Interaction between calcium and slow channel blocking drugs on atrial rate

The relationship between extracellular calcium concentration and the chronotropic effect of prenylamine, verapamil and nifedipine was studied in isolated spontaneously beating rat atria. The three slow channel blocking drugs produced a concentration-dependent decrease in atrial rate, though with different relative potencies. The order of potency for decreasing atrial rate, independently of the calcium level (1.0, 3.0, 6.0 or 9.0 mmol/l) was: verapamil > nifedipine > prenylamine. Increasing calcium from 1.0 to 6.0 and 9.0 mmol/l increased atrial rate from 251±beats·min−1 to 265±6 beats·min−1 and 285±9 beats·min−1 (mean±1 standard error) respectively (P<0.05). Despite their positive chronotropic effect high calcium levels failed to reverse the negative chronotropic effect of the slow channel blockers. Furthermore, the negative chronotropic effect of both verapamil and nifedipine was enhanced at high calcium levles. Raising calcium from 1.0 to 6.0 mmol/l in the presence of verapamil (1×10−7 mol/l) or nifedipine (3×10−7 mol/l) increased 2-fold the negative chronotropic effect of the calcium channel blockers. In addition, the concentration-effect curves for verapamil and nifedipine shifted to the left by 0.50±0.14 and 0.50±0.16 log units, respectively, when calcium increased from 1.0 to 6.0 mmol/l. The data show that increasing calcium may produce positive or negative chronotropic effects depending on whether or not the calcium channels are blocked. This paradoxical effect of calcium ions can be produced either by opposite chronotropic effects on automatic cells or by shifting the pacemaker activity to a group of cells which respond in a different way to an increment of calcium.


Introduction
Although under certain circumstances every cardiac fibre is capable of initiating the cardiac impulse, only a small number of cells actually govern the normal beating of the heart.These cells, which in the mammalian heart are located within the sinus node (West 1955;Trautwein and Dudel 1958) show characteristic action potentials with a slow diastolic depolarization and a slow maximum rate of the upstroke (Brady and Hecht 1954;Hutter and Trautwein 1956;Paes de Carvalho et al. 1959;Noma et al. 1977).The electrophysiological mechanisms underlying the diastolic depolarization in the sinus node cells are not fully established yet.However, it is generally accepted that the main ionic mechanism responsible is an inward current carried by both sodium and calcium ions through the slow channels (Kohlhardt et al. 1976;Vasalle 1977;Irisawa 1978).The sensitivity of the sinus node cells to changes in extracellular calcium concentration (Reiter and No6 1959;Seifen et al. 1964a, b;Kohlhardt et al. 1976;Lu and Mc Brooks 1969) and to substances that interfere with the slow channels (Lenfant et al. 1968;Zipes and Fischer 1974;Refsum and Landmark 1975;Kohlhardt et al. 1976) is in agreement with the role played by calcium in the slow inward current.However, the contribution of sodium ions to the slow inward current is also of major importance (Noma et al. 1977).
Among the substances that can interfere with the slow channels, specific inhibitors have been developed 9 These inhibitors share the common property of selective inhibition of the slow calcium inward current in the cardiac fibre and vascular smooth muscle (Kohlhardt et al. 1972;Fleckenstein 1977).This property is the basic feature that determines the pharmacological effects of these drugs, that is their negative chronotropic and inotropic effects, their antiarrhythmic actions and their vasodilating activities (Cranefield et al. 1974;Zipes and Fischer 1974;Fleckenstein 1975Fleckenstein , 1977;;Fleckenstein and Grtin 1975;Refsum and Landmark 1975;Refsum et al. 1976;Sugiyama et al. 1981).
