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Effects of Calcium Channel Antagonists on Ca2+Transients in Rat and Canine Cardiomyocytes
J Mol Cell Cardiol 29, 1037–1043 (1997)
Effects of Calcium Channel Antagonists on Ca2+ Transients in Rat and Canine Cardiomyocytes James Hensley, George E. Billman, J. David Johnson, Charlene M. Hohl and Ruth A. Altschuld Departments of Medical Biochemistry and Physiology, The Ohio State University College of Medicine, Columbus, OH 43210–1218, USA (Received 3 October 1996, accepted in revised form 27 November 1997) J. H, G. E. B, J. D. J, C. M. H R. A. A. Effects of Calcium Channel Antagonists on Ca2+ Transients in Rat and Canine Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1997) 29, 1037–1043. First-generation Ca2+ channel antagonists depress myocardial contractility, but many of the newer Ca2+ channel blockers have a high degree of “vascular selectivity”. This study compares the effects of the Ca2+ antagonists felodipine, amlodipine, mibefradil, verapamil and nifedipine, and the Ca2+ channel agonist, (S)(−)Bay K-8644 on Ca2+ transient amplitudes in fura-2/AM-loaded rat and canine ventricular cardiomyocytes. At 10−11 and 10−10 , felodipine increased [Ca2+]i transient amplitudes by 10–25% in field-stimulated fura-2-loaded cells from both species while at 10−6 it depressed [Ca2+]i transients by 80%. Mibefradil increased [Ca2+]i transient amplitudes by 16% at 10−11 and 10−10 and decreased the transients by 25% at 10−6 . The calcium channel agonist, (S)(−)-Bay K-8644 increased [Ca2+]i transient amplitudes at 10−10–10−6 (maximally 37% at 10−7 ) but depressed [Ca2+]i transients by 10% at 10−5 M. Nifedipine was inhibitory at all concentrations tested (10−11–10−6 ) in canine myocytes, but in rat cells, 10−10 nifedipine increased [Ca2+]i transient amplitudes by 37%. All concentrations of verapamil and amlodipine (10−11–10−6 ) depressed [Ca2+]i transients in both rat and canine myocytes. We conclude that: (1) felodipine and mibefradil may be positive rather than negative inotropes at low concentrations, which are therapeutically relevant; and (2) low concentrations of nifedipine may have a positive inotropic effect in the rat but not the dog heart. 1997 Academic Press Limited K W: Canine myocytes; Rat myocytes; Free calcium transients; Mibefradil, Verapamil; Nifedipine; Felodipine, Bay K 8644, Calcium channel antagonists.
Introduction Ca2+ channel antagonists are potent vasodilators widely used for the treatment of hypertension and angina pectoris (Opie, 1989). However, many Ca2+ blockers depress myocardial contractility and therefore must be used with caution in patients with poor cardiac function (Walsh, 1987; Multicenter diltiazem post-infarction trial research group, 1988; Boden et al., 1991; Hansen, 1991; Furberg et al., 1995). A new generation of dihydropyridine Ca2+ channel antagonists, including felodipine, amlodipine, isradipine, nicardipine and nisoldipine,
and the chemically novel Ca2+ antagonist, mibefradil, have improved vascular selectivity and could afford salutary reductions in afterload without negative inotropy (Clozel et al., 1989; Osterrieder and Holck, 1989; Perez-Vizcaino et al., 1993; Packer et al., 1996). Establishing whether or not Ca2+ channel antagonists directly affect myocardial contractility is not entirely straightforward, however. Negative inotropic effects in vivo can be masked by reflex neurohumoral responses. In addition, the binding of Ca2+ antagonists to Ca2+ channels is often voltage dependent, being one or more orders of magnitude
Please address all correspondence to: Ruth A. Altschuld, Department of Medical Biochemistry, 333 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210–1218, USA.
