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Clevidipine Blockade of L-Type Ca2+ Currents: Steady-State and Kinetic Electrophysiological Studies in Guinea Pig Ventricular Myocytes

Yi, Xiaobin; Vivien, Benoît; Lynch, Carl III

Journal of Cardiovascular Pharmacology: November 2000 - Volume 36 - Issue 5 - p 592-600
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Steady-state and transient effects of clevidipine, a rapidly degraded dihydropyridine (DHP) L-type Ca2+ channel antagonist, were examined on ICa in guinea pig ventricular myocytes. When myocytes were voltage-clamped with holding potential (VH) at −80 mV, 10 nM clevidipine decreased ICa at 0 mV by ∼30%, but >50% when VH was −40 mV. Rapid (<50 ms) perfusion switching and repeated depolarizations delivered at 0.5-2 Hz were used to determine the time constants of onset (τon) and recovery from (τoff) clevidipine inhibition of ICa. The τon and τoff were monoexponential functions of time. The τon of ICa inhibition decreased from 21.5 ± 1.2 to 9.9 ± 0.9 s when the rapidly applied [clevidipine] was increased from 10 to 100 nM at VH = −80 mV; τoff was independent of the applied [clevidipine] and was 23.9 ± 1.1 s. The dissociation constant (KD) calculated for clevidipine at VH = −80 mV was 65 ± 3 nM, similar to the IC50 of 78 nM determined in steady-state measurements. Decreasing VH to −40 mV increased τoff more than threefold to 81 ± 6 s, and KD was markedly decreased to 9.0 ± 0.8 nM (IC50, 7.1 nM at VH = −40 mV). The increased affinity at depolarized VH may contribute to the varying concentration-effect relation observed in vivo.

Department of Anesthesiology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.

Received March 2, 2000; revision accepted August 3, 2000.

Address correspondence and reprint requests to Dr. C. Lynch III at Department of Anesthesiology, Box 800710, University of Virginia Health System, Charlottesville, VA 22908-0710, U.S.A. E-mail: carllynch@virginia.edu

The dihydropyridines (DHPs) are a class of organic compounds that are important calcium channel inhibitors and modulators. They have been increasingly used in the treatment of arterial hypertension and ischemic heart disease (1-3). Their pharmacologic characteristics and clinical applications are attributed to inhibition of Ca2+ influx through L-type Ca2+ channels in the membranes of vascular smooth muscle cells and cardiomyocytes (4). In vivo and in vitro, DHP antagonists can inhibit smooth muscle contractions with accompanying improvements in coronary blood flow and/or decreases in blood pressure. In isolated cardiac myocytes or smooth muscle cells, these agents have been shown to bind to L-type Ca2+ channels and to inhibit their activity in a concentration-dependent manner. DHPs bind to L-type Ca2+ channels in a voltage-dependent manner (5,6), having a higher affinity for the inactivated state of the channel (7). Depolarization of the membrane potential strongly potentiates the inhibitory effects of DHP antagonists on ICa(8) and contraction, (9,10), respectively. For example, felodipine suppresses ICa with higher potency and selectivity in vascular smooth muscle cells compared with cardiac myocytes, which have a more negative resting potential (11-13). Méry et al. (14) investigated the time course of changes in ICa during rapid application and washout of nifedipine using a perfusion technique that allowed application or washout of the drug within <50 ms. Based on that kinetic investigation, nifedi-pine bound to the inactivated state with a ∼400-fold higher affinity than to the resting state, because of a ∼4-fold increase in the association rate constant (k1) and a ∼100-fold decrease in the dissociation rate constant (k−1) (14).

Clevidipine, an ultrashort-acting DHP vasodilator, is an effective relaxant of human internal mammary artery in the presence and in the absence of endothelium (15). It is rapidly degraded in the serum with a half-life of less than a minute, resulting in a rapid decrease in vasodilating action in vivo. However, its pharmacologic action still persists beyond the level expected based on its serum concentration (16). To determine if prolonged binding to its site of action could contribute to prolongation of the drug's effect, we determined clevidipine's non-steady-state inhibition of L-type Ca2+ currents in guinea pig ventricular myocytes using a rapid drug application and washout technique. A preliminary account of this work was presented at the Biophysical Society (17).

