in conscious dogs was unaffected by diltiazem, increased by nifedipine, and decreased ... The effects of verapamil and diltiazem, but not the effect of nifedepine,.
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Chronotropic, Inotropic, and Vasodilator Actions of Diltiazem, Nifedipine, and Verapamil A Comparative Study of Physiological Responses and Membrane Receptor Activity R.W. Millard, G. Grupp, I.L. Grupp, J. DiSalvo, A. DePover, and A. Schwartz From the Department of Pharmacology and Ceil Biophysics, Department of Internal Medicine, Division of Cardiology, and the Department of Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio SUMMARY. The three major, chemically distinct calcium channel-blocking drugs, diltiazem, nifedipine, and verapamil, produce coronary vasodilation in the conscious dog. Coronary vascular resistance was reduced by 50% with an intravenous dose of 3 /Ag/kg nifedipine, 30 /Ag/kg verapamil, and 100 /Ag/kg diltiazem. In conscious dogs, nifedipine and verapamil increased heart rate, whereas diltiazem produced a smaller increase in heart rate. The rate of left ventricular pressure development in conscious dogs was unaffected by diltiazem, increased by nifedipine, and decreased by verapamil. Tachycardia was reversed to bradycardia and consistent negative inotropic effects were demonstrated by all three drugs only after combined autonomic blockade with atropine and propranolol in conscious dogs. In isolated dog coronary artery strips contracted ex vivo with 50 ITIM potassium chloride, the ID50 for relaxation was 0.01 /AM for nifedipine, 0.02 /AM for verapamil, and 0.30 /AM for diltiazem. In isolated ex vivo hearts, all agents produced dose-dependent negative chronotropy with a 25% reduction in spontaneous heart rate achieved by 0.09 /AM nifedipine, 0.20 /AM verapamil, and 0.40 JUM diltiazem. Similarly, the rate of force development in isolated myocardial strips was 50% depressed by nifedipine, 0.03 /AM; verapamil, 0.10 JUM; and diltiazem, 0.40 /AM. On a membrane level, nifedepine, verapamil, and diltiazem interacted with a putative receptor or site associated with a calcium channel specifically labelled with [3Hlnimodipine. The specific binding to cardiac sarcolemma was competitively inhibited by nifedipine, only partly inhibited by verapamil, and was stimulated by diltiazem. The effects of verapamil and diltiazem, but not the effect of nifedepine, occurred at pharmacologically active concentrations. Considerable nonspecific binding of dihydropyridines to sarcolemma may account, at least in part, for discrepancies between their dissociation constants on purified sarcolemma and their ED50 in pharmacological effects. Diltiazem and verapamil (1 /AM) did not alter [3H]nimodipine nonspecific binding. These results strongly suggest that calcium channel-blocking drugs may have different sites of action. (Circ Res 52 (suppl I): 29-39, 1983)
FLECKENSTEIN (1977) first coined the term "calcium antagonist" to describe the cellular actions of verapamil and methoxyverapamil or D-600. Subsequently, diltiazem and nifedipine have been included in this "class" of drugs. Accumulating evidence provided by many, including Van Breemen et al. (1981), Schwartz et al. (1981), and Naylor and Grinwald (1981), has led to alternative classifications (calcium channel blockers, slow channel inhibitors, calcium entry blockers) which attempt to reflect more clearly the cellular actions of these organic compounds. At the present time, however, since mechanisms appear rather complex and poorly understood, none of the names for these drugs would appear to be more advantageous or descriptive than the original. Relaxation can be produced in isolated vascular smooth muscle by diltiazem (Van Breemen et al., 1981), nifedipine, and verapamil (Vanhoutte, 1981) by inhibition of calcium influx. The negative inotropic action of these drugs in cardiac muscle and the relaxation of vascular smooth muscle can be reversed, noncompetitively, by addition of extracellular calcium implying
