured in the ruptured (Figs. 2, 6-8) or the perforated patch configuration with 175-250 µg/ml amphotericin B in the patch pipette (Figs. 3-6). The pipette resistance was 2-5 MOmega . Seal resistance was 2-20 GOmega , and the access resistance was 6.2 ± 0.5 MOmega for ruptured patches. When using amphotericin B, experiments were started when the access resistance had decreased to less than five times the pipette resistance. The average access resistance was 15.9 ± 1.4 MOmega at the beginning of the experiments and decreased to 12.0 ± 1.5 MOmega at the end of the experiments. Cells showing a sudden drop in access resistance were discarded.

Experimental Protocols

The cell was stimulated continuously with a 200-ms depolarization from -80 to 0 mV every 5 s. This protocol allowed evaluation of ICa, tail current, and Caf-induced NCX current (INCX) under steady-state conditions. The basic protocol used to examine CICR from the SR consisted of a train of short stimulation pulses (pulse duration varied between 2 and 20 ms as specified in figures). After this, the cell was kept at -80 mV and exposed briefly to 10 mM Caf to deplete the SR of Ca2+. The stimulation protocol was then repeated and followed by a second CAF exposure (see Fig. 1). To avoid the influence of the order of the different depolarization steps, the stimulation protocol was repeated in the reverse order in some experiments. Furthermore, the short depolarization protocols were preceded by 20 regular depolarizations to assure that the cells and their SR Ca2+ content were near steady state.


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  		Fig. 1.   A and B: stimulation protocols used to elicit sarcoplasmic reticulum (SR) Ca2+ release. A: duration of a depolarization to 0 mV was increased from 2 to 20 ms in 2-ms increments. SR Ca2+ content was then cleared with 10 mM caffeine (Caf), and the short depolarizations protocol was repeated. B: cells were depolarized for 10 ms to different membrane potentials from -50 to +150 mV with increments of 40 mV or from -50 to +50 mV with increments of 20 mV. SR Ca2+ content was then cleared, and the 10-ms depolarization protocol was repeated. The second Caf application in A and B was used to evaluate the Ca2+ accumulation in the SR during the second short depolarizations protocol. In general, the SR Ca2+ content after the second Caf application was <10% of the first Caf application.

The time integral of the net Caf-sensitive tail current (Con-Caf) after a short depolarization allows quantification of the Ca2+ released from the SR triggered by the depolarization. Furthermore, the time integral of the Ca2+ current during the short depolarization gives a measure of the trigger Ca2+, and the ratio of Ca2+ released from the SR and the trigger Ca2+ gives a gain factor for the CICR. Finally, the Caf-induced INCX allows determination of the SR Ca2+ content before and after the first Caf exposure as described previously (23, 46, 47). Assuming that Ca2+ release is constant during the shortest stimulation pulse, the average Ca2+ release rate from the SR can be calculated by division of the total Ca2+ released with the time Ca2+ is released.

Because capacitive currents may interfere with ICa during very short stimulation pulses, the Ca2+ carried by ICa during a 2- and a 4-ms depolarization was obtained by assuming that Ca2+ entry increased linearly with time during the first 4 ms. Thus the net increase during the first 2 ms was obtained by subtracting the raw value obtained with a 2-ms depolarization from the raw value with a 4-ms depolarization. The value for a 4-ms depolarization was then assumed to be twice that value. For conversion of the time integrals of Ca2+ carrying currents to Ca2+ concentrations, a conversion factor of 15.4 pF/pl was used (25) and the Ca2+ accessible cell volume was considered to equal the nonmitochondrial cell volume, which in trout corresponds to 55% of the total cell volume (48).