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METHODS

Single ventricular myocytes from guinea pig heart were prepared using a modification of the methods described by Mitra (18) and Tytgat (19). Guinea pigs weighing 250-400 g were rendered unresponsive by intraperitoneal injection of sodium pentobarbital (50 mg/kg) following the guidelines of the University of Virginia Animal Care and Use Committee. The heart was rapidly excised, the aorta cannulated, and perfusion initiated at 37°C with oxygenated perfusion solution (in mM: 137 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 11.6 HEPES, 5.0 D-glucose, pH 7.4 adjusted with NaOH). Continuing oxygenation at 37°C, perfusion was switched to Ca2+-free solution followed by 5 min of the same solution with added collagenase (0.12 mg/ml; Yakult, Japan) and bovine serum albumin (0.5 mg/ml; Boehringer Mannheim, Ingelheim, Germany). After enzymatic digestion, the heart was perfused to wash away residual enzyme with storage solution (in mM: 25 KCl, 10 KH2PO4, 0.5 EGTA, 70 glutamic acid, 3.0 MgCl2, 10 D-glucose, 20 taurine, 5.0 pyruvic acid, 10 HEPES, pH 7.4 adjusted with KOH). The cell suspension was collected by mincing the well-digested portion of the heart and filtering through a nylon mesh. The filtrate containing isolated cells was incubated for 1 h in storage. Only rod-shaped cells with clear striations that remained quiescent in Tyrode's solution were used for study. Cells obtained in this manner were tolerant to Ca2+ and had normal electrophysiologic and pharmacologic properties.

The whole-cell configuration of the patch-clamp technique was used to record L-type Ca2+ current (ICa) or Ba2+ current (IBa). Before establishing the whole-cell patch-clamp configuration, cells were bathed in perfusate solution at room temperature. Patch pipettes were prepared from borosilicate glass (KIMAX-51; American Scientific, Charlotte, NC, U.S.A.) using a two-stage micropipette puller (Narishige Co. Ltd., Tokyo, Japan). Pipettes typically had resistances of ∼2 MΩ when filled with internal solution (in mM: 120 CsCl, 20 TEA-Cl, 1 CaCl2, 11 EGTA-CsOH, 10 HEPES, 5 Mg-ATP, and 0.4 GTP, pH adjusted to 7.3 with 1N HCl). Once the pipette-membrane seal was formed, the membrane ruptured, and electrophysiologic control verified, the external solution was then exchanged for ICa recording solution (in mM: 125 CsCl, 20 TEA-Cl, 2 CaCl2 or 10 BaCl2, 1 MgCl2, and 10 HEPES, pH adjusted to 7.4 with 1N CsOH). K+ currents were blocked by replacing K+ with intracellular and extracellular Cs+ and by addition of TEA+; Na+ currents were eliminated by using tetrodotoxin and Na+-free recording solutions.

Currents were recorded with an Axopatch 200A patch clamp amplifier using pClamp software (Axon Instruments, Foster City, CA, U.S.A.). The data were filtered at 2 kHz with a four-pole Bessel low-pass filter, digitized at 10 kHz, and −P/4 addition was used to compensate for leakage and capacitive currents. Peak ICa was calculated as the peak minus the baseline current at return to VH. For study of current-voltage relations, voltage steps of 10 mV were applied from the VH of −80 or −40 mV. The routine protocol for fast solution change experiments consisted of a 50-ms depolarization to 0 mV from VH of −80 mV (or −40 mV) applied at a frequency of 0.2-2 Hz. Control experiments documented that a 450-ms recovery interval was sufficient to remove any residual inactivation of ICa at VH of −80 mV. After the initial depolarization at VH of −40 mV, subsequent ICa demonstrated a persisting decline of 20-30% from ICa of the first pulse, whether the recovery interval was 0.1 up to 5 s, possibly revealing inactivation from which recovery required >10 s. Repetitive depolarizations at 0.2-2 Hz showed no difference in the amplitude of ICa.