On the basis of the assumption that the mode of action of the slow channel blockers is by interfering with the calcium inward current in the cardiac fibre, the cardiac effects of these drugs should be counteracted by increasing the calcium concentration in the media 9 This proved to be true for the negative inotropic effects of the slow channel blockers (Fleckenstein 1975;Refsum 1975).As regards their negative chronotropic effects the experimental results are controversial.Whereas the negative chronotropic effect ofverapamil on automatic Purkinje fibres (Cranefield et al. 1974) and of nifedipine on isolated rat atria (Refsum 1975) was reversed by increasing the calcium concentration, other investigators have been unable to obtain with calcium the reversal of the negative chronotropic effects of slow channel blocking drugs (Zipes and Fischer 1974;Fleckenstein 1977;Kohlhardt et al. 1976).
The present experiments were performed to study the relationship between the negative chronotropic effects of three slow channel blocking drugs: prenylamine, verapamil and nifedipine and the extracellular calcium concentration.

Material and Methods
Experiments were performed on spontaneously beating atria isolated from adult rats weighing 250-300 g.The animals were anaesthetized with sodium pentobarbitone (8 mg/kg, i.p.).The hearts were quickly excised and rinsed in saline solution.Both atria were dissected free from the adjacent tissue in an oxygenated saline solution at room temperature.Immediately the atria were placed, usually two preparations in parallel, in a 50-ml bath.One end of the atria was fixed by a clamp to a holder that had two platinum punctuate electrodes which allowed the recording of atrial electrograms.The other end was fixed, by means of a stainless steel wire, to a displacement transducer to record the spontaneous atrial contractions.Atrial rate was determined from the records of the isotonic contractions and atrial electrograms.The millimolar composition of the saline solution used was: NaC1 128.3;KC14.69;MgSOal.05;NaHCO320.2;NaH2PO4 1.05; CaC12 1.0 and dextrose 11.0.Calcium concentration in the test solution was increased to 3.0, 6.0 or 9.0 mmol/1 by adding small amounts of a concentrated solution of CaC12.The addition of CaC12 led to a concomitant increment in C1concentration and the osmolarity in the bathing solution.To be sure that the effects of increased CaClz could be ascribed to Ca 2 + and not to C1 or raised osmolarity we performed control experiments by adding appropriate amounts of NaC1.The saline solution was bubbled with a gas mixture of 5 % CO2 in 95% 0 2 and the pH of the medium was 7.38 + 0.01.All the experiments were carried out at 37 ~ C. Before any drug was added to the media the preparations were equilibrated during 60min and the saline solution was replaced every 10 rain during this period.At the end of the equilibration period the atrial rate reached a steady-state value in the majority of the preparations and remained thereafter stable for at least 3 to 4 h.Those preparations showing an unstable rate at the end of the equilibration period were discarded.
Two groups of experiments were performed.In one of them cumulative concentration-effect curves for each slow channel blocking agent were determined at 4 different concentrations of calcium in the saline solution: 1.0, 3.0, 6.0 or 9.0 mmol/1.The slow channel blockers were added to the medium in amounts that increased their final concentration in steps of 0.5 log units.Each concentration was allowed to act enough time to reach its maximal effect.This time varied from l0 to 40 rain.No significant differences were detected in the time required to reach the maximal effect between the drugs used.
In a second group of experiments the chronotropic effect of increasing calcium in the absence and in the presence of the slow channel blocking compounds was approached by two different procedures: in one of them, the calcium concentration in the medium was raised from 1.0 to 6.0 mmol/1 prior to and following 30 rain of incubating the preparations with verapamil (1 x 10-7 mol/1), nifedipine (3 x 10-v mol/l) or prenylamine (1 x 10 -6 tool/l).In another set of isolated atria, the concentration of calcium in the media was stepwise increased in the absence and in the presence of several concentrations of nifedipine.