Measurement of Intracellular Ca2+

The intracellular Ca2+ concentration was measured with the Ca2+ indicator fluo-3. Cells were incubated with 5 µM fluo-3 AM for 10-15 min at room temperature. After incubation, cells were washed and left for 30 min at room temperature before experiments were started. Fluo-3 was excited at 488 nM, and fluorescence emission was measured using a 530-nm bandpass filter. The bandpass width was ±20 nm. A sampling interval of 10 ms was used to achieve a reasonable signal-to-noise ratio. The longer sampling interval was necessary as the cell volume of trout atrial myocytes is 2-4 pl, which is about ten times less than the cell volume of mammalian cardiac myocytes. Fluorescence signals were not filtered or smoothed. Calibration of the fluo-3 signal was done by subtraction of background fluorescence and measurement of the maximal fluorescence at the end of each experiment. As long depolarizations lead to a tonic elevation of the intracellular [Ca2+] and contraction in trout cardiac myocytes (22, 26), the maximal fluorescence was obtained by depolarizing the cell for 3 s to increasingly more positive potentials until an irreversible cell contracture was achieved. The fluo-3 signal measured at irreversible cell contracture was taken as the maximal fluorescence. The intracellular [Ca2+] was then calculated from [Ca2+]i = F · Kd/(Fmax - F), where F is the measured net fluorescence, Fmax is the maximal net fluorescence, and Kd is the dissociation constant for Ca2+ for fluo-3 (46). A Kd for fluo-3 in cells of 1,100 nM (19) was used in the present study.

Measurement of Contraction

Cell shortening was recorded on videotape and analyzed offline. Cell shortening was normalized to the resting cell length to eliminate variations due to differences in cell length. In experiments with short depolarizations, the ability to elicit a visible cell shortening was determined visually on the video monitor.

Statistics

Statistical significance of the results was tested using ANOVA or Student's t-test for paired or unpaired samples.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Quantification of Ca2+ Release and Ca2+ Flux From the SR

A stimulation protocol with consecutive brief depolarizations (2- to 20-ms duration) of the cell from -80 to 0 mV was used to elicit CICR under non-steady-state conditions, and Caf was subsequently used to clear the SR Ca2+ content (see Fig. 1). The three panels in Fig. 2A show traces of the currents elicited by repolarizing the cell to -80 mV after 2-, 10-, and 20-ms depolarizations to 0 mV. Traces obtained before and after exposure to Caf are superimposed. Figure 2B shows the dependency of the amount of Ca2+ carried by the Caf-sensitive tail current on the duration of the preceding depolarization. Presumably, the decline in the net tail current with a depolarization pulse duration >10 ms is due to a decline in the Ca2+ release from the SR and Ca2+ reuptake and/or extrusion during the depolarization. The slope of the regression line through data points with depolarizations longer than 10 ms (solid line in Fig. 2B) should therefore correspond to the gross rate of Ca2+ removal from the cytosol. The slope of this regression line corresponds to a rate of removal of 33 amol · pF-1 · s-1 or 0.5 mM/s, and the dashed line represents the estimated Ca2+ removal from the cytosol as a function of the duration of the depolarization. The total SR Ca2+ release induced by the short depolarization can then be obtained by adding the Ca2+ removed from the cytosol to the Ca2+ carried by the Caf-sensitive tail current (Fig. 2B). Because a maximal SR Ca2+ release was reached with a 10-ms depolarization, this duration was used in subsequent experiments, unless otherwise stated.


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  		Fig. 2.   A-C: effect of the pulse duration on SR Ca2+ release. A: current traces elicited by a depolarization to 0 mV for 2, 10, and 20 ms followed by repolarization to -80 mV before () or immediately after (open circle ) clearing the SR Ca2+ content with 10 mM Caf. Time scale is 250 ms. B: total Ca2+ release from the SR () was obtained by adding the Ca2+ removed during depolarization (dashed line) to the Ca2+ carried by the Caf-sensitive tail current (open circle ). C: rate of bulk Ca2+ release in consecutive 2-ms intervals after onset of depolarization.

The total Ca2+ release is shown as a function of the duration of the preceding depolarization in Fig. 2B. Furthermore, the amount of Ca2+ released in consecutive 2-ms intervals was obtained by subtraction of the total Ca2+ release with a given depolarization from the subsequent depolarization. This then gives an estimate of the SR Ca2+ release rate as a function of the time elapsed after onset of depolarization. As seen from Fig. 2C, the maximal Ca2+ release rate from the SR was 4.3 ± 0.7 mM/s (n = 14) with a 2-ms depolarization, and the rate declined rapidly after depolarizing the cell.