a similarity, perhaps, of calcium channels in smooth and cardiac muscle. Electrophysiological evidence suggests that each calcium antagonist may have different actions and, possibly, different mechanisms by which. cardiac action potential effects are exerted (Lathrop et al., 1982). These effects are distinct from those produced by inorganic calcium antagonists like nickel (Ni++) which shortens the plateau phase and decreases the action potential duration (Morad and Tung, 1982). Recently, with the availability of a highly radioactive tritiated member of the nifedipine family (nitrendipine), several investigators have identified a high affinity specific binding to homogenates, microsomes, or purified sarcolemma from heart, coronary arteries, aorta, ileum, and brain (Bellemann et al., 1981; Bolger et al., 1982; DePover et al., 1982; Ehlert et al., 1982a, 1982b; Marangos et al., 1982; Murphy and Snyder, 1982; Williams and Tremble, 1982). The binding appears to be competitively inhibited by dihydropyridines, the potency of which corresponds in some experiments to their pharmacological order of po-
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Circulation Research/Suppl. I, Calcium Channel-Blocking Drugs/Vol. 52, No. 2, February 1983
tency (Bolger et al., 1982). However, the binding is only partly inhibited by verapamil (Ehlert et al., 1982a, 1982b; Marangos et al., 1982), but, surprisingly, is stimulated by diltiazem (DePover et al., 1982), suggesting that different receptor sites, or perhaps different components of a single receptor, may be occupied by members of the calcium antagonist "class." However, the discrepancy between the [3H] nitrendipine binding KD and the nitrendipine pharmacological ED50 in cardiac muscle (but not so in coronary arteries) raises very important questions about mechanisms (DePover et al., 1982). As we have indicated above, calcium antagonists— through their action at cell membranes—have significant effects on cardiac and vascular smooth muscle. Spontaneous pacemaker activity is slowed by these agents (Taira, 1979; Kawai et al., 1979), but heart rate in the intact animal is either unchanged, in some cases diminished, or, more frequently, increased (Millard et al., 1982). Similarly, force and pressure development in isolated heart strips and whole hearts is decreased by calcium antagonists (Schwartz et al., 1981), whereas, such cardiac depression is absent or transient in intact animal preparations (Millard et al., 1982). Taira (1979) reported that nifedipine was most potent and diltiazem the least potent in depressing force developments in heart muscle. The same order of potency prevails with effects on heart rate (Kawai et al., 1979). More recently, however, Henry et al. (1979) and Perez et al. (1982), using isolated guinea pig atrial preparations, presented a different order of potency for rate depression i.e., diltiazem, verapamil, and nifedipine. Thus, the negative chronotropic action of calcium antagonists is widely accepted, but the order of potency appears to be unresolved and is indeed a very important problem, particularly with respect to clinical application. Little argument exists regarding the action of these compounds on vascular smooth muscle and, thereby, on calculated vascular resistance in the intact circulation. Fleckenstein in his review (1977) indicated that, in isolated potassium-contracted bovine coronary artery strips, nifedipine is a more potent relaxant than either diltiazem or verapamil. Nagao et al. (1982) showed that diltiazem-induced relaxation was opposed by calcium added to the superfusate. Blood pressure has been shown to fall with all three compounds, but this measurement alone may be deceptive, in that it is a product of effects on cardiac pump function and the peripheral vascular resistance. In various preparations, coronary blood flow has been shown to increase after administration of diltiazem (Bache and Dymek, 1982; Lathrop et al., 1981; Millard, 1980; Schwartz et al., 1981), nifedipine (Bache and Tockman, 1982; Gross et al., 1979; Hintze and Vatner, 1982; Kawata and Kinjo, 1980), and verapamil (Lathrop et al., 1982). However, we are not aware of any study that compares the responses of resistance vessels in the intact circulation and contrasts it with responses in isolated coronary arterial strips from the conduit vessels of the same species.