Results were expressed as mean values + 1 standard error (SEM).In the cases when changes in atrial rate are given, they signify the differences between values at the end of the equilibration 1-h period and the values obtained following any given experimental procedure.Concentration-effect curves for the slow channel blockers were normalized in (log mol/ll Fig. 1.Chronotropic effect of verapamil, nifedipine and prenylamine on isolated rat atria incubated in media containing 1.0 mmol/1 of calcium.Points and vertical bars represent the mean _+ SEM.The three slow channel blockers produce a concentration-dependent decrease in atrial rate percent of the maximum effect in order to compare the relative potency of the drugs at each calcium concentration.To this purpose the response to the slow channel blockers was expressed as the percentage of the maximum decrease in rate attained in each experimental curve.Log EC10, EC3o, ECs0, ECT0 and ECg0% were obtained by interpolation and the mean + 1 standard error of the log values were calculated at each effect level.The statistical differences between groups was assessed by the analysis of variance followed by a modified "t"-test when Fvalues were significant (Wallenstein et al. 1980).The criterion for statistical significance was a P level of 0.05 or less.
Drugs Used.(+)Verapamil hydrochloride (Knoll AG, Ludwigshafen, FRG); prenylamine gluconate (Hoechst AG, Frankfurt/M., FRG) and nifedipine (Bay-a-1040, Bayer Leverkusen, FRG).The stock solutions of verapamil and prenylamine were prepared in distilled water and further diluted to the desired concentration with the saline solution for each experiment.Nifedipine was dissolved in an aqueous solution containing 2.5% (v/v) ethanol and 10% (v/v) polyethelene glycol 400.At the concentrations used the solvent for nifedipine had no detectable effect on atrial rate in control experiments.Because of the photosensitivity of nifedipine the experiments with this compound were carried out preventing the drug from light exposure.

Results
The effect of verapamil, nifedipine and prenylamine on the spontaneous rate of the atria incubated in a medium with 1.0 mmol/1 of calcium is depicted in Fig. 1.At the end of the stabilization period the mean beating frequency (basal rate) of the preparations was 251 + 6beats.min-1 (n = 28).The three slow channel blockers produced a concentrationdependent decrease of the atrial rate.This negative chronotropic effect was simultaneously detected in both the mechanical and electrical activities.None of the experiments showed evidences for excitation-contraction uncoupling.At each concentration tested the negative chronotropic effect of these drugs developed gradually until the maximal effect was attained.At the highest concentrations atrial arrest occurred in several preparations.Electrical stimulation of the quiescent preparations evoked atrial contractions indicating that the  In order to compare the relative potency of the slow channel blocking drugs their chronotropic effects were plotted as the percentage of the maximal decrease in rate evoked (Fig. 2).From these plots the log ECs0% of each drug was determined and the order of potency found was verapamil > nifedipine > prenylamine.
Figure 3 shows the concentration-effect curves for the slow channel blockers determined at different calcium concentrations, 1.0, 3.0, 6.0 and 9.0 mmol/1.In the preparations incubated with higher calcium levels, 6.0 or 9.0 mmol/1, the basal rate was significantly higher (265 +_ 6 beats, rain-1 and 285 +_ 9 beats-rain-1 respectively) than at calcium 1.0 mmol/l.No significant difference in the beating frequency was found between the atria incubated in media containing 1.0 or 3.0mmol/1 of calcium (251_+6 beats'min -1 and 242 + 8 beats, min-a respectively).On the other hand, the chronotropic response to the slow channel blockers was unaffected by the changes in calcium concentration.Despite the changes in basal rate, the maximal decrease induced by the slow channel blockers was very similar at each calcium concentration, about 230 beats, min-1.The effect of increasing extracellular calcium on the potency of slow channel inhibitors for decreasing atrial rate is shown in Fig. 4. From these plots the log ECs0o/o of each drug at every calcium concentration was determined.Irrespective of the calcium concentration, 1.0, 3.0, 6.0 or 9.0 mmol/1, the same order of potency was found: verapamil > nifedipine > prenylamine (Table 1).