Dependency of SR Ca2+ Release on Membrane Potential

To examine the dependency of SR Ca2+ release on the membrane potential, cells were depolarized from -80 mV to -50, -10, 30, 70, 110, and 150 mV for 10 ms. Figure 3 shows average intracellular Ca2+ transients (Fig. 3A) and ionic currents (Fig. 3B). Average traces in Fig. 3, A and B, were recorded before and immediately after clearing the SR Ca2+ content with a rapid Caf application. Figure 3C shows the running time integral of the Caf-sensitive part of the tail currents shown in Fig. 3B. The total amount of Ca2+ carried by the Caf-sensitive tail current 500 ms after repolarization to -80 mV was taken as a measure of the amount of Ca2+ released from the SR. Notice that a 10-ms depolarization elicited a clear Ca2+ transient at all membrane potentials above -50 mV when the SR was loaded, whereas Ca2+ transients elicited after clearance of the SR Ca2+ content were abolished or small.


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  		Fig. 3.   A-C: effect of the membrane potential on SR Ca2+ release. A: average Ca2+ transients elicited by a 10-ms depolarization to the membrane potential given above traces. Transients were recorded before (black line) or after (gray line) clearance of the SR Ca2+ content with 10 mM Caf. B: representative ionic currents recorded simultaneously with the Ca2+ transients shown in A. C: running time integral of the Caf-sensitive tail current elicited on repolarization to -80 mV in B. Time scale bars represent 500 ms in A and 300 ms in B and C.

Figure 4A shows the relationship between membrane potential during depolarization and the net increase in intracellular Ca2+ or the amount of Ca2+ released from the SR. Figure 4B summarizes the relationship between the SR Ca2+ release as obtained in Fig. 3C and the peak Ca2+ transient as obtained from Fig. 3A (n = 9). Linear regression gave a slope of 0.0099, corresponding to a buffering power of 101. 


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  		Fig. 4.   A-B: relationship between the magnitudes of SR Ca2+ release and the intracellular Ca2+ transient. A: relationship between membrane potential and the SR Ca2+ release (open circle ) or the amplitude of the Caf-sensitive Ca2+ transient elicited by a 10-ms depolarization (). B: relationship between the amount of Ca2+ released from the SR by a 10-ms depolarization and the amplitude of the resulting intracellular Ca2+ transient.

Triggering of Ca2+ Release From the SR by ICa and INCX

Figures 3 and 4 show that Ca2+ release from the SR occurs at membrane potentials near or above the reversal potential for Ca2+, where Ca2+ influx through ICa is unlikely to trigger SR Ca2+ release. Inasmuch as 10-ms depolarizations were used to elicit SR Ca2+ release, it is, however, possible that Ca2+ release at +110 and +150 mV is induced by Ca2+ entry through L-type Ca2+ channels on repolarization to -80 mV. Therefore, currents elicited by 10-ms depolarizations to -50, -10, 30, 70, 110, and 150 mV were recorded in the absence or the presence of 100 µM CdCl2 (Fig. 5A). CdCl2 abolished ICa, but as shown in Fig. 5A, it did not abolish the Caf-sensitive tail current. Figure 5B shows the average Ca2+ carried by the tail currents in the absence and the presence of 100 µM CdCl2. Notice that there was a significant Ca2+ release from the SR at membrane potentials near and above the reversal potential for Ca2+ that was not affected by CdCl2. Indeed, Ca2+ release increased monophasically with the membrane potential in the presence of CdCl2. In agreement with this, inclusion of 10 µM nifedipine (Nif) in the external medium had no effect on the Caf-sensitive tail current between 70 and 150 mV (data not shown). In the presence of 10 µM Nif or 100 µM CdCl2, the amount of Ca2+ carried by the Caf-sensitive tail current after a 10-ms depolarization to 150 mV was 1.26 ± 0.18 amol/pF (n = 14), and the corresponding SR Ca2+ load was 27.1 ± 2.6 amol/pF. Figure 5C shows the Ca2+ carried by the tail currents in the physiological range of membrane potentials with or without 100 µM CdCl2 in the extracellular medium. Notice that at 10 mV, which is near the plateau of the action potential, CdCl2 reduced the Caf-sensitive tail current to 40 ± 17% of control.


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  		Fig. 5.   A-C: dependency of SR Ca2+ release on membrane potential and L-type Ca2+ current. A: representative Caf-sensitive tail currents elicited by a preceding 10-ms depolarization to the membrane potentials given above the traces. Time scale is 250 ms. Currents were recorded without (open circle ) or with () 100 µM CdCl2. B: relationship between membrane potential and SR Ca2+ release without or with CdCl2 (n = 7). C: same relationship for physiologically relevant membrane potentials (n = 4).

