Ca2+ current and Ca2+ transients under action potential clamp in guinea pig ventricular myocytes JORGE ARREOLA, ROBERT T. DIRKSEN, RU-CHI SHIEH, DANIEL J. WILLIFORD, AND SHEY-SHING SHEU of Pharmacology, School of Medicine and Dentistry, Department University of Rochester, Rochester, New York 14642
ARREOLA, JORGE, DANIEL J. WILLIFORD,
ROBERT T. DIRKSEN, Ru-CHI SHIEH, AND SHEY-SHING SHEU. Ca2+ current
and Ca”’ transients under action potential clamp in guinea pig ventricular rnyocytes. Am. J. Physiol. 261 (Cell Physiol. 30): C393-C397, 1991.-Precise characterization of the magnitude and kinetics of transsarcolemmal Ca”’ influx during an action potential (AP) is essential for a complete understanding of excitation-contraction coupling in heart. Using a voltage-clamp protocol that simulated a physiological AP (AP clamp), we characterized the properties of the Ca”’ current (Ica) in guinea pig ventricular myocytes. The AP-generated Ioa showed a complex time course that was different from Ica generated by a square pulse. I (:a activated rapidly during the upstroke of the AP and then partially inactivated during the plateau. The fast component of Ica reached a peak value of -7.6 t 1.0 pA/pF at 2.40 & 0.30 ms after depolarization, followed by a slow component with a peak value of -2.9 t 0.4 pA/pF during the plateau. It-21 generated by an AP was composed of both L- and T-type Ca” channels. T-type Ca2’ current contributed to the fast component of Ica and L-type Ca”’ current contributed to both fast and slow components of Ica. Activation of ,&adrenoceptors enhanced Ica with a maximal effect lasting throughout the entire plateau of the AP. Measurements of cytosolic Ca2’ transients using fura- indicated that the Ica was responsible reticulum. for triggering Ca”’ release from the sarcoplasmic The AP clamp provides a new approach for investigation of the relationship between Ica and Ca”’ transients under more physiological conditions. excitation-contraction nels; calcium-induced tors; voltage clamp
coupling; L- and T-type calcium chancalcium release; heart cells; P-adrenocep-
THE FIRST STEP in excitation-contraction (EC) coupling of heart is the activation of Ca2’ channels by membrane depolarization (9,28). Ca2+current (I&, flowing through L-type Ca2’ channels, triggers Ca2’ release from sarcoplasmic reticulum (SR) and results in muscle contraction (1, 5, 9, 15, 20, 23, 28). Sequestration of Ca2+ back into the SR and efflux across the sarcolemma restores intracellular Ca2+ concentration ([ Ca2+]i) to the resting level, thereby promoting relaxation. The amplitude of Ica and the Ca2’ transient are closely related (1, 3, 5, 31). However, it is unclear whether Ica evoked by membrane depolarization is the only link to the Ca”’ transient (8,20). Several studies have examined the relationship between Ica and Ca2’ transient in single myocytes under voltage clamp using square-pulse proto0363-6143/91
$1.50 Copyright
cols (1, 3, 5, 8, 13, 30). These studies found that the voltage dependence of Ica correlated well with the relationship between the peak amplitude of the Ca2+ transients and membrane potential and that the peak amplitude of the Ca2’ transients was not altered by the duration of voltage-clamp depolarization as long as the same peak Ica was achieved. However, others have found that the voltage dependence of the depolarization-induced increase in [Ca2+]; was not the same as that of the Ica (8, 13). It has been suggested that SR Ca2’ release is proportional to lca when it is small and becomes saturated after a sufficient magnitude of lca is achieved (13). Taken together, these results suggest that, whereas the SR Ca2’ release is dependent on Ica, the membrane potential may also alter SR Ca2+ release. In mammalian heart, L-type and T-type Ca2+channels have been identified. These channels differ in their voltage dependence, pharmacology, kinetics, and conductance (2, 22, 25, 32). L-type channels activate at less negative potentials (positive to -30 mV), whereas their inactivation appears to be both voltage and Ca”+ dependent (6, 7, 26). On the other hand, T-type channels activate at more negative potentials (positive to -50 mV) and inactivate solely by a voltage-dependent process. Although the role of T-type Ica in excitation-contraction coupling is unknown, it is clear that L-type lca contributes to the action potential (AP) plateau (28) and is essential for the Ca2+-induced Ca2’ release from SR (15, 23 The properties of L-type Ca2+current and its relationship to the cytosolic Ca2+ transient have been investigated under voltage-clamp conditions using depolarizing square pulses (1, 3, 5, 8). However, Ica during an AP could behave differently (11, 12). For instance, simulation of Ica during an AP predicts rapid activation followed by a partial inactivation and reactivation such that a secondary inward “hump” of Ica occurs during the latter phase of the AP (6, 10). Attempts to test this prediction have been performed by measuring D 600-sensitive currents in single myocytes voltage clamped with their own AP waveform (11, 12), and by simultaneously recording an AP and Na’ conductance through Ca2’ channels from the same cell by using two separate electrodes (21). No study has been reported regarding the relationship between Ica during an AP and Ca2+ transients. To obtain direct information concerning the magnitude, kinetics, and the type(s) of Ca2+ channel(s) comprising Ica and its
0 1991 the American
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Society
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possible relationship to Ca2+ transients during physiological electrical activity in heart, we performed experiments in cardiomyocytes under voltage clamp using the AP as a stimulus waveform. METHODS
Single myocytes were isolated from guinea pig ventricular muscles by an enzymatic procedure described previously (29). Briefly, hearts were isolated and retrogradely perfused with a Ca2’- and antibiotic-free Joklik medium (minimal essential medium modified for suspension cultures; GIBCO, Grand Island, NY) followed by a similar solution containing 0.5 mg/ml collagenase, 0.25 mg/ml protease, 1 mg/ml albumin, and 50 PM CaC12. The heart was minced and cells were dissociated by gentle agitation in the enzyme solution. Isolated cells were stored at room temperature in Joklik medium containing 100 mg/ml albumin, 1 mM CaC12, and 10 mM glucose at pH 7.4. Ica was recorded using the whole cell voltage-clamp technique (17) with a List EPC-7 amplifier using both AP and square-pulse voltage protocols. A representative AP (Fig. 1A) was recorded from a guinea pig papillary muscle at 37°C and used in all experiments as the stimulus waveform to activate Ca2+ channels at a frequency of 0.1 Hz. The resting membrane potential, and therefore the holding potential, was -85 mV. The overshoot of the AP was 37 mV. Square pulses from -85 mV to 20 mV were applied for 200 ms at 0.1 Hz. Pipettes were l-3 MQ when filled with the following solution (in mM): 133 Csaspartate, 3 CsCl, 2 MgC12, 2.93 CaC12, 7.5 ethylene ether)-N,N,N’,N’-tetraacetic glycol-bis(P-aminoethyl acid (EGTA), 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 5 MgATP, 0.15 guanosine 5’-triphosphate (GTP), 4 phosphocreatine, and 10 U/ml creatine kinase, pH 7.30. The bath solution was the following (in mM): 140 N-methyl-D-glucamine, 2 CaC12,
E 10
PNPF
;I---
)
150
ms
100
ms
FIG. 1. Ca”’ current (I& activated by action potential (AP) and square-pulse voltage clamps at 37°C. A: AP waveform used to activate Ic‘i,. B: I~~iI activated by an AP. C: nisoldipineand N?sensitive (filled triangle and open triangle, respectively) components of Ica from B. D: square pulse used to activate Ica. E: Ica activated by a square pulse. F: nisoldipineand Ni’+ -sensitive (filled triangle and open triangle, respectively) components of Ica from E.