Studies in the intact animal indicate that heart rate is decreased by diltiazem, nifedipine, and verapamil when initial heart rate is high as during barbiturate anesthesia (Nagao et al., 1980). However, in intact conscious animal preparations, heart rate always increases after intravenous diltiazem (Franklin et al., 1980; Nakaya et al., 1981), nifedipine (Bache and Tockman, 1982; Nakaya et al., 1981), and verapamil (Nakaya et al., 1981). Only at high doses (>300 /ig/ kg, iv, bolus) of diltiazem or verapamil, where atrioventricular conduction is impaired so as to cause dissociation, does ventricular rate decline. However, even at these doses, atrial rate is not decreased. In contrast, nifedipine does not appear to cause either atrioventricular dissociation in the intact conscious preparation; neither does it slow heart rate. However, we have shown that nisoldipine, a member of the nifedipine family, can slow atrioventricular conduction time (Lathrop et al., 1982) in anesthetized dogs, with blockade of the autonomic nervous system. Thus, all of the above and more suggest that, despite the classification of diltiazem, nifedipine, and verapamil as calcium antagonists, these compounds affect cardiovascular variables in qualitatively different ways. This suggests the intriguing possibility that their cellular mechanism(s) of action may be partly or wholly distinct from one another (Fig. 1). It is certain that they all can inhibit the exchange of extracellular calcium across the sarcolemma to the intracellular space in cardiac and vascular smooth muscle. What is less clear, from increasing numbers of membrane radioligand binding/displacement studies, is whether these effects are mediated by a common receptor. Accordingly, we will present data from studies in conscious dogs treated with diltiazem, nifedipine, and verapamil, from experiments in isolated cardiac and Calcium Channel Blocking Drug HjCO.
M,CO—(/
y-C(CH,ljNCH,CH
OCH 5
CHICHjl,
Verapamil
OCH,
Diltiazem
CHjCOO^V.^-OOCCH,
Nifedipine FIGURE 1. Calcium channel-blocking drugs.
Millard et al./Vasodilation and Cardiac Inotropy by Ca++ Blockers
vascular smooth muscle, and from experiments with 3 H-nimodipine binding and displacement on cardiac muscle membranes. We hope to begin to provide a framework for understanding the cardiovascular actions of these calcium antagonists.
Methods Chronically Instrumented Dogs Two separate groups of dogs were prepared for examination of systemic hemodynamic cardiac inotropic and chronotropic and coronary vascular responses to the calcium channel-blocking drugs. In one group, five dogs (weighing 27-40 kg) were instrumented with arterial pressure catheters and left ventricular solid state microtransducers (Konigsberg, P-22) for registration of arterial and left ventricular blood pressures and left ventricle dP/dt maximum. In the second group (24-37 kg), four dogs were instrumented with precalibrated electromagnetic flow transducers (Statham, SP2200) and distal occlusive cuffs on the left circumflex coronary artery and a solid state pressure transducer in the aorta. One week after the aseptic surgery, when dogs were active, healthy, and free of infection, studies were conducted as they rested in the right lateral recumbent position while continuous recordings of heart rate, lead II of the electrocardiogram, mean and pulsatile blood pressure, mean and pulsatile coronary blood flow, and left ventricular pressure, and its first derivative dP/dt, were made on a rectilinear recorder (Gould, Mark 2800). Each calcium channel-blocking drug was injected by rapid intravenous push with incremental doses separated by 10-30 minutes. Only one calcium antagonist was given to each dog on a given day. The order in which the drugs were given was decided by a rotating sequence design. Diltiazem and verapamil upper dose range was established by appearance of atrioventricular conduction dissociation, i.e., complete AV block. Nifedipine did not have this effect in conscious dogs, and its upper dose limit was established at the level which did not decrease systemic diastolic blood pressure substantially further than the previous dose. Parasympathetic and /?-adrenergic blockade was achieved by administration of atropine (0.1-0.2 mg/kg) and propranolol (0.5 mg/kg), intravenously. Peak inotropic and chronotropic responses to diltiazem (200 jig/kg), nifedipine (50 fig/kg), and verapamil (250 Mg/kg) were obtained before and after this combined blockade of the autonomic nervous system. The effectiveness of the blockade was tested by lack of blood pressure decline and tachycardia to isoproterenol, 0.03 jug/kg. Abolition of similar effects to acetylcholine (10 /xg/kg) has been shown in conscious dogs receiving the atropine dose used (Barron and Bishop, 1981). Isolated Hearts and Cardiac Muscle Strips Hearts from heparinized (2500 U/kg) guinea pigs were rapidly excised after pentobarbital sodium anesthesia (50 mg/kg) and immediately suspended for retrograde aortic perfusion of the coronary arteries with Krebs-Henseleit balanced salt solution containing (in ITLM): 118 NaCl, 4.7 KC1, 2.5 CaCli 1.2 MgSO4, 25 NaHCO3, 11.5 glucose, 0.5 NaEDTA, pH 7.4. The total Ca++ and K+ concentrations were 2.0 and 5.9 ITIM, respectively. Spontaneous cardiac rate was stable under perfusion conditions at 37°C. Left ventricular chamber pressure was measured through an indwelling apical catheter attached to a calibrated transducer (Statham P23Db). Incremental and