0022–2828/97/031037+07 $25.00/0
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more effective when the sarcolemma is depolarized (Sun and Triggle, 1995). Because cardiac myocytes depolarize and repolarize with each heartbeat, one cannot easily predict from ligand binding to membrane fragments the concentration dependence for Ca2+ antagonist effects on functioning cardiac Ca2+ channels. There are also species differences in myocyte electrophysiology that influence the effects of individual Ca2+ channel antagonists (Godfraind et al., 1992). In the present study, we have used electrical field stimulation of isolated adult rat and canine ventricular cardiomyocytes to compare the direct cardiac effects of the Ca2+ channel antagonists felodipine, mibefradil (Ro 40-5967), verapamil, nifedipine and amlodipine, and the Ca2+ channel agonist, (S)(−)-Bay K 8644, on intracellular free Ca2+ ([Ca2+]i) transients. Isolated myocytes eliminate concerns about complex in vivo neurohumoral responses. Canine cells were investigated because they closely resemble those from human hearts, and in vivo canine experiments have been widely used for mechanistic studies of the cardiac effects of Ca2+ channel antagonists. Rat myocytes were investigated because studies of the chronic effects of Ca2+ channel antagonists are often conducted in spontaneously hypertensive rats (Brunner et al., 1991; Lonsberry et al., 1992; Lemmer et al., 1994; Bo¨hm et al., 1995) and in the closely related spontaneously hypertensive, heart failure prone SHHF/ Mccfacp rat (Radin et al., 1993; McCune et al., 1995).
Materials and Methods
with 95% O2–5% CO2-saturated buffer containing (in m): 121 NaCl, 4.85 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1 CaCl2, 25 NaHCO3, 5 sodium pyruvate. Cells were superfused with each drug concentration for 8 min and then field stimulated at 0.2 Hz (canine cells) or 0.5 Hz (rat cells). Experiments were carried out in the dark and all solutions were wrapped in aluminum foil because some Ca2+ channel antagonists are light sensitive. Stock solutions of verapamil and Bay K 8644 were prepared in absolute ethanol. Stock solutions of nifedipine, felodipine, and amlodipine were prepared in dimethyl sulfoxide. Stock solutions of mibefradil were prepared in distilled water. Steady-state fura-2 fluorescence data were collected at 30 points per second with a PTI Filterscan. Sixteen consecutive steady-state [Ca2+]i transients were signal averaged for each experimental condition. The [Ca2+]i-dependent fluorescence ratio signals were not calibrated because 10–30% of the fura-2 free anion partitions into the mitochondria (Davis et al., 1987). This precludes accurate estimation of cytosolic free Ca2+ ion concentrations. However, changes in the amplitudes of intracellular fura-2 ratio transients reflect accurately changes in cytosolic Ca2+ transient amplitudes. When cytosolic [Ca2+]i indicators are selectively quenched with Mn2+ in rat myocytes, the cells continue to twitch in response to electrical stimulation, but the Ca2+sensitive mitochondrial signal does not exhibit beat to beat oscillations, indicating little contribution to whole cell [Ca2+]i transients (Miyata et al., 1991). Data were analyzed using single factor analysis of variance followed by the Scheffe´ test (DawsonSaunders and Trapp, 1990).
Isolated myocytes Ventricular myocytes were isolated from adult male Sprague–Dawley rats and adult mongrel dogs of either sex using previously described collagenase perfusion methods (Wimsatt et al., 1990; Hohl and Altschuld, 1991).
Fura-2 AM measurements Cells were loaded with 2 l fura-2/AM for 5 min followed by a 45–60-min post-incubation at room temperature to ensure complete hydrolysis of the acetoxymethyl ester groups and generate the Ca2+sensitive fura-2 free anion. Cells were loaded into a plexiglass superfusion chamber and allowed to attach to the bottom glass coverslip. After 10 min, superfusion at 2 ml/min, 37°C, pH 7.4, was begun
Results Figure 1 shows typical signal-averaged [Ca2+]i transients for canine myocytes incubated with varying concentrations of felodopine and nifedipine. In Figure 2 we present dose–response curves for the effects of the Ca2+ channel antagonists and agonist on relative [Ca2+]i transient amplitudes in rat and canine myocytes. Felodipine (a) significantly augmented [Ca2+]i transient amplitudes at low concentrations (10−11–10−9 ) in canine cells but became inhibitory as the concentration was raised. Low concentrations of felodipine also increased average Ca2+ transient amplitudes in rat cells, but when the dose–response curves were subjected to analysis of variance (ANOVA), the only statistically significant effect was that observed at 10−6 . By contrast, the new non-dihydropyridine Ca2+ channel antagonist, mibefradil, elicited nearly identical responses
Ca2+ Antagonists and Cardiomyocytes
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in rat and canine myocytes (b). Mibefradil had a small but significant stimulatory effect at low concentrations (10−11–10−10 ) and at 10−6 was the weakest negative inotrope of any of the Ca2+ channel antagonists investigated. Verapamil depressed [Ca2+]i transient amplitudes over a wide concentration range (10−11–10−6 ) in both rat and canine myocytes (c) and the canine cells were significantly more sensitive to the negative inotropic effects of the drug. Like verapamil, amlodipine depressed [Ca2+]i transient amplitudes throughout the concentration range examined, but there was a minimal species difference (d). The effects of nifedipine, on the other hand, were strongly species dependent (e). Nifedipine gave a biphasic dose– response curve for rat myocytes, significantly augmenting [Ca2+]i transient amplitudes at 10−11–10−9 but inhibiting [Ca2+]i transients at higher concentrations. By contrast, in canine myocytes, nifedipine had only an inhibitory effect. Like felodipine, mibefradil, and nifedipine (in the rat), the Ca2+ channel agonist, (S)(−)-Bay K 8644, also gave biphasic dose–response curves (f) with maximal stimulation at 10−7 and 20% inhibition at 10−5 . Unlike felodipine and nifedipine, however, the effects of (S)(−)-Bay K 8644 on Ca2+ transient amplitudes in rat and canine myocytes were nearly identical.