CURRENT
AND
CA')+ TRANSIENTS
5 CsCl, 1 MgC12, 10 HEPES, 10 glucose, and 0.05 tetrodotoxin (TTX), pH 7.40. Ica was recorded 8-10 min after obtaining the whole cell configuration. Cesium and TTX were used to block K+ and Na’ currents, respectively. lca was filtered at 1 kHz by an eight-pole low-pass Bessel filter (Frequency Devices) and sampled at 12 bits A/D resolution with pCLAMP software (Axon Instruments, Burlingame, CA). The capacitative currents in our nisoldipine-sensitive I ca records obtained from both action potential and square-pulse protocols are subtracted out through the use of P/N linear leak subtraction or nisoldipine-insensitive current subtraction. Thus nisoldipineinsensitive currents, including capacitative currents, are removed. Ca2+ transients were recorded using the Ca2’-sensitive dye fura-2. The single-electrode voltage-clamp technique (34) was used to record Ica and Ca2+ transients elicited by the AP waveform previously described. Myocytes were loaded with 3 PM fura-2/acetoxymethyl ester (AM) for 10 min at room temperature and then incubated at room temperature for >45 min to allow for dye conversion. These cells were then transferred to the recording chamber on the stage of an inverted microscope equipped with epifluorescence illumination. Electrode resistances were 20-30 MQ when filled with 3 M KCl. All experiments were carried out at 37°C in a bath solution containing the following (in mM): 140 NaCl, 5 KCl, 2 CaC12, 1 MgC12, 5 HEPES, 10 glucose, and 0.05 TTX, pH 7.40. The dye in the cell was alternately (200 Hz) excited at 340- and 380-nm wavelengths of light generated by a Deltascan illumination system (Photon Technology International). Emission fluorescence at 500 nm was detected with a photon-counting photomultiplier tube (R928, Hamamatsu, Japan). The fluorescence ratio (340/ 380 nm) was calculated as a measure of Ca2+ concentration. RESULTS
Whole cell Ica was recorded at room temperature (23°C) and 37°C with either a voltage protocol that simulated an AP (Fig. 1A) or a square-step depolarization (Fig. 10). On initial depolarization by an AP clamp, Ica activated rapidly and then inactivated partially (Fig. 1B). Ica was maintained during the plateau of the AP. This complex time course of Ica generated by an AP was different from that generated by a square pulse. Ica activated by a square pulse showed a more simple time course (Fig. 1E) than that generated by an AP. The main difference was the sustained current generated by the AP and not the square pulse. Pharmacological dissection revealed the presence of both T-type (open triangle) and L-type (filled triangle) Ca2’ channels during the fast peak of the Ica activated by the action potential (Fig. 1C) and square pulse (Fig. 1F) protocols. Nisoldipine (10m6 M) was used to isolate L-type Ca2’ channels, and the residual current was characterized as a T-type Ca2’ current by its sensitivity to Ni”+ (4 X 10B5 M) (26). To confirm the AP-generated currents as Ica, the effects of ,&adrenergic stimulation and temperature on Ica were studied. Isoproterenol (10B6 M) increased the peak Ica activated by an AP by 69 t 16% (Fig. 2A) and by a square pulse by 60 t 10% (Fig. 2B) at 37OC. The isopro-
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150
100
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2. Modulation of Iczl by isoproterenol and temperature. Ica was elicitec 3 by either an AP clamp (A, C, and E) or a square voltage step in Fig. 1, before (open circle) and after 1 W, D, and F) as described PM isoproterenol (filled circle) at either 37°C (A and B) or room temperature (C and 0). Effect of temperature alone on Ica activated by an AP or square pulse is shown in E and F, respectively (filled square, 37°C; open square, room temperature). PIG.
terenol-induced increase in Ica was sustained during the plateau of the AP (Fig. 2A). At room temperature, the sustained increase in Ica by isoproterenol was even more pronounced under the AP clamp (74 t 15%) than the square-pulse clamp (Fig. 2, C and D, respectively). The effects of temperature alone on Ica generated by an AP (Fig. 2E) and a square-pulse clamp (Fig. 2F) were also investigated. The inactivation of the AP-generated Ica was slower at room temperature (Fig. 2E, open square), and, therefore, Ica during the plateau of the AP was noticeably sustained. On the other hand, the fast peak of Ica at room temperature was slower and smaller than that at 37°C (Table 1). The specific membrane conductance (Fig. 3A) to Ca2’ (gc,) during the control AP clamp at 37°C (filled square) and at room temperature (open square) was calculated using the Ohm equation with a reversal potential for lca of +70 mV, determined experimentally. At 37°C gca was 195.4 t 23.8 and 40.9 t 6.9 pS/pF (n = 4) at the fast peak of Ica and at 20 mV during the plateau of the AP (53 ms later), respectively. The corresponding values at room temperature were 145.7 t 16.4 and 70.9 t 5.2 pS/ pF. The lower temperature produced a decrease in gca during the upstroke of the AP and an increase in gc, during the plateau of AP. It has been shown that Ca”’ influx through Ca”’ channels in heart triggers Ca2’- induced Ca2’ release from the SR (l-3). Therefore, we calculated the exact Ca2’ influx via Ca2+ channels during an AP by integrating the APTABLE
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c395
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DISCUSSION
The precise knowledge of Ica during the AP has interested cardiac physiologists for years. However, due to technical limitations until now, lca during an AP could only be modeled from data derived from activation and
1. Properties and modulation of Ica elicited by an action potential and voltage step at two temperatures Action Room
are means
ms
Potential
Clamp
Square
temperature
Control
Fast peak, pA/pF Time to fast peak, Slow peak, pA/pF Flux, lo-" mol/pF Values
AND