1-31
cumulative concentrations of the calcium channel-blocking drugs were separately added to the perfusion reservoir, and the effects of one drug examined in each heart. Strips of right ventricular and right atrial muscle were taken from guinea pig heart and from canine atrial and ventricular trabeculae and suspended in a water-jacketed temperature-controlled bath containing 50 ml of balanced Krebs-Henseleit solution at pH 7.4, 37°C for guinea pig strips, 35°C for dog trabeculae. Preload tension was set at 1 g. Tension was measured with a linear displacement force transducer (Grass FT.03C). After a 90-minute equilibration period, cumulative dose-response curves were constructed from force decrement and decline in dF/dt produced by the calcium-blocking drugs. The strips were paced at a rate of 180 beats/min (guinea pig) and 90 beats/min (dog). Data were collected on five atrial and five ventricular trabecular strips from dog heart for each drug and for five atrial and ventricular strips of guinea pig heart. Each dose was allowed 15 or 60 minutes of equilibration time, during which responses were registered before the next higher dose was added to the bathing solution. Coronary Artery Vascular Muscle Arterial smooth muscle strips were prepared from canine circumflex coronary arteries according to methods described previously (DiSalvo and Schmidt, 1976; Berner et al., 1980; Silver et al., 1982). Briefly, the circumflex artery was dissected from the heart within 15-30 minutes after the animal was killed with an intravenous overdose of pentobarbital sodium and was carefully freed of adhering myocardial and adipose tissue. Four to eight strips measuring 68 mm long and 2-4 mm wide were cut from a circular section of the artery and mounted in separate muscle chambers for recording isometric tension (Grass FT.03C). The strips were stretched passively to optimal length by imposing a resting tension of 4 g. Each strip was incubated in a balanced salt solution (BSS, 20 ml) maintained at 37°C, pH 7.3-7.4, and equilibrated with 95% O2, 5% CO2. The composition of BSS (in mM) was NaCl, 130; KC1, 4.7; KH2PO4, 1.18; MgSO4-7H2O, 1.17; CaCl2-2H2O, 1.6; NaHCO3, 14.9; CaNa2 versenate, 0.026; and dextrose, 5.5. After 90 minutes of equilibration, the strips were challenged three or four times with 50 mM KG, a dose that produced 90-95% of the maximal increase in isometric force attainable with KG. As in previous studies reported from this laboratory (DiSalvo and Schmidt, 1976; Berner et al., 1980; Silver et al., 1982), strips in which repeated responses to KG did not agree within 10% of each other or which failed to attain 90% of the expected response (1.95 g isometric tension) were omitted from further study. Accordingly, 24 of the 61 strips prepared were discarded. Strips then were contracted with 50 mM KG for 15 minutes (maximal isometric force was developed in 7-10 minutes) and cumulative dose-response data were obtained for verapamil (0.1 run to 30 /IM), diltiazem (0.1 nM to 30 fiM), and nifedipine (1.0 nM to 10 JUM). Each strip was exposed to only one Ca++ antagonist; however, in any given experiment the relaxant effects of the antagonists were studied in strips from the same heart. Experiments with nifedipine were performed in the presence of a sodium light source to avoid photolysis-induced degradation (Ebel et al., 1978). Relaxation in each strip was expressed as a percentage decrease in isometric force produced with reference to the 50 mM KG response set as the baseline to contracted level. Results are expressed as means ± 1 SE. Dose-response curves were constructed by logistic fit according to the computer program developed by DeLean et al. (1978). This analysis permits simultaneous analysis of
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Circulation Research/Suppl. I, Calcium Channel-Blocking DrugsA'ol. 52, No. 2, February 1983
all collected data points in terms of minimal and maximal responses, steepness and ED50. Statistical significance of differences in ED50 and steepness (slope) between drugs was assessed with multiple regression analysis.