Clevidipine, amlodipine, and felodipine were gifts from Astra Hässle, Molndal, Sweden. A stock solution of 10 mM DHP agent in ethanol was diluted in recording solution to obtain the final needed concentration just before each experiment (protected from light). In control studies, an ethanol concentration of ≤2 mM, equivalent to the highest diluent concentration used, had no effect on ICa. The method of rapid solution changes is a modification from Méry et al. (14). Once a voltage-clamp seal was formed, the outlet tube array of a rapid solution changer device (SF-77 Perfusion Fast-Step; Warner Instrument Corp., Grand Haven, MI, U.S.A.) was positioned over the cell. Cardiomyocytes were thus maintained in a continuously flowing stream of buffer that was free of or included DHPs. When the outlet tube array was rapidly shifted laterally by 0.6-0.8 mm, the stream of solution superfusing the cell was changed. The resulting exchange time of solution around the myocyte was <50 ms (data not shown), as determined by changes in holding current when the perfusing K+ concentration was increased from 5 to 10 mM.

Data are presented as mean percentage of control ± SEM for n cells studied. The difference between means was tested for statistical significance of drug effect by paired or unpaired Student's t test as appropriate for the experimental design and comparison. The steady-state drug concentration that decreased peak ICa by 50% (IC50) was determined by a nonlinear least-squares fit (SigmaPlot, SPSS Inc., Chicago, IL, U.S.A.) to the Hill equation: EQUATION (1) where n is the slope factor.

After drug application, the peak ICa of each succeeding depolarization decreased at an exponential rate given by Equation (2) where ICa, t is the amplitude of ICa at time t after drug application, A is the component of ICa blocked by drug, τon is the time constant of block onset for each successive depolarization, and ICa,u is the remaining unblocked ICa after drug application. Likewise, with drug washout, ICa was restored with an exponential time course, which could be defined by Equation (3) where t is the time after the drug washout was initiated, and τoff is the time constant for the recovery of ICa with each successive depolarization. The τon and τoff were calculated by nonlinear least-squares fit to Eqs. 2 and 3, respectively. The drug dissociation rate (k−1) is defined simply by k−1 = 1/τoff. From τon, the association (k1) rate can then be calculated from the definition of τon (1/τon = k1 · [drug] + k−1). By rearrangement, the dissociation constant (KD = k−1/k1) can also be expressed as functions of τon and τoff: Equation (4)

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RESULTS

Steady-state clevidipine effects on ICa

Peak ICa in these ventricular myocytes was typically 200-500 pA and demonstrated a reversal to outward currents at 50-60 mV. Figure 1 shows the current-voltage relation of peak ICa at different holding potentials. At a holding potential of −80 mV (Fig. 1A), 10 nM clevidipine reduced ICa to 71% of the control value. In contrast, when the myocyte was clamped at −40 mV (Fig. 1B), depression of currents by clevidipine was far more profound (25% of control). Somewhat lower-amplitude ICa was typically observed when VH was −40 mV and appears to represent some ongoing inactivation at the more positive holding potential. Figure 2 compares the mean inhibition of ICa observed at a test potential of 0 mV from a holding potential of −80 or −40 mV in the presence of 10 nM clevidipine, 10 nM felodipine, and 10 nM amlodipine. All three compounds showed voltage-dependent blockade of cardiac L-type calcium channels consistent with the DHP drug class effect. At each potential, the potency of clevidipine did not differ significantly from amlodipine or felodipine. Myocyte exposure to clevidipine, amlodipine, and felodipine in the chamber perfusate inhibited ICa within ∼1 min, and reached a steady state of inhibition in 3 min. Inhibition of peak ICa by these three drugs reversed on washout, the extent of recovery depending on VH, concentration, and duration of drug application. For 10 nM clevidipine, recovery to 36 ± 5% (n = 4, VH = −40 mV) and 76 ± 7% (n = 4, VH = −80 mV) of the control value was obtained after 5 min of continuous washout.

FIG. 1

FIG. 1

FIG. 2

FIG. 2

To determine the concentration and voltage dependence of ICa inhibition, steady-state concentration-response curves (Fig. 3) for clevidipine-induced effects were determined at VH of −80 and −40 mV. For VH of −40 mV instead of −80 mV, the concentration-response curve was shifted to lower concentrations (Fig. 3), and the calculated IC50 of clevidipine for inhibition of ICa was reduced from 78.8 to 7.1 nM.