Discussion There are two novel and important results of this study. First, the newer Ca2+ channel antagonists,
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mibefradil and felodipine, did not depress [Ca2+]i transient amplitudes at therapeutically relevant concentrations in isolated rat or canine ventricular cardiomyocytes. In fact, low concentrations of these drugs had slight stimulatory effects, which reached statistical significance for canine cells treated with felodipine and for both rat and canine cells treated with mibefradil. Second, there are species differences in the responses of field stimulated adult mammalian ventricular cardiomyocytes to some Ca2+ channel antagonists, especially nifedipine. Our data with felodipine are in agreement with other studies demonstrating a positive inotropic effect of this Ca2+ channel blocker in dogs. In vivo studies by Cheng et al. (1994) have shown that felodipine (but not nifedipine or amlodipine) exhibits a significant positive inotropic effect in the presence of autonomic blockade in canine myocardium. Further, Pettersson et al. (1987) have shown a positive inotropic effect with felodipine in the isovolumically contracting canine heart. Our studies suggest that the positive inotropic effects reported by these investigators are due to an elevation of peak systolic [Ca2+]i. Positive inotropic effects of mibefradil have not previously been described, but most in vitro studies have investigated higher drug concentrations. In vivo, mibefradil is reported only to be devoid of negative inotropic effects (Clozel et al., 1991; Portegies et al., 1991). As expected, the first generation Ca2+ channel antagonist, verapamil, depressed [Ca2+]i transient amplitudes in both rat and canine myocytes throughout the concentration range investigated. This phenylalkylamine has minimal vascular selectivity and is capable of precipitating congestive heart failure when myocardial contractility is otherwise compromised (Walsh, 1987; Godfraind et al., 1992). The inhibitory effects of nifedipine on [Ca2+]i transient amplitudes in canine myocytes were also predictable on the basis of its negative inotropic effects demonstrated in vivo in both awake and anesthetized dogs (Cheng et al., 1994; Pagel et al., 1994). The augmentation of rat myocyte [Ca2+]i transients by 10−11–10−9 nifedipine was unanticipated, but it might explain the opposite cardiac effects of nifedipine and verapamil observed in the SHHF/Mcc-facp rat. Antihypertensive doses of nifedipine cause a marked regression of cardiac hypertrophy in these heart failure-prone, obese, hypertensive animals (Radin et al., 1993), but comparable blood pressure reduction with verapamil exacerbates cardiac hypertrophy and precipitates decompensation and premature death from congestive heart failure (Park et al., 1996).