TABLE 1
Heart Rate Maxima and Arterial Blood Pressure Minima after Intravenous Diltiazem, Nifedipine, and Verapamil in Conscious Dogs Heart rate change (beats/ min)
Mean arterial blood pressure change (mm Hg)
Baseline (n = 17)
85 + 4
106 ± 7
Diltiazem (/ig/kg, n = 5) 3 30 300
1 ± 1 13 ± 4 63 ± 8
-3 ± 3 -12 ± 3 -31 ± 5
Nifedipine (/ig/kg, n = 6) 0.1 1 10
4 ±4 18 ± 3 77 ± 11
-1 ± 1 -8 ±3 -32 ± 4
Verapamil (jug/kg, n = 5) 3 30 300
4± 3 24 ± 7 83 ± 9
-1 ± 1 -14 ± 2 -42 ± 5
[3H]Nimodipine Binding to Cardiac Sarcolemma Purified sarcolemma was prepared from dog heart according to the method of Van Alstyne et al. (1980). [3H] Nimodipine specific binding (160 Ci/mmol) was assayed by a membrane filtration technique using Whatmann® GF/F glass fiber filters, as described previously (DePover et al., 1982). Since, by this technique, the bulk of nonspecific binding to sarcolemma is washed out, we have estimated the nonspecific binding by gel filtration technique. G25 Sephadex 80 X 5 mm columns were equilibrated with 10 ml of buffer containing [3H]nimodipine and 10~6 M unlabeled nimodipine or nifedipine. Samples (0.1 ml) containing protein and [3H]nimodipine were filtered and radioactivity was measured in 15-20 samples of eluate. Radioactivity peaks were obtained at elution times identical to that of dextran blue as a standard protein. Drug Sources and Solutions Diltiazem (gift from Marion Laboratories, Inc.) and verapamil (gift from Knoll) were dissolved in physiological salt solution and further diluted as needed for each experiment. Nifedipine (gift from Pfizer Pharmaceuticals) was dissolved in 15% polyethelene glycol:70% saline by volume and protected from light throughout preparation and use. [3H]Nimodipine and nimodipine were gifts of Miles Laboratory (Dr. A. Scriabine). Statistical Methods Employed Dose-response data obtained in isolated tissues are presented as semilog plots and resulted in sigmoid curves. The data points presented on these curves represent mean ± SE of at least four experiments (dogs). The data were submitted to a program which assigned a best fit polynominal line to the data, calculated the ED50 or ID50 with confidence limits, and determined the slope with confidence limits of the most linear and steepest portion of the dose-response relation. This analysis was obtained by the logistic fit computer program of DeLean et al. (1978). Differences in the ED50 or ID50 values were examined using multiple linear regression analysis with P < 0.05 established as the criterion for rejection of the null hypothesis. Data from conscious dogs were examined by multivariate analysis to determine when changes from baseline values occurred. Intergroup (drug) and intragroup (autonomic blockade) data were treated with ANOVA, and Duncan's test was applied. Again, statistical significance was achieved when P became < 0.05.
Results Heart Rate, Left Ventricular dP/dt, and Blood Pressure in Conscious Dogs All doses of the three drugs produced transient tachycardia and dose-dependent reductions in systemic arterial blood pressure (Table 1). Equivalent systemic arterial hypotension of 8-12 mm Hg was obtained with diltiazem (30 /ig/kg), nifedipine (1 jug/ kg), and verapamil (30 jig/kg). These reductions in mean blood pressure after rapid intravenous administration were transient. Blood pressure returned to
All values are expressed as mean ± SE. All drugs injected intravenously over 10-15 seconds.