generated Ica over time in control and after isoproterenol (lo-” M) (Fig. 3B). Isoproterenol increased the total Ca2’ flux by 70% during the AP. The time course of the Ca2’ flux was not significantly altered by isoproterenol. We calculated the increase in Ca2’ concentration/cell due to lca during an AP to be equal to 16 PM (4.01 x lo-l6 mol/ 25 pl; Ref. 8). This value was much greater than previous measurements (using aequorin, fura-2, or indo-1) of the peak Ca2’ transient (0.5-3.0 PM), which includes not only the Ca”+ flowing through Ca2+ channels but also released Ca”+ from SR. This suggeststhat Ca”+ might be localized highly in a restricted area (e.g., underneath the sarcolemmal membrane) and/or Ca2+-buffering systems or extrusion mechanisms are sufficiently fast to maintain lower intracellular Ca”+ concentration levels. Table 1 summarizes various properties of both APand square-pulse-generated Ica before and after ,&adrenoceptor stimulation at room temperature and 37°C. Although the effect of isoproterenol on the fast peak of lca was qualitatively similar with an AP or a squarepulse clamp, only the AP clamp produced a slower peak that was preferentially enhanced by both isoproterenol and temperature. The release of Ca”+ from the SR is dependent on Ica. The Ca”+ fluorescence signal (Fig. 4, A and B) elicited by an AP clamp at 37°C (closed circle) was abolished by nisoldipine (lo-” M) (open circle). Thus the membrane potential change alone was unable to activate the release of Ca2’ from intracellular stores. Stimulation of ,&adrenergic receptors by isoproterenol (10D6M) enhanced the Ca” transient by 56 t 18% and decreased the 50% relaxation time from 0.41 t 0.05 s to 0.22 t 0.04 s (Fig. 4B; control, closed circle; Iso, closed square). The isoproterenol-enhanced Ca”+ transient was also blocked by nisoldipine (open circle). Single-electrode voltageclamped cells in which Ca2’ transients were studied also showed a nisoldipine-sensitive inward current (Fig. 4C) with a similar configuration but possessing a more dramatic plateau phase than that seen in the whole cell voltage-clamp experiments (Figs. 1 and 2). This could be due to other Ca2+-activated overlapping currents; such as K+ or Na’-Ca”’ exchange currents (14, 19), that are present in single microelectrode voltage-clamp experiments.
10 P A/P
CURRENT
5.77t0.56 5.00~1.10 3.5220.30 2.36t0.22
t SE; n = 4 experiments.
IS0
37°C (lo-”
8.58k0.94 3.60t0.60 6.12t0.70 4.13t0.50
Time
M)
Control
IS0
7.62k1.03 2.4OkO.30 2.88t0.45 1.75t0.30
to slow peak
Room (lo-"
12.8721.32 2.2OkO.20 5.43tl.18 3.1OkO.71 (20
mV)
M)
Control
Pulse 37°C
temperature IS0
(lo-”
M)
Control
IS0
(lo-
6.73kO.90 6.12t1.24
12.24t1.22 4.25t0.76
8.34t1.50 0.75kO.10
13.33k2.08 0.63kO.10
2.75t0.32
5.25t0.83
2.35k0.32
3.64t0.64
was 56 ms. ISO,
M)
isoproterenol.
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C396
ACTION
POTENTIAL-GENERATED
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‘i‘ 3000 $ E
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CON x 3
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-
I = 0 < 0
5 El I -90
1000
i?
-
W
TIME
(ms)
0 I
I
0
200
TIME
I
400
(ms)
FIG. 3. Effect of temperature and isoproterenol on Ca”’ conductance (g(J and Ca”’ influx. A: effect of temperature on the gca during an AP clamp at either room temperature (open square) or 37°C (filled square). using Ohm equation with a reversal potential of 70 gc. THwas calculated mV determined experimentally. B: effect of isoproterenol (ISO, 1 PM) on the Ca” flux via nisoldipineand Ni”‘-sensitive Ca” channels during an AP clamp at 37°C. CON, control.
400 PA
100
ms
l
0
1 s
4. AP clamp-generated Ca” transients and current measured with single microelectrode voltage-clamp technique. A: furafluorescence ratio before (filled circle) and after addition of 1 PM nisoldipine (open circle). B: furafluorescence ratio in control (filled circle), after 1 PM isoproterenol (filled square), and after 1 PM isoproterenol and 1 FM nisoldipine (open circle). C: nisoldipine-sensitive current from same experiment shown in A. Nisoldipine-insensitive currents were subtracted. Fluorescence ratio units are dimensionless. FIG.
inactivation curves generated from square pulses. Di Francesco and Noble (10) and Campbell and Giles (6) have proposed mathematical models that simulate Ica during an AP. Several characteristics predicted by these models are manifested in our recordings of Ica. These include fast inactivation, sustained phase during the plateau, and a possible reactivation during the AP plateau. A D 600-sensitive lca during an AP clamp has been shown previously (11, 12); however, the true lca in these experiments is difficult to quantify due to the presence of Na’-, K+-, and Ca2+-dependent currents. On the other hand, Na+ currents through single L-type calcium channels have been reported during the AP of atria1 cells incubated in low Ca2+ solution using a double-electrode patch-clamp technique (21). Our experiments allowed for the isolation of whole cell
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AND
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nisoldipine-sensitive Ica resulting from an AP in the absence of any other overlapping currents. However, the lca obtained in the whole cell experiments may lack a component of Ca2+ -dependent inactivation via Ca2’ release from the sarcoplasmic reticulum. It is conceivable that the Ca2+-dependent inactivation contributes to the differences seen between the AP-generated Ica observed with whole cell patch clamp (Figs. 1 and 2) and single microelectrode voltage clamp (Fig. 4C). The Ica generated by an AP has a sustained phase during the plateau of the AP, which can be explained by an increase in the driving force for Ca2’, as a result of the repolarization after the peak of the action potential as well as a slow or partial inactivation and a voltage-dependent reactivation of channels at plateau potentials (6). The reactivation of Ica has been proposed in atria1 and Purkinje cells to partially explain the U-shaped voltage dependence of the lca inactivation curve at potentials positive to 10 mV (6, 7). Therefore, the physiological loa during the AP plateau is the result of a delicate balance between the activation, inactivation, and reactivation of Ca2+ channels as well as the driving force for Ca2’. This complex course of Ica generated by an AP is clearly different from that generated by a square pulse. It has also been reported that both L- and T-type Ca2’ channels are present in guinea pig ventricular cells (2, 22, 25, 32). We report here that T-type Ca2+ channels are activated along with L-type channels during the AP to contribute to the fast peak of Ica during EC coupling. It would appear that the current via T-type Ca2+ channels alone is an inadequate trigger for cardiac EC coupling, because block of L-type Ica can virtually eliminate the Ca”+ transient. Although SR Ca2+ is released by Ica generated by square pulses, Ica during an AP clamp is presumably the only signal to trigger Ca2+ release under physiological conditions. The absence of an intracellular Ca2+ transient after nisoldipine indicates that a change in membrane voltage alone or the presence of other ionic