FIG. 3

FIG. 3

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Kinetic determination of clevidipine effects

Figure 4 illustrates a typical experiment showing the effect on ICa of abrupt application and washout of clevidipine in myocytes. ICa was elicited at a frequency of 1 Hz by depolarizing the cell to 0 mV for 50 ms from a holding potential of −80 mV. With the rapid application of 200 nM clevidipine, ICa decreased with a monoexponential time course, achieving a steady-state reduction to 30% of control. On washout, ICa recovered to control level as a single exponential function of time (Fig. 4D and E). Fitting the decline in peak ICa to Eqs. 2 and 3 yielded values for τon of 3.9 s and τoff of 24.3 s; another experiment with 200 nM clevidipine gave values of 2.7 and 18.1 s, respectively. From these time constants, values were calculated for k1 and k−1 of 1.3 × 106/s/M and 0.048/s, respectively. Using Eq. 4, the calculated KD for 200 nM clevidipine application and washout is 62 nM.

FIG. 4

FIG. 4

When a lower concentration of 30 nM clevidipine was applied, the onset of ICa inhibition occurred at a slower rate (τon of 12.8 ± 1.9 s; n = 6), whereas the washout of inhibition did not differ (τoff of 19.8 ± 2.9 s; n = 6). Increasing clevidipine concentration to 1,000 nM inhibited ICa by 78 ± 3% (n = 3) and, as anticipated, ICa inhibition developed at a faster rate than at lower concentrations (τon = 2.1 ± 0.1 s; n = 3). However, ICa recovered with a time course (τoff = 30.3 ± 3.0 s; n = 3), giving an average dissociation rate (k−1) of 0.034 ± 0.003/s (n = 3) for VH of −80 mV, similar to values observed during the washout of other concentrations.

When VH was altered between successive applications of drug (Fig. 5), the kinetics of clevidipine inhibition were dramatically altered. Although the effect onset rate of 200 nM clevidipine did not differ appreciably between VH of −80 and −40 mV, the ICa inhibitory effect declined at a markedly slower rate on rapid washout at the more depolarized VH. As in the steady-state experiments, fractional depression of ICa by clevidipine was more pronounced when the holding potential was at −40 mV instead of −80 mV (85% vs. 56% depression, respectively).

FIG. 5

FIG. 5

The various kinetic variables derived from studies of clevidipine at differing VH and drug concentrations are summarized in Fig. 6. For experiments in cells in which VH was −40 mV, the τon of ICa inhibition accelerated with increasing concentrations of clevidipine, as observed with VH at −80 mV. As predicted from the definition of τon where 1/τon = k1 × [drug] + k−1, 1/τon is clearly a function of [clevidipine]. In contrast, the time course of the recovery (τoff) from washout was independent of the drug concentration. At VH of −40 mV, τoff (81 ± 6 s; n = 16) was >3 times longer than for VH of −80 mV (24.1 ± 1.1 s; n = 34), so that the calculated dissociation rate (k−1) decreased from 0.046 ± 0.004/s to 0.013 ± 0.001/s. In four paired experiments in which VH was changed between separate clevidipine applications (as shown in Fig. 5), τoff was sixfold longer when the VH was −40 mV. Based on the τoff and τon values, the calculated association rate (k1) also changed with the more depolarized VH, increasing from 7.9 ± 7 × 105/s/M to 1.8 ± 33 × 106/s/M. For each VH, the calculated KD at varying concentrations remained approximately constant because of these changes of τon and τoff. The kinetically determined dissociation constant (KD) for clevidipine agrees quite closely with the IC50 determined from the classic steady-state experiments. For VH of −80 or −40 mV, the KD values determined from experiments at all concentrations were 70 ± 5 nM (n = 35) or 9.0 ± 0.8 nM (n = 16), compared with IC50 values of 79 or 7.1 nM, respectively. However, the fractional depression by any given clevidipine or nifedipine concentration with steady-state application was typically somewhat greater than the maximal inhibition achieved during the 20-60 s presence of drug rapid application.