baseline values (106 ± 7 mm Hg) within 30 minutes at these doses. At the times at which blood pressure was minimal, heart rate was elevated from a baseline of 85 ± 4 beats/min, by 13 beats with diltiazem, by 18 beats with nifediepine, and by 24 beats with verapamil. When doses of all three drugs were increased 10-fold, blood pressures fell by 30-40 mm Hg and heart rate increased by 60-80 beats/min. At these doses, diltiazem and nifedipine reduced blood pressure equivalently, while verapamil had a more potent effect. Heart rate was increased more by nifedipine than diltiazem, at equal blood pressures, and was increased most by verapamil. At these higher dose levels, the rate of left ventricular pressure development, dP/dt, was transiently depressed by nifedipine (-137 ± 80 mm Hg/sec), slightly but significantly (P < 0.05) decreased by diltiazem (—211 ± 43 mm Hg/ sec), and considerably depressed (P < 0.5) by verapamil (—1039 ± 136 mm Hg/sec) from baseline of 2639 ± 80 mm Hg/sec. Coronary Blood Flow in Conscious Dogs (Fig. 2) Dose-dependent blood flow increases in the coronary circulation followed intravenous rapid injection of all three calcium-blocking drugs. Nifedipine was the most potent, having its half maximal effect on blood flow at a dose of slightly more than 1 jug/kg. The basal blood flow in the left circumflex coronary artery (45 ± 4 ml/min) was increased to a maximum of 228 ± 22% at a dose of nifedipine, 10 /xg/kg body weight. Diltiazem and verapamil increased this baseline blood flow by 164 ± 36% and 161 ± 17% at doses of 300 /ig/kg body weight. Higher doses of these two drugs produced significant atrioventricular conduction delays and occasional atrioventricular conduction blockade. The half-maximal dose of diltiazem or flow
Millard et al./Vasodilalion and Cardiac Inotropy by Ca ++ Blockers TABLE 2
250 r~
Half Maximal Inhibition of Force Developed [ID 50 (MM)1 i n Isolated Dog Atrial and Ventricular Trabecular Muscles as a Function of Exposure Time to Diltiazem, Nifedipine, and Verapamil
Coronary Blood Flow (Baseline=45 ±4 ml/min) 200 -
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Conscious Dog (n=4)
Exposure time 15 min 100
Atria (n = 3-4)
-
Diltiazem Nifedipine Verapamil 0.1
1 3 10 30 Intravenous Dose (yg/kg)
100
300
FIGURE 2. Coronary biood flow responses to intravenous diltiazem, nifedipine, and verapami/ (VP) in the conscious dog. Data are mean values ± SE (n = 4).
was slightly more than 30 jug/kg body weight, whereas that of verapamil was at or slightly less than 30 ju,g/kg body weight. Doubling of baseline coronary blood flow was achieved with 1-2 /xg nifedipine/kg body weight, 30-40 jug verapamil/kg body weight, and 3050 jug diltiazem/kg body weight. Thus, while all may have a nearly equivalent capability of improving coronary blood flow, nifedipine is approximately 20 to 30 times more potent than either verapamil or diltiazem. Inhibition of Contractile Force (IC50) in Isolated Cardiac Muscle
The negative inotropic responses to nifedipine, verapamil, and diltiazem were compared in isolated atrial and ventricular trabecular muscles of the dog at 30°C and 1 Hz stimulation rate in Krebs-Henseleit solution (2.0 mM Ca++). All agents decreased contractile force (see Table 2) in atria and ventricles with the order of potency: nifedipine > verapamil > diltiazem. When 15- and 60-minute exposures of the cumulative doses of the agents in atrial trabeculae were compared, in Table 2, only nifedipine revealed a time dependence. This observation strongly suggests that the dihydropyridines may have multiple sites of action, and that these sites may not be restricted to the sarcolemma, but may, in fact, be located within the myocardial cell or, perhaps, on the inside surface of the sarcolemma. An alternate explanation involves a diffusional requirement in this superfused tissue preparation where drug penetration to the central myocytes of the myocardial tissue requires more time than does action at myocytes on the exposed perimeter of the tissue. In guinea pig atrial and ventricular strips, nifedipine was most potent, I50 0.045 /XM, closely followed by verapamil (0.1 JUM); diltiazem was the least potent (0.9 JUM); the atria were slightly more sensitive than the ventricles. A comparison of the negative inotropic responses in non-working guinea pig hearts perfused at 37°C
0.415±0.092 0.76±0.16 0.215±0.12
Ventricles (n = 4 ) 0.93±0.51
60 min Atria (n = 3-4)
Ventricles (n = 3-4)
0.33±0.16 0.09+0.014 0.14+0.11
0.43+0.12 0.14±0.05 0.17±0.09
ID50 values for dog atrial and ventricular trabeculae exposed to drugs for 15 and 60 minutes at each dose. Values in JIM, expressed as mean ± SE, were obtained from the average results of three or four experiments.