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The deleterious response to verapamil was assumed to be associated with its negative inotropic effects, but this hypothesis had been difficult to reconcile with the positive response to nifedipine in the same animal model. The present demonstration of a positive rather than negative inotropic effect of low concentrations of nifedipine in the rat could explain this apparent discrepancy. This hypothesis is currently being tested using in vivo echocardiography of SHHF/Mcc-facp rats treated with antihypertensive doses of nifedipine or verapamil. The large species difference between the responses of rat and canine myocytes to nifedipine was especially striking, but significant species differences were also observed with verapamil and felodipine. Each of these compounds was more effective in reducing [Ca2+]i transients in canine than in rat myocytes. It is not clear whether these differences can be attributed to structural differences in the -type Ca2+ channels. However, dihydropyridine binding to isolated cardiac sarcolemmal fragments is very similar across species (Janis et al., 1987) and exhibits a complex voltage dependency (Sanguinetti and Kass, 1984; Godfraind et al., 1992). Resting membrane potentials in the rat and canine myocytes prepared in our laboratory are both close to −80 mV, but the rat cells have an abbreviated action potential (APD50=20–30 ms, Li et al., 1989). Thus, even though the rat myocytes in the present study were paced more rapidly than the canine cells, to reflect the more rapid in vivo heart rate of the rat, the brief action potential could have limited the time available for depolarization-induced Ca2+ antagonist binding to the rat myocyte Ca2+ channels. By extension, the prolonged canine action potential (APD50=300–400 ms; Freeman and Li, 1991) could have favored the antagonistic form of binding of some of the voltage-sensitive Ca2+ channel modulators. Thus, action potential duration may be an important factor in determining the effects of varied concentrations of nifedipine, felodipine and verapamil on cardiac [Ca2+]i transients. Effects of the Ca2+ channel activator, (S)(−)-Bay K 8644, were nearly identical in rat and canine myocytes, consistent with the observation that binding of this dihydropyridine is not influenced by membrane potential in cardiac myocytes (Ferrante
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et al., 1989). Inhibition of [Ca2+]i transients in both species at high concentrations of (S)(−)-Bay K 8644 is in agreement with previous reports (Kass, 1987; Bechem and Hoffmann, 1993). Effects of mibefradil were also the same in rat and canine myocytes. Nevertheless, the effects of mibefradil on inward Ca2+ currents in guinea-pig myocytes are strongly influenced by the experimentally imposed holding potential (Liang-min and Osterrieder, 1991). At a membrane holding potential of −80 mV, the IC50 is 12 l, a value comparable to that shown in Figure 2 for field-stimulated rat and canine myocytes. However, the IC50 declines >50fold when the holding potential is brought to a moderately depolarized value of −50 mV (Liangmin and Osterrieder, 1991). Thus, while mibefradil may be insensitive to action potential duration, it is extraordinarily sensitive to resting membrane potential. Bezprozvanny and Tsien (1995) concluded that the strong voltage dependency of mibefradil most likely results from a preferential binding to the open and inactivated state of the type Ca2+ channel. The fact that dihydropyridines can have opposing effects on myocardial contractility depending on concentration was first reported by Thomas et al. (1984). The stimulatory effect at low drug concentrations is probably analogous to the increases in -type Ca2+ current seen after a brief exposure of isolated myocytes to D600 (McDonald et al., 1989). In that study, increases in -type Ca2+ current were attributed to a stimulation of single Ca2+ channel currents with longer openings and fewer blanks. In the present study, we used the pure stimulatory S)(−) enantiomer of Bay K 8644 (Ferrante et al., 1989) and mibefradil exists as a single enantiomer (Clozel et al., 1991). Thus, the dual effects on Ca2+ transient amplitudes cannot be attributed to the use of racemic compounds where different enantiomers have differing effects on Ca2+ channels. Based on our results and those of others (for a review, see McDonald et al., 1989) a dual effect of compounds that bind to cardiac Ca2+ channels appears to be a relatively common occurrence that can be overlooked when a narrow range of drug concentrations is used (see Perez-Vizcaino et al., 1993, for example). Recent site-directed mutagenesis studies of -type
Figure 2 Cumulative dose–response curves for: (a) felodipine; (b) mibefradil; (c) verapamil; (d) amlodipine; (e) nifedipine; and (f) (S)-Bay K-8644 in rat (Χ) and canine (Φ) myocytes. [Ca2+]i transient amplitudes were expressed as percentage of control for each myocyte prior to data analysis. All data are mean±... for 6–10 myocytes in each group. (The standard error bars for mibefradil are smaller than the symbols at some points.) ∗ Indicates that the [Ca2+]i transient amplitude was significantly different from its respective control. Statistically significant species differences were observed at 10−8 felodipine, 10−11–10−8 verapamil, 10−8 amlodipine and 10−11–10−7 nifedipine.
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Ca channels have defined the IVS6 segment of the a1 subunit as being critical for the agonist and antagonist effects of dihydropyridines (Schuster et al., 1996). Three amino acids Tyr1485, Met1486 and Ile1493 are required for high affinity block by dihydropyridines, whereas only the first two amino acids, Tyr1485 and Met1486 are required for stimulation of the Ca channel by Bay K 8644. Because of the similarity of agonist and antagonist binding sites, it is not surprising that one compound can exhibit both agonist and antagonist effects depending on concentration. Three amino acids adjacent to that for the dihydropyridine block are required for high affinity block by the phenylalkylamines, but mutation of neither the dihydropyridine nor the phenylalkylamine sites affects the affinity of the channel for mibefradil. Other pharmacologically relevant regulatory site(s) must also exist on -type Ca channels (Schuster et al., 1996).