currents during an AP does not directly cause the release of SR Ca2’ in either the absence or presence of ,8-adrenoceptor stimulation. These results do not rule out a possible modulatory effect of membrane potential on Ca2’ release, but recent experiments suggest that SR Ca2’ release occurs by a Ca2+ trigger, independent of membrane potential (23, 24). Therefore, Ca2+-induced Ca2+ release from the SR is triggered by the influx of Ca2’ via Ca2+ channels during the AP. It is possible that the fast component of Ica is responsible for the Ca2+-induced Ca2+ release, whereas the sustained slow component is involved in reloading the SR, as has been proposed previously (15, 16). Therefore, changes in the AP that modify the fast component of Ica would have an immediate effect on the Ca2+ transient. An alternative hypothesis is that SR Ca2+ release is under the continuous control of Ica and does not proceed in a regenerative manner (13). This would imply an even closer association between lca and the Ca2’ transient. The effect of isoproterenol on Ica during the AP agrees with previous reports that show a ,&adrenergic enhancement of both Ica and Ca2+ transients (5,26,33). Furthermore, the sustained increase in Ic., during the plateau of
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ACTION
POTENTIAL-GENERATED
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the AP by ,&adrenoceptor stimulation may contribute to the increase in the Ca*’ transient. An increase or reactivation of L-type Ca*+ window current during the plateau of the AP has been implicated in the genesis of early afterdepolarizations in heart (18)) which occur frequently during catecholamine exposure (27). In summary, Ica has been recorded during the physiological AP. This complex current is intimately involved in cardiac EC coupling. The AP clamp provides the methodology to study the relationship of loa to intracellular events I.eading to myocardial cell contraction. We thank L. White for preparation of the typescript. This study was supported by National Heart, Lung, and Blood Institute Grant HL-33333, a Grant-in-Aid from the American Heart Association, and National Institute for Drug Abuse Training Grant DA-07232. J. Arreola has a fellowship from the American Heart Association New York Affiliate 88-120F. S.-S. Sheu is an Established Investigator of the American Heart Association. Address for reprint requests: S.-S. Sheu, Dept. of Pharmacology, School of Medicine and Dentistry, Univ. of Rochester, 601 Elmwood Ave., Rochester, NY 14642. Received
15 February
1991; accepted
in final
form
13 May
14.
15.
16.
17.
18.
19. 20.
1991.
REFERENCES 1. BARCENAS-RUIZ, L., AND W. G. WIER. Voltage dependence of intracellular [Ca’+] transients in guinea pig ventricular myocytes. Circ. Res. 61: 148-154, 1987. 2. BEAN, B. P. Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51: 367-384, 1989. 3. BEUCKELMANN, D. J., AND W. G. WIER. Mechanism of release of calcium from sarcoplasmic reticulum of guinea pig cardiac cells. J. Physiol. Lond. 405: 233-255, 1988. 4. BLINKS, J. R., W. G. WIER, P. HESS, AND F. G. PRENDERGAST. Measurements of Ca”’ concentration in living cells. Prog. Biophys. Mol. Biol. 40: 1-114, 1982. 5. CALLEWAERT, G., L. CLEEMAN, AND M. MORAD. Epinephrine enhances Ca”’ current-regulated Ca” release and Ca” reuptake in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA 85: 20092013, 1988. 6. CAMPBELL, D. L., AND W. GILES. Calcium currents. In: Calcium and the Heart, edited by G. A. Langer. New York: Raven, 1990, p. 27-83. 7. CAMPBELL, D. L., W. R. GILES, J. R. HUME, AND E. F. SHIBATA. Inactivation of calcium current in bull-frog atria1 myocytes. J. Physiol. Lond. 403: 287-315, 1988. 8. CANNELL, M. B., J. R. BERLIN, AND W. J. LEDERER. Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science Wash. DC 238: 1419-1423, 1987. 9. CHAPMAN, R. A. Control of cardiac contractility at the cellular level. Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H535-H552, 1983. 10. DIFRANCESCO, D., AND D. NOBLE. A model of cardiac electrical activity incorporating ionic pumps and concentration changes. Philos. Trans. R. Sot. Lond. B Biol. Sci. 307: 353-398, 1985. 11. DOERR, T., R. DENGER, AND W. TRAUTWEIN. Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp. Pfluegers Arch. 413: 599-603, 1989. 12. DOERR, T., R. DENGER, A. DOERR, AND W. TRAUTWEIN. Ionic currents contributing to the action potential in single ventricular myocytes of the guinea pig studied with action potential clamp. Pfluegers Arch. 416: 230-237, 1990. 13. DUBELL. W. H.. AND S. R. HOUSER. Voltage and beat denendence
21.
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28. 29.
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TRANSIENTS
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of Ca”’ transient in feline ventricular myocytes. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H746-H759, 1989. EGAN, T. M., D. NOBLE, S. J. NOBLE, T. POWELL, A. J. SPINDLER, AND V. W. TWIST. Sodium-calcium exchange during the action potential in guinea pig ventricular cells. J. Physiol. Lond. 411: 639661,1989. FABIATO, A. Calcium-induced release of calcium from cardiac sarcoplasmic reticulum. Am. J. Physiol. 245 (Cell Physiol. 14): Clc14, 1983. FABIATO, A. Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum in a skinned canine cardiac Purkinje cell. J. Gen. Physiol. 85: 291-320,1985. HAMILL, 0. P., A. MARTY, E. NEHER, B. SAKMANN, AND F. J. SIGWORTH. Improved patch clamp techniques for high resolution current recording from cell and cell-free membrane patches. Pfluegers Arch. 391: 85-100, 1981. JANUARY, C. T., AND J. M. RIDDLE. Early afterpolarizations: mechanism of induction and block. A role for L-type Ca”’ current. Circ. Res. 64: 977-990, 1989. KIMURA, J., A. NOMA, AND H. IRISAWA. Na-Ca exchange current in mammalian heart cells. Nature Lond. 319: 596-597, 1986. LEDERER, W. J., J. R. BERLIN, N. M. COHEN, R. W. HADLEY, D. M. BERS, AND M. B. CANNELL. Excitation-contraction coupling in heart cells. Roles of the sodium-calcium exchange, the calcium current, and the sarcoplasmic reticulum. Ann. NY Acad. Sci. 588: 190-206,199O. MAZZANTI, M., AND L. J. DEFELICE. Regulation of the Na-conducting Ca channel during the cardiac action potential. Biophys. J. 51: 115-121, 1987. MITRA, R., AND M. MORAD. Two types of calcium channels in guinea pig ventricular myocytes. Proc. Natl. Acad. Sci. USA 83: 5340-5344,1986. NABAUER, M., G. CALLEWAERT, L. CLEEMANN, AND M. MORAD. Regulation of calcium release is gated by calcium, not gating charge, in cardiac myocytes. Science Wash. DC 244: 800-803, 1989. NIGGLI, E., AND W. J. LEDERER. Voltage-independent calcium release in heart muscle. Science Wash. DC 250: 565-568, 1990. NILIUS, B., P. HESS, J. B. LANSMAN, AND R. W. TSIEN. A novel type of cardiac calcium channel in ventricular cells. Nature Lond. 316: 443-446, 1985. PELZER, D., S. PELZER, AND T. F. MCDONALD. Properties and regulation of calcium channels in muscle cells. Rev. Physiol. Biochem. Pharmacol. 