When the effect of increasing calcium on the chronotropic action of slow channel blocking compounds was analyzed, it was found that increased calcium did not alter the negative chronotropic effect of prenylamine (Fig. 4).Similarly, the increment of calcium from 1.0 to 3.0 mmol/1 neither modified the chronotropic effect of verapamil nor the effect of nifedipine (Fig. 4).Furthermore, when calcium was increased to 6.0 mmol/1 a shift to the left of the concentration-effect curve for verapamil and nifedipine was detected (Fig. 4).When calcium was raised to 9.0 mmol/l a similar effect was detected for verapamil and nifedipine, although of a lesser magnitude than at calcium 6.0 mmol/1 (Fig. 4).At 9.0 mmol/1 the shift of the concentration-effect curve was only statistically significant at the EC10, ECao and ECs0 % in the case of nifedipine.
To further explore the shift of the concentration-effect curves for the slow channel blocking drugs the effect of increasing calcium from 1.0 to 6.0 mmol/1 was tested in the absence and presence ofequi-effective concentrations of these compounds.For each drug, the concentration chosen was that which produced a decrease in rate similar to the increase in frequency induced by raising calcium from 1.0 to 6.0 mmol/1.Figure 5 shows the results of the experiments with nifedipine and verapamil.In the absence of any slow channel blocker the increment in calcium from 1.0 to 6.0mmol/1 produced an average increment in rate of about 40 beats 9 min-1 (Fig. 5).The positive chronotropic effect of calcium was reversed when the excess of the ion was removed from the medium.When atrial rate had returned to its control value,  3. Concentration-effect curves were normalized in percentage of the maximum effect (see Methods).* indicates differences statistically significant compared to the values at 1.0 mmol/1 of calcium.Some standard errors (horizontal bars) are enclosed within their respective symbols.The increase in calcium to 6.0 mmol/1 shifts the concentration-effect curves for verapamil and nifedipine to the left suggesting a potentiating effect of the increase in calcium concentration Fig. 5. Effect of increasing the calcium concentration on spontaneous rate in the absence and in the presence of slow channel blockers.Results are expressed as the mean + SEM (vertical bars) of changes in atrial rate (see Methods).The shaded areas indicate the incubation period in a medium with 6.0 mmol/1 of calcium chloride.Notice that the negative chronotropic effect of both verapamil and nifedipine was potentiated when calcium was increased from 1.0 to 6.0mmol/1 Notice that the rate-accelerating effect of calcium is shifted to the right by nifedipine.A negative chronotropic effect of calcium is also detected in the presence of high concentrations of nifedipine the preparations were incubated with nifedipine 3 x 10-7 mol/1 (n = 6), verapamil I x 10-7mol/1 (n=6) or prenylamine 1 x 10 -6 mol/1 (n = 8, data not shown).At the end of 30 min Of incubation the negative chronotropic effect of the slow channel blockers had reached their maximum.The decrease in atrial rate obtained was of 47 + 7 beats, min-1 with nifedipine, 52 + 15 beats, min-1 with verapamil (Fig. 5) and 54_+ 18 beats-min -1 with prenylamine (data not shown).
The increment of calcium to 6.0 mmol/1 in the presence of prenylamine did not modify the chronotropic effect of the drug.On the other hand, the increment of calcium enhanced the chronotropic action of both verapamil and nifedipine to an extent that their negative chronotropic effect were doubled (Fig. 5).Control experiments showed that the effects have to be ascribed to the action of calcium since elevation of the chloride concentration and changes of the osmolarity of a similar extent had no effect on atrial rate.