Dependency of SR Ca2+ Release on INa and Intracellular Na+

The effects of the pipette [Na+] on SR Ca2+ release was examined by comparing the Caf-sensitive tail currents elicited by a brief depolarization to different membrane potentials with 10 or 16 mM Na+ in the patch pipette. Figure 6A summarizes the dependency of the Ca2+ released from the SR on the membrane potential of the preceding depolarization obtained from five cells with 10 mM Na+ and seven cells with 16 mM Na+ in the patch pipette. Figure 6B shows the corresponding relationship between the membrane potential during depolarization and cell shortening. The pipette [Na+] significantly affected the relationship between membrane potential and SR Ca2+ release (P < 0.001) or contraction (P < 0.001). The effects of the pipette [Na+] on SR Ca2+ release and contraction were, however, opposite. Thus 16 mM pipette Na+ significantly increased SR Ca2+ release, whereas cell shortening was significantly reduced at membrane potentials above -30 mV. Importantly, however, the average SR Ca2+ content (32.5 ± 3.6 amol/pF) with 10 mM pipette [Na+] was not significantly different from that with 16 mM Na+ (33.7 ± 4.4 amol/pF).


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  		Fig. 6.   A and B: dependency of SR Ca2+ release on membrane potential and pipette Na+ concentration ([Na+]). A: relationship between the amount of Ca2+ released from the SR and membrane potential. B: relationship between cell shortening and membrane potential during the depolarization. Values in A and B were obtained with 10 mM Na+ (filled symbols; n = 5) or 16 mM Na+ (open symbols; n = 7) in the patch pipette.

To examine if INa is able to induce or enhance reverse mode NCX-induced SR Ca2+ release, tail currents after brief depolarizations were recorded in the absence or the presence of TTX. In the absence of TTX (Fig. 7A), INa was activated at -50 mV and strongly activated at -30 mV. The corresponding tail currents were, however, small or absent. In contrast, a 10-ms depolarization from -80 to 10 mV elicited both INa and ICa and a strong tail current. Figure 7B shows superimposed TTX-sensitive current traces elicited by a 10-ms depolarization from -80 to -50, -30 and 10 mV. There was no TTX-sensitive tail current at any of these membrane potentials, and no significant TTX sensitive-tail current was observed at any membrane potential between -50 and +50 mV (n = 6). The results in Fig. 7, A and B, were obtained with 10 mM Na+ in the patch pipette. In contrast, Fig. 7C shows TTX-sensitive tail currents recorded at -30 and 10 mV with 16 mM Na+ in the patch pipette. Figure 7D summarizes the voltage dependency of the Ca2+ carried by the TTX-sensitive tail current with 16 mM Na+ in the patch pipette (n = 6).


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  		Fig. 7.   A-D: effect of Na current (INa) on SR Ca2+ release. A: representative current traces elicited by a 10-ms depolarization to -50, -30, and 10 mV in the absence of TTX with 10 mM pipette [Na+]. B and C: superimposed TTX-sensitive current traces recorded with 10 and 16 mM Na+ in the patch pipette, respectively. Time scale in A-C is 100 ms. D: dependency of the amount of Ca2+ carried by the TTX-sensitive tail current on the membrane potential with 16 mM Na+ in the patch pipette (n = 6).

Gain of Ca2+ Release From the SR Elicited by ICa and INCX

To examine the gain of SR Ca2+ release induced by either ICa or reverse-mode INCX, ICa was first inhibited with Nif to separate the SR Ca2+ release induced by ICa and reverse mode NCX. Figure 8A shows superimposed current traces elicited by a 2 ms depolarization to 0 mV in the absence (Con) or the presence of 5 or 10 µM Nif. Notice that the tail current after a 2-ms depolarization was significantly reduced, but not abolished, by Nif. Indeed, a weak contraction of the cell was elicited by a 2-ms depolarization both in the presence and the absence of Nif in all five cells examined (not shown). Furthermore, the difference between the current traces with a short depolarization in the absence and presence of Nif should give the ICa during depolarization and the net tail current should give the SR Ca2+ release triggered by ICa. Figure 8B shows the net current trace for a 20-ms depolarization. Figure 8C summarizes the average Ca2+ carried by ICa and the net tail current (Con-Nif) as a function of the duration of the depolarization (n = 5). The Nif-sensitive tail current corresponded to 61 ± 14% of the total tail current. The ratio of SR Ca2+ release to ICa Ca2+ influx gave an average gain of 16.0 ± 4.7 with a 2-ms depolarization.