114: 107-207, 1990. PRIORI, S. G., AND P. B. CORR. Mechanisms underlying early and delayed after depolarizations induced by catecholamines. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H1796-H1805, 1990. REUTER, H. Properties of two membrane inward current in the heart. Annu. Rev. Physiol. 41: 413-424, 1979. SHEU, S. S., V. K. SHARMA, AND S. P. BANERJEE. Measurements of cytosolic free calcium concentration in isolated rat ventricular myocytes with quin 2. Circ. Res. 55: 830-834, 1984. SPURGEON, H. A., M. D. STERN, G. BAARTZ, S. RAFFAELI, R. G. HANSFORD, A. TALO, E. LAKATTA, AND M. C. CAPOGROSSI. Simultaneous measurements of Ca’+, contraction, and potential in cardiac myocytes. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H574-H586,1990. TRAUTWEIN, W., AND J. HESCHELER. Regulation of cardiac L-type calcium current by phosphorylation and G proteins. Annu. Rev. Physiol. 52: 257-274, 1990. TSIEN, R. W. Calcium channels in excitable cell membranes. Annu. Rev. Physiol. 45: 341-358, 1983. WIER, W. G. Cytoplasmic [ Ca”] in mammalian ventricle: dynamic control by cellular processes. Annu. Rev. Physiol. 52: 467-485, 1990. WILSON, W. A., AND M. M. GOLDNER. Voltage clamping with a single microelectrode. J. Neurobiol. 6: 411-422, 1975.
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ARREOLA, JORGE, DANIEL J. WILLIFORD,
ROBERT T. DIRKSEN, Ru-CHI SHIEH, AND SHEY-SHING SHEU. Ca2+ current
and Ca”’ transients under action potential clamp in guinea pig ventricular rnyocytes. Am. J. Physiol. 261 (Cell Physiol. 30): C393-C397, 1991.-Precise characterization of the magnitude and kinetics of transsarcolemmal Ca”’ influx during an action potential (AP) is essential for a complete understanding of excitation-contraction coupling in heart. Using a voltage-clamp protocol that simulated a physiological AP (AP clamp), we characterized the properties of the Ca”’ current (Ica) in guinea pig ventricular myocytes. The AP-generated Ioa showed a complex time course that was different from Ica generated by a square pulse. I (:a activated rapidly during the upstroke of the AP and then partially inactivated during the plateau. The fast component of Ica reached a peak value of -7.6 t 1.0 pA/pF at 2.40 & 0.30 ms after depolarization, followed by a slow component with a peak value of -2.9 t 0.4 pA/pF during the plateau. It-21 generated by an AP was composed of both L- and T-type Ca” channels. T-type Ca2’ current contributed to the fast component of Ica and L-type Ca”’ current contributed to both fast and slow components of Ica. Activation of ,&adrenoceptors enhanced Ica with a maximal effect lasting throughout the entire plateau of the AP. Measurements of cytosolic Ca2’ transients using fura- indicated that the Ica was responsible reticulum. for triggering Ca”’ release from the sarcoplasmic The AP clamp provides a new approach for investigation of the relationship between Ica and Ca”’ transients under more physiological conditions. excitation-contraction nels; calcium-induced tors; voltage clamp
coupling; L- and T-type calcium chancalcium release; heart cells; P-adrenocep-
THE FIRST STEP in excitation-contraction (EC) coupling of heart is the activation of Ca2’ channels by membrane depolarization (9,28). Ca2+current (I&, flowing through L-type Ca2’ channels, triggers Ca2’ release from sarcoplasmic reticulum (SR) and results in muscle contraction (1, 5, 9, 15, 20, 23, 28). Sequestration of Ca2+ back into the SR and efflux across the sarcolemma restores intracellular Ca2+ concentration ([ Ca2+]i) to the resting level, thereby promoting relaxation. The amplitude of Ica and the Ca2’ transient are closely related (1, 3, 5, 31). However, it is unclear whether Ica evoked by membrane depolarization is the only link to the Ca”’ transient (8,20). Several studies have examined the relationship between Ica and Ca2’ transient in single myocytes under voltage clamp using square-pulse proto0363-6143/91
$1.50 Copyright
cols (1, 3, 5, 8, 13, 30). These studies found that the voltage dependence of Ica correlated well with the relationship between the peak amplitude of the Ca2+ transients and membrane potential and that the peak amplitude of the Ca2’ transients was not altered by the duration of voltage-clamp depolarization as long as the same peak Ica was achieved. However, others have found that the voltage dependence of the depolarization-induced increase in [Ca2+]; was not the same as that of the Ica (8, 13). It has been suggested that SR Ca2’ release is proportional to lca when it is small and becomes saturated after a sufficient magnitude of lca is achieved (13). Taken together, these results suggest that, whereas the SR Ca2’ release is dependent on Ica, the membrane potential may also alter SR Ca2+ release. In mammalian heart, L-type and T-type Ca2+channels have been identified. These channels differ in their voltage dependence, pharmacology, kinetics, and conductance (2, 22, 25, 32). L-type channels activate at less negative potentials (positive to -30 mV), whereas their inactivation appears to be both voltage and Ca”+ dependent (6, 7, 26). On the other hand, T-type channels activate at more negative potentials (positive to -50 mV) and inactivate solely by a voltage-dependent process. Although the role of T-type Ica in excitation-contraction coupling is unknown, it is clear that L-type lca contributes to the action potential (AP) plateau (28) and is essential for the Ca2+-induced Ca2’ release from SR (15, 23 The properties of L-type Ca2+current and its relationship to the cytosolic Ca2+ transient have been investigated under voltage-clamp conditions using depolarizing square pulses (1, 3, 5, 8). However, Ica during an AP could behave differently (11, 12). For instance, simulation of Ica during an AP predicts rapid activation followed by a partial inactivation and reactivation such that a secondary inward “hump” of Ica occurs during the latter phase of the AP (6, 10). Attempts to test this prediction have been performed by measuring D 600-sensitive currents in single myocytes voltage clamped with their own AP waveform (11, 12), and by simultaneously recording an AP and Na’ conductance through Ca2’ channels from the same cell by using two separate electrodes (21). No study has been reported regarding the relationship between Ica during an AP and Ca2+ transients. To obtain direct information concerning the magnitude, kinetics, and the type(s) of Ca2+ channel(s) comprising Ica and its