Figure 6 shows the relationship between stepwise increments of extracellular calcium concentration and atrial rate in the absence and presence of nifedipine.In the absence of nifedipine a decrease in atrial rate was detected in several preparations when calcium was raised to 3.0 mmol/1, whereas no changes in the beating frequency were observed in the remaining ones.Thus, the increment of calcium to 3.0 mmol/1 had no detectable chronotropic effect in the averaged data.Further increments in calcium concentration to 6.0 and 9.0 mmol/1 produced a concentration-dependent increase of the spontaneous rate.In the presence of low concentrations of nifedipine (1 x 10-lo and 3 x l 0-9 mol/1) the rate-increasing effect of increasing calcium was shifted to the right, so that greater concentrations of calcium were required to produce similar increments in atrial rate.The negative chronotropic effect of elevating calcium to 3.0 mmol/1 was again detected, although the decrease in rate observed was not statistically significant.In the presence of a higher concentration of nifedipine (1 x 10 8 mol/1) the negative chronotropic effect of calcium 3.0mmol/1 was more evident and it was even accentuated at calcium 6.0mmol/1.On the other hand the rightwards shift of the rate increasing effect of calcium was still greater.

Discussion
In our experiments, in which atrial activity was considered to originate in the sinus node region, prenylamine, verapamil and nifedipine produced a concentration-dependent decrease in spontaneous rate.This negative chronotropic effect of the slow channel blocking compounds was not reversed by increasing the calcium concentration in the media.Furthermore, an enhancement of the negative chronotropic effect of verapamil and nifedipine was detected when calcium was raised over 3.0mmol/1.This effect was evidenced by the leftwards shift of the concentration-effect curves for these slow channel blockers when calcium was raised to 6.0 or 9.0 mmol/l, and also by the enhancement of their slowing effect when an excess of calcium was added to the media.
The decrease in atrial rate induced by the slow channel blocking drugs was interpreted as the result of a direct inhibitory action on sinus node automaticity.However, diverse mechanisms such as sinus node exit block, could account for the slowing in rate induced by the slow channel blockers and without intracellular recording of sinus node action potentials, we can only be sure that the spontaneous atrial rate decreased.However, the fact that the slowing effect of the slow channel blocking compounds developed gradually and, on the other hand, the lack of configurational changes in the electrograms reasonably strengthened the idea that the frequency of beating could be considered an index of sinus node automaticity.
Although the three slow channel blocking drugs produced their negative chronotropic effect in a concentration related manner, different potencies for decreasing the atrial rate were detected among the drugs studied.In our experiments, verapamil was the most potent agent for slowing the rate, prenylamine the weakest one and nifedipine was intermediate.It has been previously reported that nifedipine was more potent than verapamil for decreasing av-node automaticity (Kohlhardt and Haap 1981) and more potent than verapamil and prenylamine for decreasing cardiac contractility and for vasodilating the blood vessels (Fleckenstein 1977).In addition the observation that electrical stimulation evoked mechanical responses in the quiescent atrial preparations suggests that the slow channel blocking compounds impair sinus node automaticity at lower concentrations than those at which they suppress cardiac contractility.However, the possibility of the existence of an alteration in atrial conductibility, as it was mentioned above, cannot be absolutely ruled out from our results.