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  		Fig. 8.   A-F: effect of nifedipine and NiCl2 on SR Ca2+ release. A: representative current traces elicited by a 2-ms depolarization to 0 mV in the absence (Con) or the presence (Nif) of 10 µM nifedipine. B: net Nif-sensitive current elicited by a 20-ms depolarization to 0 mV. C: amount of Ca2+ carried by the Nif-sensitive current during depolarization (ICa) and repolarization (SR). D: current traces elicited by a 2-ms depolarization to 0 mV in the presence of Nif without (Nif) or with (NiCl2) 10 mM NiCl2. E: net Nif-insensitive but NiCl2-sensitive current elicited by a 20-ms depolarization to 0 mV. F: amount of Ca2+ carried by the Nif-insensitive but NiCl2-sensitive current during depolarization (INCX) and repolarization (SR). Values were obtained from 5 cells.

Because a saturating dose of Nif (5 or 10 µM) was unable to abolish contraction and tail currents after a short depolarization, 10 mM NiCl2 was added to Nif to inhibit NCX. Figure 8D shows superimposed current traces obtained with a 2-ms depolarization to 0 mV in the presence of Nif before (Nif) and after addition of 10 mM NiCl2. NiCl2 abolished contractions in all experiments (not shown). The difference between the current traces represents the Ca2+ carried by the NCX during depolarization and repolarization. Figure 8E shows the net current for a 20-ms depolarization. Figure 8F shows the Ca2+ carried by the NCX during depolarization and repolarization as a function of the length of the depolarization (n = 5). The gain for the NCX-induced Ca2+ release after a 2-ms depolarization was 14.8 ± 7.1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Quantification and Characterization of SR Ca2+ Release

The present work has examined mechanisms involved in triggering Ca2+ release from the SR in trout atrial myocytes through measurements of intracellular Ca2+ transients and quantification of Caf-sensitive tail currents elicited by short stimulation pulses. The quantification of Ca2+ release from the SR using this approach is not useful in mammalian myocytes, as the NCX in these cells cannot compete as effectively with SR Ca2+ uptake as it can in trout myocytes (2, 26).

It may be appropriate to justify the assumptions on which this approach relies. Thus the following is assumed. 1) The Caf-sensitive part of the tail current elicited on repolarization to -80 mV is exclusively due to extrusion of Ca2+ released from the SR by the NCX. 2) Rapid Caf applications are able to effectively clear the SR Ca2+ content, and the SR does not reload significantly during the execution of the stimulation protocol after Caf exposure. 3) Repolarization to -80 mV after a short depolarization does not give rise to any significant Ca2+ release from the SR caused by Ca2+ influx through noninactivated L-type Ca2+ channels.

With respect to the first assumption, it is likely that the Caf-sensitive tail current is exclusively due to Ca2+ extrusion by the NCX because of the following. 1) Rapid Caf application in Na+- and Ca2+-free solution activates a strong cell contracture but no ionic current in trout cardiac myocytes while reintroduction of extracellular Na+ activates an inward ionic current and relaxes the cell contracture (22). If other Ca2+-activated currents were important, these currents should be detected during Caf exposure, as this causes a much larger increase in the intracellular [Ca2+] than that elicited by a 10-ms depolarization. 2) Potassium currents were eliminated by omission of potassium in the pipette and bath solutions. With the perforated patch configuration, some potassium could be present in the cell at the beginning of the experiments despite the pipette solution used. A Ca2+-activated K+ current would, however, be expected to be small at -80 mV. 3) Inclusion of 10 or 30 µM of the Cl--current inhibitor SITS had no effect on the ionic current in trout atrial myocytes (unpublished observations).

The amount of Ca2+ released from the SR could, however, be underestimated because substitution of K+ with Cs+ has been reported to reduce both intracellular Ca2+ transients and SR Ca2+ content (18) and slow SR Ca2+ uptake and release (30). Although these effects of Cs+ could affect the present quantification of SR Ca2+ release, Cs+ is not expected to have selective effects on reverse-mode NCX or ICa-induced Ca2+ release from the SR if it acts through inhibition of SR K+ channels (30).