0 1991 the American
Physiological
Society
c393
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c394
ACTION
POTENTIAL-GENERATED
CA"
possible relationship to Ca2+ transients during physiological electrical activity in heart, we performed experiments in cardiomyocytes under voltage clamp using the AP as a stimulus waveform. METHODS
Single myocytes were isolated from guinea pig ventricular muscles by an enzymatic procedure described previously (29). Briefly, hearts were isolated and retrogradely perfused with a Ca2’- and antibiotic-free Joklik medium (minimal essential medium modified for suspension cultures; GIBCO, Grand Island, NY) followed by a similar solution containing 0.5 mg/ml collagenase, 0.25 mg/ml protease, 1 mg/ml albumin, and 50 PM CaC12. The heart was minced and cells were dissociated by gentle agitation in the enzyme solution. Isolated cells were stored at room temperature in Joklik medium containing 100 mg/ml albumin, 1 mM CaC12, and 10 mM glucose at pH 7.4. Ica was recorded using the whole cell voltage-clamp technique (17) with a List EPC-7 amplifier using both AP and square-pulse voltage protocols. A representative AP (Fig. 1A) was recorded from a guinea pig papillary muscle at 37°C and used in all experiments as the stimulus waveform to activate Ca2+ channels at a frequency of 0.1 Hz. The resting membrane potential, and therefore the holding potential, was -85 mV. The overshoot of the AP was 37 mV. Square pulses from -85 mV to 20 mV were applied for 200 ms at 0.1 Hz. Pipettes were l-3 MQ when filled with the following solution (in mM): 133 Csaspartate, 3 CsCl, 2 MgC12, 2.93 CaC12, 7.5 ethylene ether)-N,N,N’,N’-tetraacetic glycol-bis(P-aminoethyl acid (EGTA), 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 5 MgATP, 0.15 guanosine 5’-triphosphate (GTP), 4 phosphocreatine, and 10 U/ml creatine kinase, pH 7.30. The bath solution was the following (in mM): 140 N-methyl-D-glucamine, 2 CaC12,
E 10
PNPF
;I---
)
150
ms
100
ms
FIG. 1. Ca”’ current (I& activated by action potential (AP) and square-pulse voltage clamps at 37°C. A: AP waveform used to activate Ic‘i,. B: I~~iI activated by an AP. C: nisoldipineand N?sensitive (filled triangle and open triangle, respectively) components of Ica from B. D: square pulse used to activate Ica. E: Ica activated by a square pulse. F: nisoldipineand Ni’+ -sensitive (filled triangle and open triangle, respectively) components of Ica from E.
CURRENT
AND
CA')+ TRANSIENTS
5 CsCl, 1 MgC12, 10 HEPES, 10 glucose, and 0.05 tetrodotoxin (TTX), pH 7.40. Ica was recorded 8-10 min after obtaining the whole cell configuration. Cesium and TTX were used to block K+ and Na’ currents, respectively. lca was filtered at 1 kHz by an eight-pole low-pass Bessel filter (Frequency Devices) and sampled at 12 bits A/D resolution with pCLAMP software (Axon Instruments, Burlingame, CA). The capacitative currents in our nisoldipine-sensitive I ca records obtained from both action potential and square-pulse protocols are subtracted out through the use of P/N linear leak subtraction or nisoldipine-insensitive current subtraction. Thus nisoldipineinsensitive currents, including capacitative currents, are removed. Ca2+ transients were recorded using the Ca2’-sensitive dye fura-2. The single-electrode voltage-clamp technique (34) was used to record Ica and Ca2+ transients elicited by the AP waveform previously described. Myocytes were loaded with 3 PM fura-2/acetoxymethyl ester (AM) for 10 min at room temperature and then incubated at room temperature for >45 min to allow for dye conversion. These cells were then transferred to the recording chamber on the stage of an inverted microscope equipped with epifluorescence illumination. Electrode resistances were 20-30 MQ when filled with 3 M KCl. All experiments were carried out at 37°C in a bath solution containing the following (in mM): 140 NaCl, 5 KCl, 2 CaC12, 1 MgC12, 5 HEPES, 10 glucose, and 0.05 TTX, pH 7.40. The dye in the cell was alternately (200 Hz) excited at 340- and 380-nm wavelengths of light generated by a Deltascan illumination system (Photon Technology International). Emission fluorescence at 500 nm was detected with a photon-counting photomultiplier tube (R928, Hamamatsu, Japan). The fluorescence ratio (340/ 380 nm) was calculated as a measure of Ca2+ concentration. RESULTS
Whole cell Ica was recorded at room temperature (23°C) and 37°C with either a voltage protocol that simulated an AP (Fig. 1A) or a square-step depolarization (Fig. 10). On initial depolarization by an AP clamp, Ica activated rapidly and then inactivated partially (Fig. 1B). Ica was maintained during the plateau of the AP. This complex time course of Ica generated by an AP was different from that generated by a square pulse. Ica activated by a square pulse showed a more simple time course (Fig. 1E) than that generated by an AP. The main difference was the sustained current generated by the AP and not the square pulse. Pharmacological dissection revealed the presence of both T-type (open triangle) and L-type (filled triangle) Ca2’ channels during the fast peak of the Ica activated by the action potential (Fig. 1C) and square pulse (Fig. 1F) protocols. Nisoldipine (10m6 M) was used to isolate L-type Ca2’ channels, and the residual current was characterized as a T-type Ca2’ current by its sensitivity to Ni”+ (4 X 10B5 M) (26). To confirm the AP-generated currents as Ica, the effects of ,&adrenergic stimulation and temperature on Ica were studied. Isoproterenol (10B6 M) increased the peak Ica activated by an AP by 69 t 16% (Fig. 2A) and by a square pulse by 60 t 10% (Fig. 2B) at 37OC. The isopro-
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ACTION
POTENTIAL-GENERATED
CA’+
F
r
C 10 P AIPF
E
F-y---c
5 nA
150
100
ms
ms
2. Modulation of Iczl by isoproterenol and temperature. Ica was elicitec 3 by either an AP clamp (A, C, and E) or a square voltage step in Fig. 1, before (open circle) and after 1 W, D, and F) as described PM isoproterenol (filled circle) at either 37°C (A and B) or room temperature (C and 0). Effect of temperature alone on Ica activated by an AP or square pulse is shown in E and F, respectively (filled square, 37°C; open square, room temperature). PIG.