The most striking finding in our study is related to the interaction between calcium ion and the slow channel blockers on sinus node automaticity.The latter have the common property of directly inhibiting the transmembrane calcium influx through the slow channels (Kohlhardt et al. 1972;Fleckenstein 1977).Therefore, their pharmacological effects can be reversed either by increasing the extracellular calcium concentration or by increasing calcium influx, i.e. via/3-adrenoceptor stimulation.The concentration-effect curve for calcium on cardiac contractility is shifted to the right by the slow channel blocking drugs (Bristow and Green 1977), indicating that greater calcium concentrations are required to obtain similar inotropic levels in the presence of the slow channel inhibitors.Similar findings have been reported in vascular smooth muscle (Kanamori et al. 1981).As regards atrial automaticity there are controversial data in the available literature.The attenuation of the slowing effect of nifedipine on atrial rate (Refsum 1975) and of the inhibitory action of verapamil on automatic Purkinje fibres (Cranefield et al. 1974) when extracellular calcium is elevated has been already reported.In contrast to these findings, Fleckenstein (1977) and Kohlhardt et al. (1976) failed to reverse with calcium the inhibitory action of the calcium-channel blockers on sinus node automaticity.However, the same investigators could reverse the negative chronotropic effect of the slow channel blocking compounds by promoting an increase in the calcium inward current with catecholamines (Fleckenstein 1977;Kohlhardt et-al. 1976).In our experiments the increment of calcium (tom 1.0 to 3.0 mmol/1 failed to reverse the negative chronotropic effect of the slow channel blocking compounds.Higher increments in calcium concentration to 6.0 or 9.0 mmol/1 not only failed in reversing their negative chronotropic effect but also enhanced it.These results are in agreement with those reported by Zipes and Fischer (1974) who found that the infusion of 2 mg of calcium chloride or calcium gluconate into the sinus node artery failed to reverse the chronotropic effect of the slow channel inhibitory agents, whereas the infusion of larger amounts of calcium depressed sinus node automaticity even more than the slow channel blockers did alone (Zipes and Fischer 1974).The fact is that calcium ions and the slow channel blocking compounds produced just opposite chronotropic effects when used independently but they interacted synergistically, at least within certain calcium levels, when used together.This paradoxical effect of calcium is clearly illustrated by the experiments in which the same increase in calcium either accelerated or slowed the atrial rate depending on the absence or on the presence of the slow channel blocking drugs (Fig. 5).
Several alternative hypothesis should be analyzed in order to explain this paradoxical effect of calcium: 1)The chronotropic effect of calcium would depend on the beat to beat interval length: calcium ion should be positive chronotropic on fast preparations and conversely, it would act as a negative chronotropic agent on slow preparations.Therefore, the increment of calcium in the absence of slow channel blocking compounds increases the rate of beating, but when the rate has been previously slowed down by the slow channel blockers the same increment in calcium slows the rate even more.It is unlikely that this possibility would play a role in our results since an opposite relationship between the chronotropic action of calcium and cycle length has been reported in the rabbit sinus node (Op't Hofet al. 1980).2) The increment of extracellular calcium should enhance the automaticity of certain sinus node cells but would decrease the automaticity of others.The slow channel blockers would shift the pacemaker activity within the sinus node from the former to the latter ones.Therefore, calcium ions in the presence of the slow channel blockers would produce a negative chronotropic effect.Differences in the sensitivity to calcium increase have been reported in the sinus node cells of the rabbit (Mackaay et al. 1980a) and also, in several circumstances, the shift of the pacemaker activity within the sinus node has been already documented (Lu 1970;Mackaay et al. 1980a, b). 