with Krebs-Henseleit solution containing 2 mM calcium shows that nifedipine and verapamil have an ID50 of 0.03 and 0.10 JUM, respectively. It is important to note that diltiazem is considerably less potent, the ID50 being 0.40 JUM. This relationship is also seen when the negative chronotropic response to the three drugs is compared (Table 3). However, at equal negative chronotropic effects (25% reduction), the negative inotropic effects of all three drugs are more evident than the negative chronotropic action in this preparation (Fig. 3), although, as above, diltiazem is still the least potent of the three. Thus, the negative inotropic effect assessed as the reduction of force generated by isolated strips of guinea pig atrium and ventricle and by the depression of the rate of left ventricular pressure development, dP/dt, is dose-dependent and preparation-dependent. It is important to consider the type of preparation when one compares chronotropic effects of Ca++ block obtained in vitro. For instance, in the Langendorff preparation, chronotropic responses can be produced in the SA node or by interference with AV conduction or both, whereas right atrial preparations reflect only effects on the SA node or extranodal foci. It is possible that the difference of preparations explains the difference between our observations and those of Henry et al. (1979) and, more recently, of Perez et al. (1982), who worked with spontaneously beating right atrial preparations. In all cases, the negative inotropic effects of the three drugs occur at the lowest doses in non-working Langendorff-perfused whole hearts. Isolated ventricular strips are the most resistant to the negative inotropic actions of the drugs, whereas atrial responses are intermediate in sensitivity. Diltiazem has similar negative inotropic dose-dependency in whole hearts and in isolated atria. Atrial and ventricular strips are similarly sensitive to verapamil. The increased sensitivity of the whole heart to the inotropic action of the calcium-blocking drugs may result from a more efficient distribution of the drug to the myocytes by capillaries of the coronary circulation rather
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Circulation Research/SuppJ. I, Calcium Channel-Blocking Drugs/Vol. 51, No. 2, February 1983 TABLE 3
Effects of Different Ca ++ -Blocking Agents (/IM) on Heart Rate, Contractility, and Coronary Flow of Saline-Perfused Isolated Work-Performing Guinea Pig Hearts Verapamil (/IM) (n = 5 )
Diltiazem (/IM) (n = 5 )
Nifedipine (/IM) (n = 4 )
Heart rate-inhibiting dose (I25)
0.20 ± 0.02
0.40 ± 0.04
0.085 ± 0.003
Left ventricular (dP/dt)-inhibiting dose (I50)
0.096 + 0.07
0.40 ± 0.06
0.033 ± 0.003
Coronary flow increase (ED50)
0.062 ± 0.01
0.12 ± 0.06
0.015 ± 0.004
All values are expressed as mean ± SE. All determinations were carried out using 2.0 mM Ca ++ . I25 = 25% reduction in heart rate; I50 = 50% reduction in dP/dt; ED50 = 50% increase in coronary blood flow.
than by the diffusion route required in the atrial and ventricular strips. This diffusion aspect may also explain why atrial strips appear more sensitive than ventricular strips to all three calcium blockers.
Langendorf Guinea Pig Heart (37°C) Heart Rate 100 80 60 40 o o CD
•33 in CO
m
20 0
[3H]Nimodipine Binding to Dog Heart Sarcolemma As previously reported for [3H]nitrendipine (DePover et al., 1982), [3H]nimodipine binding to cardiac sarcolemma occurred at high affinity sites (Fig. 5). The dissociation constant (KD) was estimated from Scatchard plots (Scatchard, 1949) to be 0.18 JUM, which was similar to [3H]nitrendipine KD. The specific binding of 0.18 nM [3H]nimodipine (i.e., binding displaceable by 10"6 M nimodipine) was also completely inhibited by nifedipine (I50 = 3 nM) and inhibited 55% by verapamil, but was stimulated by diltiazem. The effective concentrations of verapamil and diltiazem were respectively in the range of 10~8 to 10~6 M and 10"7 to 10"5 (data not shown).
LV dP/dt 100 r
o a>
0. in CO in