With respect to the second assumption, we previously showed that reapplication of Caf after clearance of the SR Ca2+ content does not elicit an inward current (22). Furthermore, the Ca2+ released from the SR by the second Caf exposure after a train of short depolarizations was typically <10% of that of the first Caf application (see Fig. 1). In agreement with this, Fig. 3A shows that a 10-ms depolarization is unable to cause a significant increase in the intracellular [Ca2+] after the SR Ca2+ has been depleted by the first Caf exposure.

As shown in Fig. 4, the Caf-sensitive tail currents elicited at membrane potentials near or above the reversal potential for Ca2+ are not diminished in the presence of 100 µM CdCl2, whereas ICa is abolished at all membrane potentials. It is therefore unlikely that the recorded Caf-sensitive tail currents are due to triggering of Ca2+ release from the SR by Ca2+ entry through noninactivated L-type Ca2+ channels on repolarization.

Thus it seems reasonable to use the Caf-sensitive tail current elicited by a brief depolarization as a measure of the Ca2+ released from the SR. To obtain a measure of the total Ca2+ released from the SR, it was assumed that the decline in the Caf-sensitive tail current with depolarizations lasting 12-20 ms is due to Ca2+ removal from the cytosol during the depolarization (see Fig. 2). This gave a gross rate of Ca2+ removal from the cytosol of 33 amol · pF-1 · s-1 or 0.5 mM/s during the depolarization, which is about three times the maximal SR Ca2+ uptake rate reported in trout ventricular myocytes at room temperature (22). This suggests that the SR Ca2+ uptake is faster in atrial myocytes and/or that Ca2+ extrusion by the NCX accounts for the difference between the two values. The latter seems plausible, because it was recently found that SR Ca2+ uptake accounts for ~40% of the total Ca2+ removal from the cytosol in trout atrial myocytes (24, 26).

The total Ca2+ released from the SR was obtained by the addition of the Ca2+ removed from the cytosol during depolarization to the Ca2+ carried by the Caf-sensitive tail current. Division of the total SR Ca2+ release by the period of time when Ca2+ release is activated gave the average rate of SR Ca2+ release. The maximal measurable rate of release obtained with this approach was 4.3 ± 0.7 mM/s. This value represents the global rate of SR Ca2+ release, and, contrary to measurements based on fluorescent Ca2+ indicators, it does not rely on assumptions about passive Ca2+ buffering. It is nevertheless similar to values reported in mammalian cells using approaches that are based on measurements of changes in the free [Ca2+] (5, 41, 42). Although the gain factor may vary with specific experimental conditions, SR Ca2+ load and the parameters used to calculate the gain, a value ~15 for calcium-induced calcium release from the SR is within the range of values reported in mammalian cells using either total (39) or peak Ca2+ fluxes (33). In agreement with data from the mammalian heart (16), Ca2+ release from the SR also ceased rapidly after the onset of depolarization and appeared to be complete within 10 ms (see Fig. 2). Furthermore, the gross Ca2+ removal from the cytosol during a 10-ms depolarization amounts to 0.33 amol/pF or 26% of the total release. This value is also within the range of values reported for active Ca2+ buffering by the SR in mammalian cardiac myocytes (3, 27, 28).

Ca2+ Sources Triggering Ca2+ Release from the SR

ICa-induced Ca2+ release from the SR. Both electrophysiological work and studies of the spatial arrangement of L-type Ca2+ channels, NCX, and ryanodine receptors favor a greater efficiency of ICa in triggering Ca2+ release in mammalian cardiac myocytes (4, 35, 37, 38, 40). Furthermore, the Ca2+ flux rate for the L-type Ca2+ channel is ~1,000 times greater than that of the NCX (4, 20, 21), making it physically difficult to arrange a large number of exchangers rather than a single or a few L-type Ca2+ channels in the junctional cleft. The present results confirm the importance of the ICa in the activation of Ca2+ release from the SR in trout atrial myocytes. Indeed, with a pipette [Na+] of 10 mM, the relationship between membrane potential during a 10-ms depolarization and the Caf-sensitive tail current or cell shortening was similar to the bell-shaped relationship between voltage and ICa amplitude. Furthermore, INa was unable to trigger or enhance SR Ca2+ release. This agrees with previous data from mammalian cardiac myocytes (35-38, 40).