FIG. 6

FIG. 6

To provide control for clevidipine effect, the classic DHP nifedipine was rapidly applied and washed out in a similar fashion. Figure 7A is a concentration-response curve of nifedipine for inhibition of ICa at a holding potential of −80 mV. Nifedipine (IC50, 60 nM) was of similar potency to clevidipine (IC50, 79 nM) on ICa for VH of −80 mV. Figure 7B shows the effect of a rapid application and washout of 100 nM nifedipine on ICa in a guinea pig ventricular myocyte. At −80 mV, time constants for 100 nM nifedipine (τon, 9.3 s; and τoff, 32.5 s) were only slightly greater than those of clevidipine, giving a calculated KD of 40.1 nM.

FIG. 7

FIG. 7

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DISCUSSION

The DHPs act selectively on the voltage-dependent L-type Ca2+ channels and have a distinct binding site(s) on the α1 subunit, the principal subunit that contains the voltage-gated channel pore (4). As observed with other DHPs, clevidipine caused a clear depression of L-type Ca2+ channel current in guinea pig myocytes. The calculated IC50 of clevidipine of 76 nM at −80 mV was similar to that of nifedipine, the IC50 of which in our study was 60 nM, and for which a value of 63 nM had previously been reported for guinea pig myocytes (20). A prominent feature of the DHPs is their selective depression of vascular smooth muscle contractions compared with myocardial contractions. This property has been attributed to the voltage-dependent binding and block of L-type Ca2+ channels. With increasing depolarization, the binding affinity for the channel and the degree of inhibition become greater (5,6,21-24). DHP antagonists such as nifedipine bind more avidly to Ca2+ channels when cell membranes are depolarized (7,23,25,26). Clevidipine also shows voltage-dependent inhibition of L-type Ca2+ channels typical of DHPs, becoming threefold to sixfold more potent when the holding potential was raised to −40 mV from −80 mV. This differential potency was similar to that observed for felodipine and amlodipine. Some DHPs may act as agonists at more hyperpolarized potentials (−80 mV or less) and antagonists at more depolarized potentials. For example, nitrendipine enhanced guinea pig myocyte ICa when the holding potential was −50 mV or less and the stimulation rate was 0.06 Hz; however, at more positive holding potential, nitrendipine was only inhibitory (27). We saw no evidence of a stimulatory action of clevidipine when the holding potential was −80 mV and the stimulation rate was decreased to 0.2 Hz, suggesting that this DHP has minimal potency at any stimulatory DHP site(s).

The kinetic study of onset and offset rates provided important confirmation of the steady-state results. Whereas the offset time constant (τoff) reflects the simple dissociation of the channel-drug complex, the onset time constant (τon) reflects both the concentration-dependent association (k1 × [clevidipine]) and the dissociation rate (k−1, 1/τoff). As anticipated, the onset of ICa depression with drug application was clearly concentration dependent, whereas the rate of offset was not. The results of this study are similar to those reported regarding effects of nifedipine on ICa in frog ventricular myocytes by Méry et al. (14). However, in our studies using guinea pig ventricular myocytes, the rapid application and washout of 100 nM nifedipine had a slower onset (τon of 9.3 vs. 4.8 s in frog) and washout (τoff of 32.5 vs. 21.5 s in frog), and a somewhat higher calculated KD (40 nM) than the value (27 nM) obtained from frog ventricular myocytes (14). This discrepancy may be due to the different holding potential (−80 vs. −70 mV), as well as species and cell structural differences. Based on Eq. 4, the KD of 40 nM calculated for nifedipine is at −80 mV, similar to the IC50 of 60 nM determined for ICa inhibition by equilibrium application. These results have been interpreted as the DHP having a lower binding affinity to the resting state and a higher affinity binding to the inactivated state, assuming that the depolarization shifted a fraction of Ca2+ channels from resting to inactivated states.

The offset time constant (τoff) for clevidipine was slightly faster than nifedipine (18-20 s), giving a higher estimated KD of 70 nM. This value is compatible with the equilibrium studies that demonstrate that at −80 mV, clevidipine had an IC50 of 78 nM, which was also slightly greater than that for nifedipine.