Acknowledgement These studies were supported in part by NIH grants HL36240 and HL48835, and DK-33727, and by gifts from Merck and Hoffmann LaRoche.
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Effects of Calcium Channel Antagonists on Ca2+ Transients in Rat and Canine Cardiomyocytes James Hensley, George E. Billman, J. David Johnson, Charlene M. Hohl and Ruth A. Altschuld Departments of Medical Biochemistry and Physiology, The Ohio State University College of Medicine, Columbus, OH 43210–1218, USA (Received 3 October 1996, accepted in revised form 27 November 1997) J. H, G. E. B, J. D. J, C. M. H R. A. A. Effects of Calcium Channel Antagonists on Ca2+ Transients in Rat and Canine Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1997) 29, 1037–1043. First-generation Ca2+ channel antagonists depress myocardial contractility, but many of the newer Ca2+ channel blockers have a high degree of “vascular selectivity”. This study compares the effects of the Ca2+ antagonists felodipine, amlodipine, mibefradil, verapamil and nifedipine, and the Ca2+ channel agonist, (S)(−)Bay K-8644 on Ca2+ transient amplitudes in fura-2/AM-loaded rat and canine ventricular cardiomyocytes. At 10−11 and 10−10 , felodipine increased [Ca2+]i transient amplitudes by 10–25% in field-stimulated fura-2-loaded cells from both species while at 10−6 it depressed [Ca2+]i transients by 80%. Mibefradil increased [Ca2+]i transient amplitudes by 16% at 10−11 and 10−10 and decreased the transients by 25% at 10−6 . The calcium channel agonist, (S)(−)-Bay K-8644 increased [Ca2+]i transient amplitudes at 10−10–10−6 (maximally 37% at 10−7 ) but depressed [Ca2+]i transients by 10% at 10−5 M. Nifedipine was inhibitory at all concentrations tested (10−11–10−6 ) in canine myocytes, but in rat cells, 10−10 nifedipine increased [Ca2+]i transient amplitudes by 37%. All concentrations of verapamil and amlodipine (10−11–10−6 ) depressed [Ca2+]i transients in both rat and canine myocytes. We conclude that: (1) felodipine and mibefradil may be positive rather than negative inotropes at low concentrations, which are therapeutically relevant; and (2) low concentrations of nifedipine may have a positive inotropic effect in the rat but not the dog heart. 1997 Academic Press Limited K W: Canine myocytes; Rat myocytes; Free calcium transients; Mibefradil, Verapamil; Nifedipine; Felodipine, Bay K 8644, Calcium channel antagonists.
Introduction Ca2+ channel antagonists are potent vasodilators widely used for the treatment of hypertension and angina pectoris (Opie, 1989). However, many Ca2+ blockers depress myocardial contractility and therefore must be used with caution in patients with poor cardiac function (Walsh, 1987; Multicenter diltiazem post-infarction trial research group, 1988; Boden et al., 1991; Hansen, 1991; Furberg et al., 1995). A new generation of dihydropyridine Ca2+ channel antagonists, including felodipine, amlodipine, isradipine, nicardipine and nisoldipine,
and the chemically novel Ca2+ antagonist, mibefradil, have improved vascular selectivity and could afford salutary reductions in afterload without negative inotropy (Clozel et al., 1989; Osterrieder and Holck, 1989; Perez-Vizcaino et al., 1993; Packer et al., 1996). Establishing whether or not Ca2+ channel antagonists directly affect myocardial contractility is not entirely straightforward, however. Negative inotropic effects in vivo can be masked by reflex neurohumoral responses. In addition, the binding of Ca2+ antagonists to Ca2+ channels is often voltage dependent, being one or more orders of magnitude
Please address all correspondence to: Ruth A. Altschuld, Department of Medical Biochemistry, 333 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210–1218, USA.
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more effective when the sarcolemma is depolarized (Sun and Triggle, 1995). Because cardiac myocytes depolarize and repolarize with each heartbeat, one cannot easily predict from ligand binding to membrane fragments the concentration dependence for Ca2+ antagonist effects on functioning cardiac Ca2+ channels. There are also species differences in myocyte electrophysiology that influence the effects of individual Ca2+ channel antagonists (Godfraind et al., 1992). In the present study, we have used electrical field stimulation of isolated adult rat and canine ventricular cardiomyocytes to compare the direct cardiac effects of the Ca2+ channel antagonists felodipine, mibefradil (Ro 40-5967), verapamil, nifedipine and amlodipine, and the Ca2+ channel agonist, (S)(−)-Bay K 8644, on intracellular free Ca2+ ([Ca2+]i) transients. Isolated myocytes eliminate concerns about complex in vivo neurohumoral responses. Canine cells were investigated because they closely resemble those from human hearts, and in vivo canine experiments have been widely used for mechanistic studies of the cardiac effects of Ca2+ channel antagonists. Rat myocytes were investigated because studies of the chronic effects of Ca2+ channel antagonists are often conducted in spontaneously hypertensive rats (Brunner et al., 1991; Lonsberry et al., 1992; Lemmer et al., 1994; Bo¨hm et al., 1995) and in the closely related spontaneously hypertensive, heart failure prone SHHF/ Mccfacp rat (Radin et al., 1993; McCune et al., 1995).