Reverse-mode NCX-induced Ca2+ release from the SR. In contrast to this, INa enhanced SR Ca2+ release with 16 mM Na+ in the patch pipette (see Fig. 7). This agrees with the data of Refs. 31 and 32. Furthermore, a large SR Ca2+ release at -30 mV caused a weak cell shortening, whereas a similar SR Ca2+ release at +30 mV caused a strong cell shortening (see Fig. 6, B and C). In addition, the similar gain factors obtained for Nif-insensitive but NiCl2-sensitive SR Ca2+ release and the Nif-sensitive SR Ca2+ release suggest that reverse-mode NCX may be as efficient in eliciting SR Ca2+ release as ICa. Finally, 10-ms depolarizations to membrane potentials near or above the reversal potential for Ca2+ elicited both intracellular Ca2+ transients, cell shortening, and a significant SR Ca2+ release that persisted in the presence of CdCl2 or Nif. This excludes a possible triggering of SR Ca2+ release by ICa tail currents elicited on repolarization from these positive membrane potentials to -80 mV (see Fig. 5).

These results are different from results obtained in mammalian cardiac myocytes both with respect to the gain (38, 40) and the time course of NCX-induced SR Ca2+ release (33, 40). Because triggering of Ca2+ release is more efficient at higher SR Ca2+ loads and shows hypersensitivity when the SR is saturated with Ca2+ (3, 18, 23), it was important to verify that the SR Ca2+ release was obtained at physiologically relevant SR Ca2+ loads. This was assured by discarding Ca2+-overloaded cells with spontaneous inward currents and contractions at -80 mV. More cells were discarded with 16 mM pipette [Na+] than with 10 mM pipette [Na+], and this may account for the similar SR Ca2+ loads with the two pipette [Na+] in this study. Furthermore, the average SR Ca2+ loads used in this study were similar to that obtained by continuously stimulating myocytes with 200-ms depolarizations (22-24).

Modulation of SR Ca2+ Release and Contraction by the NCX

It has recently been reported (15) and previously suggested (32) that the NCX does not directly activate Ca2+ release from the SR, but determines the gain of the ICa-induced Ca2+ release through a regulation of the [Ca2+] in the microdomain that includes the SR Ca2+ release channels. Furthermore, there is evidence in favor of an enhancement of the NCX activity by ICa acting on the catalytic Ca binding site of the NCX (49). The observed enhancement of the Caf-sensitive tail current at -30 and -10 mV with a change in the pipette [Na+] from 10 to 16 mM (Fig. 6) does support a role of the NCX activity in regulating the gain of Ca2+-induced Ca2+ release. The SR Ca2+ release at -30 mV does not, however, result in a corresponding enhancement of cell shortening. Furthermore, 10-ms depolarizations to membrane potentials between -30 and 50 mV induce an SR Ca2+ release that is significantly larger with 16 than with 10 mM pipette [Na+], whereas the corresponding cell shortening is significantly smaller (see Fig. 6). Therefore, the main effect of impeding Ca2+ extrusion by the NCX or favoring Ca2+ entry through the NCX seems neither to be a potentiation of ICa-induced Ca2+ release from the SR nor an enhancement of the NCX activity through interaction of ICa with the catalytic Ca2+-binding site of the NCX.

Thus NCX-induced SR Ca2+ release seems more likely to account for these results. However, if a tight coupling between the sarcolemmal Ca2+ transporting protein and the ryanodine receptor is required for calcium-induced calcium release, the turnover rate of the exchanger would also have to be larger in trout myocytes for reverse-mode NCX to induce Ca2+ release. In this respect, maximal NCX velocities in sarcolemmal vesicles from dog and trout ventricle were similar at 21°C (45). The calculated maximal NCX rate for trout using isolated myocytes was, however, in the upper range of mammalian values (21, 26) and at physiological intracellular [Ca2+], the NCX rate was 10-20 times faster than mammalian values (2, 26). Thus it is conceivable that the threshold for NCX-induced SR Ca2+ release may be reached faster and require fewer exchangers in trout than in mammalian cardiac myocytes.