terenol-induced increase in Ica was sustained during the plateau of the AP (Fig. 2A). At room temperature, the sustained increase in Ica by isoproterenol was even more pronounced under the AP clamp (74 t 15%) than the square-pulse clamp (Fig. 2, C and D, respectively). The effects of temperature alone on Ica generated by an AP (Fig. 2E) and a square-pulse clamp (Fig. 2F) were also investigated. The inactivation of the AP-generated Ica was slower at room temperature (Fig. 2E, open square), and, therefore, Ica during the plateau of the AP was noticeably sustained. On the other hand, the fast peak of Ica at room temperature was slower and smaller than that at 37°C (Table 1). The specific membrane conductance (Fig. 3A) to Ca2’ (gc,) during the control AP clamp at 37°C (filled square) and at room temperature (open square) was calculated using the Ohm equation with a reversal potential for lca of +70 mV, determined experimentally. At 37°C gca was 195.4 t 23.8 and 40.9 t 6.9 pS/pF (n = 4) at the fast peak of Ica and at 20 mV during the plateau of the AP (53 ms later), respectively. The corresponding values at room temperature were 145.7 t 16.4 and 70.9 t 5.2 pS/ pF. The lower temperature produced a decrease in gca during the upstroke of the AP and an increase in gc, during the plateau of AP. It has been shown that Ca”’ influx through Ca”’ channels in heart triggers Ca2’- induced Ca2’ release from the SR (l-3). Therefore, we calculated the exact Ca2’ influx via Ca2+ channels during an AP by integrating the APTABLE
CA’+
c395
TRANSIENTS
DISCUSSION
The precise knowledge of Ica during the AP has interested cardiac physiologists for years. However, due to technical limitations until now, lca during an AP could only be modeled from data derived from activation and
1. Properties and modulation of Ica elicited by an action potential and voltage step at two temperatures Action Room
are means
ms
Potential
Clamp
Square
temperature
Control
Fast peak, pA/pF Time to fast peak, Slow peak, pA/pF Flux, lo-" mol/pF Values
AND
generated Ica over time in control and after isoproterenol (lo-” M) (Fig. 3B). Isoproterenol increased the total Ca2’ flux by 70% during the AP. The time course of the Ca2’ flux was not significantly altered by isoproterenol. We calculated the increase in Ca2’ concentration/cell due to lca during an AP to be equal to 16 PM (4.01 x lo-l6 mol/ 25 pl; Ref. 8). This value was much greater than previous measurements (using aequorin, fura-2, or indo-1) of the peak Ca2’ transient (0.5-3.0 PM), which includes not only the Ca”+ flowing through Ca2+ channels but also released Ca”+ from SR. This suggeststhat Ca”+ might be localized highly in a restricted area (e.g., underneath the sarcolemmal membrane) and/or Ca2+-buffering systems or extrusion mechanisms are sufficiently fast to maintain lower intracellular Ca”+ concentration levels. Table 1 summarizes various properties of both APand square-pulse-generated Ica before and after ,&adrenoceptor stimulation at room temperature and 37°C. Although the effect of isoproterenol on the fast peak of lca was qualitatively similar with an AP or a squarepulse clamp, only the AP clamp produced a slower peak that was preferentially enhanced by both isoproterenol and temperature. The release of Ca”+ from the SR is dependent on Ica. The Ca”+ fluorescence signal (Fig. 4, A and B) elicited by an AP clamp at 37°C (closed circle) was abolished by nisoldipine (lo-” M) (open circle). Thus the membrane potential change alone was unable to activate the release of Ca2’ from intracellular stores. Stimulation of ,&adrenergic receptors by isoproterenol (10D6M) enhanced the Ca” transient by 56 t 18% and decreased the 50% relaxation time from 0.41 t 0.05 s to 0.22 t 0.04 s (Fig. 4B; control, closed circle; Iso, closed square). The isoproterenol-enhanced Ca”+ transient was also blocked by nisoldipine (open circle). Single-electrode voltageclamped cells in which Ca2’ transients were studied also showed a nisoldipine-sensitive inward current (Fig. 4C) with a similar configuration but possessing a more dramatic plateau phase than that seen in the whole cell voltage-clamp experiments (Figs. 1 and 2). This could be due to other Ca2+-activated overlapping currents; such as K+ or Na’-Ca”’ exchange currents (14, 19), that are present in single microelectrode voltage-clamp experiments.
10 P A/P
CURRENT
5.77t0.56 5.00~1.10 3.5220.30 2.36t0.22
t SE; n = 4 experiments.
IS0
37°C (lo-”
8.58k0.94 3.60t0.60 6.12t0.70 4.13t0.50
Time
M)
Control
IS0
7.62k1.03 2.4OkO.30 2.88t0.45 1.75t0.30
to slow peak
Room (lo-"
12.8721.32 2.2OkO.20 5.43tl.18 3.1OkO.71 (20
mV)
M)
Control
Pulse 37°C
temperature IS0
(lo-”
M)
Control
IS0
(lo-
6.73kO.90 6.12t1.24
12.24t1.22 4.25t0.76
8.34t1.50 0.75kO.10
13.33k2.08 0.63kO.10
2.75t0.32
5.25t0.83
2.35k0.32
3.64t0.64
was 56 ms. ISO,
M)
isoproterenol.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
C396
ACTION
POTENTIAL-GENERATED
CA’+
‘i‘ 3000 $ E
IS0
CON x 3
w -60
-
I = 0 < 0
5 El I -90
1000
i?
-
W
TIME
(ms)
0 I
I
0
200
TIME
I
400
(ms)
FIG. 3. Effect of temperature and isoproterenol on Ca”’ conductance (g(J and Ca”’ influx. A: effect of temperature on the gca during an AP clamp at either room temperature (open square) or 37°C (filled square). using Ohm equation with a reversal potential of 70 gc. THwas calculated mV determined experimentally. B: effect of isoproterenol (ISO, 1 PM) on the Ca” flux via nisoldipineand Ni”‘-sensitive Ca” channels during an AP clamp at 37°C. CON, control.
400 PA
100
ms
l
0
1 s
4. AP clamp-generated Ca” transients and current measured with single microelectrode voltage-clamp technique. A: furafluorescence ratio before (filled circle) and after addition of 1 PM nisoldipine (open circle). B: furafluorescence ratio in control (filled circle), after 1 PM isoproterenol (filled square), and after 1 PM isoproterenol and 1 FM nisoldipine (open circle). C: nisoldipine-sensitive current from same experiment shown in A. Nisoldipine-insensitive currents were subtracted. Fluorescence ratio units are dimensionless. FIG.