Materials and Methods
with 95% O2–5% CO2-saturated buffer containing (in m): 121 NaCl, 4.85 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1 CaCl2, 25 NaHCO3, 5 sodium pyruvate. Cells were superfused with each drug concentration for 8 min and then field stimulated at 0.2 Hz (canine cells) or 0.5 Hz (rat cells). Experiments were carried out in the dark and all solutions were wrapped in aluminum foil because some Ca2+ channel antagonists are light sensitive. Stock solutions of verapamil and Bay K 8644 were prepared in absolute ethanol. Stock solutions of nifedipine, felodipine, and amlodipine were prepared in dimethyl sulfoxide. Stock solutions of mibefradil were prepared in distilled water. Steady-state fura-2 fluorescence data were collected at 30 points per second with a PTI Filterscan. Sixteen consecutive steady-state [Ca2+]i transients were signal averaged for each experimental condition. The [Ca2+]i-dependent fluorescence ratio signals were not calibrated because 10–30% of the fura-2 free anion partitions into the mitochondria (Davis et al., 1987). This precludes accurate estimation of cytosolic free Ca2+ ion concentrations. However, changes in the amplitudes of intracellular fura-2 ratio transients reflect accurately changes in cytosolic Ca2+ transient amplitudes. When cytosolic [Ca2+]i indicators are selectively quenched with Mn2+ in rat myocytes, the cells continue to twitch in response to electrical stimulation, but the Ca2+sensitive mitochondrial signal does not exhibit beat to beat oscillations, indicating little contribution to whole cell [Ca2+]i transients (Miyata et al., 1991). Data were analyzed using single factor analysis of variance followed by the Scheffe´ test (DawsonSaunders and Trapp, 1990).
Isolated myocytes Ventricular myocytes were isolated from adult male Sprague–Dawley rats and adult mongrel dogs of either sex using previously described collagenase perfusion methods (Wimsatt et al., 1990; Hohl and Altschuld, 1991).
Fura-2 AM measurements Cells were loaded with 2 l fura-2/AM for 5 min followed by a 45–60-min post-incubation at room temperature to ensure complete hydrolysis of the acetoxymethyl ester groups and generate the Ca2+sensitive fura-2 free anion. Cells were loaded into a plexiglass superfusion chamber and allowed to attach to the bottom glass coverslip. After 10 min, superfusion at 2 ml/min, 37°C, pH 7.4, was begun
Results Figure 1 shows typical signal-averaged [Ca2+]i transients for canine myocytes incubated with varying concentrations of felodopine and nifedipine. In Figure 2 we present dose–response curves for the effects of the Ca2+ channel antagonists and agonist on relative [Ca2+]i transient amplitudes in rat and canine myocytes. Felodipine (a) significantly augmented [Ca2+]i transient amplitudes at low concentrations (10−11–10−9 ) in canine cells but became inhibitory as the concentration was raised. Low concentrations of felodipine also increased average Ca2+ transient amplitudes in rat cells, but when the dose–response curves were subjected to analysis of variance (ANOVA), the only statistically significant effect was that observed at 10−6 . By contrast, the new non-dihydropyridine Ca2+ channel antagonist, mibefradil, elicited nearly identical responses
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in rat and canine myocytes (b). Mibefradil had a small but significant stimulatory effect at low concentrations (10−11–10−10 ) and at 10−6 was the weakest negative inotrope of any of the Ca2+ channel antagonists investigated. Verapamil depressed [Ca2+]i transient amplitudes over a wide concentration range (10−11–10−6 ) in both rat and canine myocytes (c) and the canine cells were significantly more sensitive to the negative inotropic effects of the drug. Like verapamil, amlodipine depressed [Ca2+]i transient amplitudes throughout the concentration range examined, but there was a minimal species difference (d). The effects of nifedipine, on the other hand, were strongly species dependent (e). Nifedipine gave a biphasic dose– response curve for rat myocytes, significantly augmenting [Ca2+]i transient amplitudes at 10−11–10−9 but inhibiting [Ca2+]i transients at higher concentrations. By contrast, in canine myocytes, nifedipine had only an inhibitory effect. Like felodipine, mibefradil, and nifedipine (in the rat), the Ca2+ channel agonist, (S)(−)-Bay K 8644, also gave biphasic dose–response curves (f) with maximal stimulation at 10−7 and 20% inhibition at 10−5 . Unlike felodipine and nifedipine, however, the effects of (S)(−)-Bay K 8644 on Ca2+ transient amplitudes in rat and canine myocytes were nearly identical.