Model for Ca2+-Induced Ca2+ Release in Trout Atrial Myocytes

To explain the present experimental results, a model for CICR is proposed where both ICa and reverse-mode NCX can trigger SR Ca2+ release. The model assumes that both L-type Ca2+ channels and the NCX are closely associated with ryanodine receptors, but not with the same receptors.

The implications of this model are that Ca2+ release from the SR triggered by ICa can occur as described in mammals. Additionally, Ca2+ release from the SR triggered by reverse-mode NCX will, due to the proximity of the ryanodine receptor and the Na+/Ca2+ exchangers that trigger the Ca2+ release, reverse the direction of these exchangers during the Ca2+ release. This results in extrusion of part of the released Ca2+ across the sarcolemma by the Na+/Ca2+ exchangers before it reaches the myofilaments, thereby reducing the efficiency of the NCX-induced Ca2+ release in raising the bulk cytosolic [Ca2+] and activating cell shortening. Indeed, detection of reverse-mode NCX-induced Ca2+ release with fluorescent Ca2+ indicators that measures bulk cellular Ca2+ will underestimate NCX-induced Ca2+ release because of this phenomenon.

At 10 mV, the direction of the NCX will change from Ca2+ entry to Ca2+ extrusion if SR Ca2+ release increases the local [Ca2+] above 1.1 and 5.4 µM with a pipette [Na+] of 10 and 16 mM, respectively. Assuming that local SR Ca2+ release takes place in the space that is not taken up by mitochondria or myofibrils (15% of a total cell volume of 3 pl), a Ca2+ release of 31 and 42 amol Ca2+ with 10 and 16 mM pipette [Na+] gives a local [Ca2+] of 103 and 139 µM, respectively. This is 63 and 17 times more than the [Ca2+] required to reverse the NCX to the forward mode, suggesting that NCX-induced SR Ca2+ release does reverse the NCX to the forward mode at physiologically relevant membrane potentials.

The efficiency of the Ca2+ released from the SR in raising bulk Ca2+ and activating contraction will, therefore, depend on the fraction of Ca2+ release that is induced by reverse-mode NCX. Thus the same amount of Ca2+ released from the SR is expected to result in a smaller contraction with 16 than with 10 mM pipette Na+ (as observed in Fig. 6), because it favors reverse-mode NCX-induced Ca2+ release from the SR. This phenomenon is analogous to a reduction of the Ca2+ transient by active SR Ca2+ buffering during SR Ca2+ release (2, 27). Such a reversal of the NCX by SR Ca2+ release may not be unique to trout atrial myocytes, because overlapping distributions of NCX and ryanodine receptors have been described in adult mammalian cardiac myocytes (17). The phenomenon is, however, expected to be more pronounced in trout because of the prominent NCX (24, 26) and lower ICa density (25) in these cells.

In conclusion, the present results show that both ICa and reverse-mode NCX can trigger Ca2+ release from the SR and the gain of this release is similar for the two trigger sources. The relative importance of the two trigger sources depends critically on the intracellular [Na+] and membrane potential. The upper estimate for NCX-induced SR Ca2+ release at 10 mV is 40% of the total Ca2+ release, whereas its contribution during the more positive action potential peak should be larger. Furthermore, reverse-mode NCX-induced Ca2+ release from the SR is less efficient in elevating the intracellular [Ca2+] and activating contraction than ICa-induced Ca2+ release. This may occur because a larger fraction of the Ca2+ released from the SR by reverse-mode NCX is extruded from the cell before it reaches the myofilaments.


    ACKNOWLEDGEMENTS

This work was supported by a grants from The Spanish Ministry of Education and Culture (Incorporación de Doctores) to L. Hove-Madsen and Generalitat de Catalunya (FPI grant) to A. Llach and (MAR97-402-C02) to L. Tort, and the National Sciences and Engineering Research Council of Canada to G. F. Tibbits.


    FOOTNOTES

Address for reprint requests and other correspondence: L. Hove-Madsen, Laboratorio de Fisiología Celular, Cardiología, Hospital de Sant Pau, 08025 Barcelona, Spain (E-mail: lhove{at}hsp.santpau.es).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 16, 2003;10.1152/ajpregu.00404.2002

Received 8 July 2002; accepted in final form 7 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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