3) The increment of extracellular calcium may simultaneously produce two opposite effects: a rate-increasing and a ratedecreasing effect.The rate-increasing effect is due to the increment in the slope of diastolic depolarization when extracellular calcium raises (Seifen et al. 1964b;Kohlhardt et al. 1976).On the other hand the rate-decreasing effect may be the result of a neutralization of fixed charges on the external surface of the membrane by calcium ion (Langer and Frank 1972).The neutralization of external negative charges in the close vicinity of the ionic channels may result in an alteration of their gating mechanisms so that a change in the threshold potential can be suspected when extracellular calcium concentration changes.When calcium raises, a rise in the threshold would occur and consequently the atrial rate would decrease.The resulting chronotropic effect of any given increase in calcium would therefore depend on the relative magnitude of the two calcium actions, rate-increasing and rate-decreasing, which would be both calcium-concentration dependent.The rate increasing effect should operate at low calcium-concentrations generally masking the negative chronotropic effect of calcium, which although being present, would be weak at this level.At higher calcium concentrations the rate-increasing effect would reach its maximum and the negative chronotropic effect of calcium would become predominant.This supposition is supported by the experimental 9 data reported by Schaer (1964) and Seifen et al. (1964a) who have demonstrated that the increment of calcium beyond 10 mmol/1 produces a negative chronotropic effect following a rate-increasing action.In addition, Seifen et al. (1964a) reported a rise in the threshold potential of the pacemaker cells at calcium 10 mmol/1.Since the rate-increasing effect of calcium is related to the slow inward calcium current, it would be sensitive to the action of the slow channel blocking compounds.On the other hand, the negative chronotropic effect of calcium would depend on its action on the external surface of the membrane and would be rather insensitive to slow channel blockers.Therefore, in the presence of slow channel blocking drugs the rate-increasing effect of calcium shifts to higher calcium levels (Fig. 6) and so the ratedecreasing effect is unmasked.Consequently the addition of calcium in the presence of slow channel blockers enhances the negative chronotropic action of these drugs.4) The change in extracellular calcium concentration may alter the binding of the slow channel blocking drugs to their specific binding sites.Relevant to this are the binding studies of Glossmann et al. (1982) showing that calcium promotes the binding of labeled nimodipine to guinea-pig heart membranes.If the binding of the compounds used in our study have the same requirements for calcium as nimodipine, the increase in calcium concentration in the presence of slow channel inhibitors would enhance the negative chronotropic effects of these compounds despite the positive chronotropic action of calcium ions.
In any case the chronotropic response to altering calcium concentration seems to be paradoxical after slow channels blockade.Seifen E, Schaer H, Marshall JM (1964b)

Fig. 2 .
Fig. 2. Relative potency of verapamil, nifedipine and prenylamine to decrease atrial rate.Results were taken from the experiments shown in Fig. 1.Concentration-effect curves were normalized in percentage of the maximum effect (see Methods).Some standard errors (horizontal bars) are enclosed within their respective symbols.(i) Verapamil, (A) nifedipine, (e) prenylamine.Histograms on the right show the mean + SEM (vertical bars) of the maximal decrease in rate induced by the slow channel blocking drugs.Verapamil is the most potent agent for decreasing the rate whereas prenylamine is the weakest one Fig.3A--C.Chronotropic effect of verapamil, nifedipine and prenylamine at different extracellular calcium concentrations.Concentration-effect curves were determined at 4 values of extracellular calcium concentration (mmol/l): 1 9 (e), 3.0 (v1), 6.0 (A) and 9.0 (~).Points and vertical bars represent the mean -+ SEM.Panels: (A)Verapamil, (B)nifedipine and (C)prenylamine.Numbers in parentheses indicate the number &experiments.Although the basal atrial rate is increased at high calcium concentrations, the maximal decrease in rate induced by the slow channel blockers does not change with increasing calcium

Fig. 4 .
Fig. 4. Effect of increasing the calcium concentration on the negative chronotropic action of the slow channel blocking drugs.Results were taken from the experiments shown in Fig.3.Concentration-effect curves were normalized in percentage of the maximum effect (see Methods).* indicates differences statistically significant compared to the values at 1.0 mmol/1 of calcium.Some standard errors (horizontal bars) are enclosed within their respective symbols.The increase in calcium to 6.0 mmol/1 shifts the concentration-effect curves for verapamil and nifedipine to the left suggesting a potentiating effect of the increase in calcium concentration

Fig. 6 .
Fig.6.Chronotropic effects of cumulative increments of calcium in the absence and in the presence of nifedipine.Results are expressed as mean _+ SEM (vertical bars) of changes in atrial rate.* indicates statistically significant difference from the control value.(e, no nifedipine, n = 6), (A, nifedipine I x 10-10 mol/l, n = 8), (I, nifedipine 3 x 10 -9 mol/1, n = 10), (O, nifedipine 1 x/0-Smol/1, n = 8).Notice that the rate-accelerating effect of calcium is shifted to the right by nifedipine.A negative chronotropic effect of calcium is also detected in the presence of high concentrations of nifedipine