Unlike frog ventricular cells, guinea pig ventricular myocytes have a complex t-tubular network, which can result in a diffusional delay of drug delivery to Ca2+ channels located in the depths of the t-tubular network in myocytes (28). However, the association rate of drug for the channels is sufficiently slow that the delay introduced by diffusion in the t-tubular network should introduce little error into the calculation of the kinetic variables, at least at concentrations <1,000 mM. The close agreement between the IC50 derived from the steady-state measurements and the kinetically derived KD also suggests that diffusional delay introduces minimal error. With the sustained bath application, there was usually a greater fractional depression for any drug concentration than seen with rapid application, suggesting that a slowly developing component of inhibition may be present but not observed with brief application. However, if a small degree of rundown occurred during the 15- to 20-min steady-state experiments, which was not typically observed with rapid application of ≤1,000 nM clevidipine, this would be interpreted as greater apparent inhibition, and also artificially decrease the IC50.

Because the resting potential of vascular smooth muscle cells is less negative than that of cardiac myocytes, this voltage dependence may in part explain the selectivity of clevidipine in depressing contractility of vascular smooth muscle (15,29). However, the different DHP sensitivities of cardiac and vascular L-type Ca2+ channels (α1C-a and α1C-b subunits, respectively) may be caused at least in part by the tissue-specific expression of alternatively spliced IS6 segments of the α1C gene (30). In addition, Hu et al. (31) indicated that the more potent nisoldipine inhibition of smooth muscle versus cardiac L-type Ca2+ channels is not attributable to differences in channel inactivation or activation. They suggested that intrinsic, gating-independent DHP-receptor binding affinity difference must be invoked to explain the isoform-specific sensitivity of the DHP block. Nevertheless, because voltage-dependent DHP effects are present in both cardiac and vascular smooth muscle, we used cardiac myocytes as our model for this study.

Most DHPs are metabolized in vivo by the liver, resulting in a metabolic half-life of 2-5 h for oral administration. Even intravenously administered nicardipine has a half-life of 45 min, far exceeding τoff, even at depolarized potentials. In contrast, clevidipine has an ultrashort half-life in various species (20 s, rat; 22 s, dog; 12 s, rabbits; 96 s, human) because of its degradation by serum esterases or rapid glucuronidation (16,32). Slightly longer half-lives are observed in vitro (33), with values in the range of τoff observed in this study when VH was −40 mV. At the less polarized potentials of many tissues (i.e., vascular smooth muscle), the slowed rate of dissociation of clevidipine from its L-type channel receptor site (τoff, ∼80 s) will decrease the rate of drug diffusion back into the serum. When infusion of the drug is stopped, slower return to the circulating blood will decrease the rate at which it is available for hydrolysis or metabolism to its inactive metabolite, possibly extending its duration of its pharmacologic action. The similarity in time constants for drug unbinding and hydrolysis or metabolic clearance may have important implications for the pharmacodynamics of the drug in vivo. For example, the blood pressure response to intravenously administered clevidipine concentration shows distinct hysteresis. In a study in dogs, as the blood concentration was slowly increased by drug infusion, a 20% decrease in mean arterial pressure (MAP) occurred at a serum concentration of 100 nM, with a 25% MAP decrease at 300 nM. On suddenly discontinuing the drug, when the blood concentration declined to 100 nM clevidipine, MAP was still decreased by 24%. Over ∼1.2 min, [clevidipine] decreased to ∼25 nM, and the MAP decrease at that time was ∼19%, similar to that observed with the earlier 100 nM concentration (32). This hysteresis required a model in which an effect compartment was interposed to produce a delay in onset. Instead, slowed diffusion from the receptor could also explain the lack of equilibrium between concentration and effect. Because clevidipine is so rapidly hydrolyzed or cleared from the serum, its avid binding to and slowed dissociation from L-type Ca2+ channels of less polarized cells may contribute to its pharmacodynamic profile in vivo.

Acknowledgment: This study was supported by NIH grant R01-31144 and a grant from Astra Hässle to CL3. We thank Hans Ericsson and Margereta Nordlander of Astra Hässle, Mölndal, Sweden, for helpful discussion.

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Keywords:

Dihydropyridine; Calcium channels; Pharmacodynamics; Dissociation constant; Guinea pig heart

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