Discussion There are two novel and important results of this study. First, the newer Ca2+ channel antagonists,
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mibefradil and felodipine, did not depress [Ca2+]i transient amplitudes at therapeutically relevant concentrations in isolated rat or canine ventricular cardiomyocytes. In fact, low concentrations of these drugs had slight stimulatory effects, which reached statistical significance for canine cells treated with felodipine and for both rat and canine cells treated with mibefradil. Second, there are species differences in the responses of field stimulated adult mammalian ventricular cardiomyocytes to some Ca2+ channel antagonists, especially nifedipine. Our data with felodipine are in agreement with other studies demonstrating a positive inotropic effect of this Ca2+ channel blocker in dogs. In vivo studies by Cheng et al. (1994) have shown that felodipine (but not nifedipine or amlodipine) exhibits a significant positive inotropic effect in the presence of autonomic blockade in canine myocardium. Further, Pettersson et al. (1987) have shown a positive inotropic effect with felodipine in the isovolumically contracting canine heart. Our studies suggest that the positive inotropic effects reported by these investigators are due to an elevation of peak systolic [Ca2+]i. Positive inotropic effects of mibefradil have not previously been described, but most in vitro studies have investigated higher drug concentrations. In vivo, mibefradil is reported only to be devoid of negative inotropic effects (Clozel et al., 1991; Portegies et al., 1991). As expected, the first generation Ca2+ channel antagonist, verapamil, depressed [Ca2+]i transient amplitudes in both rat and canine myocytes throughout the concentration range investigated. This phenylalkylamine has minimal vascular selectivity and is capable of precipitating congestive heart failure when myocardial contractility is otherwise compromised (Walsh, 1987; Godfraind et al., 1992). The inhibitory effects of nifedipine on [Ca2+]i transient amplitudes in canine myocytes were also predictable on the basis of its negative inotropic effects demonstrated in vivo in both awake and anesthetized dogs (Cheng et al., 1994; Pagel et al., 1994). The augmentation of rat myocyte [Ca2+]i transients by 10−11–10−9 nifedipine was unanticipated, but it might explain the opposite cardiac effects of nifedipine and verapamil observed in the SHHF/Mcc-facp rat. Antihypertensive doses of nifedipine cause a marked regression of cardiac hypertrophy in these heart failure-prone, obese, hypertensive animals (Radin et al., 1993), but comparable blood pressure reduction with verapamil exacerbates cardiac hypertrophy and precipitates decompensation and premature death from congestive heart failure (Park et al., 1996).