inactivation curves generated from square pulses. Di Francesco and Noble (10) and Campbell and Giles (6) have proposed mathematical models that simulate Ica during an AP. Several characteristics predicted by these models are manifested in our recordings of Ica. These include fast inactivation, sustained phase during the plateau, and a possible reactivation during the AP plateau. A D 600-sensitive lca during an AP clamp has been shown previously (11, 12); however, the true lca in these experiments is difficult to quantify due to the presence of Na’-, K+-, and Ca2+-dependent currents. On the other hand, Na+ currents through single L-type calcium channels have been reported during the AP of atria1 cells incubated in low Ca2+ solution using a double-electrode patch-clamp technique (21). Our experiments allowed for the isolation of whole cell
CURRENT
AND
CA2+ TRANSIENTS
nisoldipine-sensitive Ica resulting from an AP in the absence of any other overlapping currents. However, the lca obtained in the whole cell experiments may lack a component of Ca2+ -dependent inactivation via Ca2’ release from the sarcoplasmic reticulum. It is conceivable that the Ca2+-dependent inactivation contributes to the differences seen between the AP-generated Ica observed with whole cell patch clamp (Figs. 1 and 2) and single microelectrode voltage clamp (Fig. 4C). The Ica generated by an AP has a sustained phase during the plateau of the AP, which can be explained by an increase in the driving force for Ca2’, as a result of the repolarization after the peak of the action potential as well as a slow or partial inactivation and a voltage-dependent reactivation of channels at plateau potentials (6). The reactivation of Ica has been proposed in atria1 and Purkinje cells to partially explain the U-shaped voltage dependence of the lca inactivation curve at potentials positive to 10 mV (6, 7). Therefore, the physiological loa during the AP plateau is the result of a delicate balance between the activation, inactivation, and reactivation of Ca2+ channels as well as the driving force for Ca2’. This complex course of Ica generated by an AP is clearly different from that generated by a square pulse. It has also been reported that both L- and T-type Ca2’ channels are present in guinea pig ventricular cells (2, 22, 25, 32). We report here that T-type Ca2+ channels are activated along with L-type channels during the AP to contribute to the fast peak of Ica during EC coupling. It would appear that the current via T-type Ca2+ channels alone is an inadequate trigger for cardiac EC coupling, because block of L-type Ica can virtually eliminate the Ca”+ transient. Although SR Ca2+ is released by Ica generated by square pulses, Ica during an AP clamp is presumably the only signal to trigger Ca2+ release under physiological conditions. The absence of an intracellular Ca2+ transient after nisoldipine indicates that a change in membrane voltage alone or the presence of other ionic currents during an AP does not directly cause the release of SR Ca2’ in either the absence or presence of ,8-adrenoceptor stimulation. These results do not rule out a possible modulatory effect of membrane potential on Ca2’ release, but recent experiments suggest that SR Ca2’ release occurs by a Ca2+ trigger, independent of membrane potential (23, 24). Therefore, Ca2+-induced Ca2+ release from the SR is triggered by the influx of Ca2’ via Ca2+ channels during the AP. It is possible that the fast component of Ica is responsible for the Ca2+-induced Ca2+ release, whereas the sustained slow component is involved in reloading the SR, as has been proposed previously (15, 16). Therefore, changes in the AP that modify the fast component of Ica would have an immediate effect on the Ca2+ transient. An alternative hypothesis is that SR Ca2+ release is under the continuous control of Ica and does not proceed in a regenerative manner (13). This would imply an even closer association between lca and the Ca2’ transient. The effect of isoproterenol on Ica during the AP agrees with previous reports that show a ,&adrenergic enhancement of both Ica and Ca2+ transients (5,26,33). Furthermore, the sustained increase in Ic., during the plateau of
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ACTION
POTENTIAL-GENERATED
CA’)+ CURRENT
the AP by ,&adrenoceptor stimulation may contribute to the increase in the Ca*’ transient. An increase or reactivation of L-type Ca*+ window current during the plateau of the AP has been implicated in the genesis of early afterdepolarizations in heart (18)) which occur frequently during catecholamine exposure (27). In summary, Ica has been recorded during the physiological AP. This complex current is intimately involved in cardiac EC coupling. The AP clamp provides the methodology to study the relationship of loa to intracellular events I.eading to myocardial cell contraction. We thank L. White for preparation of the typescript. This study was supported by National Heart, Lung, and Blood Institute Grant HL-33333, a Grant-in-Aid from the American Heart Association, and National Institute for Drug Abuse Training Grant DA-07232. J. Arreola has a fellowship from the American Heart Association New York Affiliate 88-120F. S.-S. Sheu is an Established Investigator of the American Heart Association. Address for reprint requests: S.-S. Sheu, Dept. of Pharmacology, School of Medicine and Dentistry, Univ. of Rochester, 601 Elmwood Ave., Rochester, NY 14642. Received
15 February
1991; accepted
in final
form
13 May
14.
15.
16.
17.
18.
19. 20.
1991.
REFERENCES 1. BARCENAS-RUIZ, L., AND W. G. WIER. Voltage dependence of intracellular [Ca’+] transients in guinea pig ventricular myocytes. Circ. Res. 61: 148-154, 1987. 2. BEAN, B. P. Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51: 367-384, 1989. 3. BEUCKELMANN, D. J., AND W. G. WIER. Mechanism of release of calcium from sarcoplasmic reticulum of guinea pig cardiac cells. J. Physiol. Lond. 405: 233-255, 1988. 4. BLINKS, J. R., W. G. WIER, P. HESS, AND F. G. PRENDERGAST. Measurements of Ca”’ concentration in living cells. Prog. Biophys. Mol. Biol. 40: 1-114, 1982. 5. CALLEWAERT, G., L. CLEEMAN, AND M. MORAD. Epinephrine enhances Ca”’ current-regulated Ca” release and Ca” reuptake in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA 85: 20092013, 1988. 6. CAMPBELL, D. L., AND W. GILES. Calcium currents. In: Calcium and the Heart, edited by G. A. Langer. New York: Raven, 1990, p. 27-83. 7. CAMPBELL, D. L., W. R. GILES, J. R. HUME, AND E. F. SHIBATA. Inactivation of calcium current in bull-frog atria1 myocytes. J. Physiol. Lond. 403: 287-315, 1988. 8. CANNELL, M. B., J. R. BERLIN, AND W. J. LEDERER. Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science Wash. DC 238: 1419-1423, 1987. 9. CHAPMAN, R. A. Control of cardiac contractility at the cellular level. Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H535-H552, 1983. 10. DIFRANCESCO, D., AND D. NOBLE. A model of cardiac electrical activity incorporating ionic pumps and concentration changes. Philos. Trans. R. Sot. Lond. B Biol. Sci. 307: 353-398, 1985. 11. DOERR, T., R. DENGER, AND W. TRAUTWEIN. Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp. Pfluegers Arch. 413: 599-603, 1989. 12. DOERR, T., R. DENGER, A. DOERR, AND W. TRAUTWEIN. Ionic currents contributing to the action potential in single ventricular myocytes of the guinea pig studied with action potential clamp. Pfluegers Arch. 416: 230-237, 1990. 13. DUBELL. W. H.. AND S. R. HOUSER. Voltage and beat denendence
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31.
32. 33.
34.
AND
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TRANSIENTS
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