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The deleterious response to verapamil was assumed to be associated with its negative inotropic effects, but this hypothesis had been difficult to reconcile with the positive response to nifedipine in the same animal model. The present demonstration of a positive rather than negative inotropic effect of low concentrations of nifedipine in the rat could explain this apparent discrepancy. This hypothesis is currently being tested using in vivo echocardiography of SHHF/Mcc-facp rats treated with antihypertensive doses of nifedipine or verapamil. The large species difference between the responses of rat and canine myocytes to nifedipine was especially striking, but significant species differences were also observed with verapamil and felodipine. Each of these compounds was more effective in reducing [Ca2+]i transients in canine than in rat myocytes. It is not clear whether these differences can be attributed to structural differences in the -type Ca2+ channels. However, dihydropyridine binding to isolated cardiac sarcolemmal fragments is very similar across species (Janis et al., 1987) and exhibits a complex voltage dependency (Sanguinetti and Kass, 1984; Godfraind et al., 1992). Resting membrane potentials in the rat and canine myocytes prepared in our laboratory are both close to −80 mV, but the rat cells have an abbreviated action potential (APD50=20–30 ms, Li et al., 1989). Thus, even though the rat myocytes in the present study were paced more rapidly than the canine cells, to reflect the more rapid in vivo heart rate of the rat, the brief action potential could have limited the time available for depolarization-induced Ca2+ antagonist binding to the rat myocyte Ca2+ channels. By extension, the prolonged canine action potential (APD50=300–400 ms; Freeman and Li, 1991) could have favored the antagonistic form of binding of some of the voltage-sensitive Ca2+ channel modulators. Thus, action potential duration may be an important factor in determining the effects of varied concentrations of nifedipine, felodipine and verapamil on cardiac [Ca2+]i transients. Effects of the Ca2+ channel activator, (S)(−)-Bay K 8644, were nearly identical in rat and canine myocytes, consistent with the observation that binding of this dihydropyridine is not influenced by membrane potential in cardiac myocytes (Ferrante
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et al., 1989). Inhibition of [Ca2+]i transients in both species at high concentrations of (S)(−)-Bay K 8644 is in agreement with previous reports (Kass, 1987; Bechem and Hoffmann, 1993). Effects of mibefradil were also the same in rat and canine myocytes. Nevertheless, the effects of mibefradil on inward Ca2+ currents in guinea-pig myocytes are strongly influenced by the experimentally imposed holding potential (Liang-min and Osterrieder, 1991). At a membrane holding potential of −80 mV, the IC50 is 12 l, a value comparable to that shown in Figure 2 for field-stimulated rat and canine myocytes. However, the IC50 declines >50fold when the holding potential is brought to a moderately depolarized value of −50 mV (Liangmin and Osterrieder, 1991). Thus, while mibefradil may be insensitive to action potential duration, it is extraordinarily sensitive to resting membrane potential. Bezprozvanny and Tsien (1995) concluded that the strong voltage dependency of mibefradil most likely results from a preferential binding to the open and inactivated state of the type Ca2+ channel. The fact that dihydropyridines can have opposing effects on myocardial contractility depending on concentration was first reported by Thomas et al. (1984). The stimulatory effect at low drug concentrations is probably analogous to the increases in -type Ca2+ current seen after a brief exposure of isolated myocytes to D600 (McDonald et al., 1989). In that study, increases in -type Ca2+ current were attributed to a stimulation of single Ca2+ channel currents with longer openings and fewer blanks. In the present study, we used the pure stimulatory S)(−) enantiomer of Bay K 8644 (Ferrante et al., 1989) and mibefradil exists as a single enantiomer (Clozel et al., 1991). Thus, the dual effects on Ca2+ transient amplitudes cannot be attributed to the use of racemic compounds where different enantiomers have differing effects on Ca2+ channels. Based on our results and those of others (for a review, see McDonald et al., 1989) a dual effect of compounds that bind to cardiac Ca2+ channels appears to be a relatively common occurrence that can be overlooked when a narrow range of drug concentrations is used (see Perez-Vizcaino et al., 1993, for example). Recent site-directed mutagenesis studies of -type
Figure 2 Cumulative dose–response curves for: (a) felodipine; (b) mibefradil; (c) verapamil; (d) amlodipine; (e) nifedipine; and (f) (S)-Bay K-8644 in rat (Χ) and canine (Φ) myocytes. [Ca2+]i transient amplitudes were expressed as percentage of control for each myocyte prior to data analysis. All data are mean±... for 6–10 myocytes in each group. (The standard error bars for mibefradil are smaller than the symbols at some points.) ∗ Indicates that the [Ca2+]i transient amplitude was significantly different from its respective control. Statistically significant species differences were observed at 10−8 felodipine, 10−11–10−8 verapamil, 10−8 amlodipine and 10−11–10−7 nifedipine.
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Ca channels have defined the IVS6 segment of the a1 subunit as being critical for the agonist and antagonist effects of dihydropyridines (Schuster et al., 1996). Three amino acids Tyr1485, Met1486 and Ile1493 are required for high affinity block by dihydropyridines, whereas only the first two amino acids, Tyr1485 and Met1486 are required for stimulation of the Ca channel by Bay K 8644. Because of the similarity of agonist and antagonist binding sites, it is not surprising that one compound can exhibit both agonist and antagonist effects depending on concentration. Three amino acids adjacent to that for the dihydropyridine block are required for high affinity block by the phenylalkylamines, but mutation of neither the dihydropyridine nor the phenylalkylamine sites affects the affinity of the channel for mibefradil. Other pharmacologically relevant regulatory site(s) must also exist on -type Ca channels (Schuster et al., 1996).
Acknowledgement These studies were supported in part by NIH grants HL36240 and HL48835, and DK-33727, and by gifts from Merck and Hoffmann LaRoche.
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