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Mecanismos de la acción vasoselectiva de dos nuevas dihidropiridinas:
Elgodipina y Oxodipina

Galán Loipa; Talavera Karel; Vassort Guy; Alvarez Julio

Instituto de Cardiología y Cirugía Cardiovascular
La Habana, Cuba
U-390 INSERM Montpellier, Francia

Abstract
Introduction
Objectives
Methods
Results
Discussion
Conclusions
References

Abstract
Introduction: There is still a need for more vascular selective calcium antagonists due to the potentially life-threatening effects of their cardiac depressant action. Elgodipine and oxodipine are second generation dihydropyridines that are known to strongly depress smooth muscle contraction. However, their mechanisms of action on calcium channels have not been fully characterized.
Objectives: The present study was undertaken to compare the pharmacological properties of elgodipine and oxodipine and to characterize their mechanisms of action on calcium channels in isolated rat ventricular cardiomyocytes.
Material and Methods: Contractions were recorded on KCl-depolarized rat and rabbit abdominal aortic rings and on electrically-stimulated rat right ventricular strips. Patch-clamp recordings of the whole-cell calcium currents were done in rat cultured neonatal ventricular myocytes and in enzymatically rat isolated adult ventricular myocytes.
Results: Elgodipine and oxodipine depressed aortic contraction with stronger potency than cardiac contraction. The vascular selectivity index (IC50 ventricular/IC50 aorta) was greater for elgodipine (680) than for oxodipine (72). Elgodipine and oxodipine decreased the L-type Ca current (ICaL) of neonatal ventricular myocytes with IC50 of 0.33 and 0.24 µM respectively. Oxodipine was mor e potent on the T-type Ca current (ICaT) than elgodipine (IC50 = 0.41 vs 2.18 µM). Both compounds were less potent in inhibiting ICaL of adult cardiomyocytes. Elgodipine and oxodipine increased their potency of action on ICaT and ICaL by at least one order of magnitude when the cells were partially depolarized. Elgodipine in addition exhibited a frequency-dependent block of both currents.
Discussion and Conclusions: Both dihydropyridines are highly vascular selective and the underlying mechanism of this vascular selectivity is the voltage-dependent block of calcium currents. In the case of elgodipine, which shows a vascular selectivity higher than that of oxodipine, its use-dependent inhibition of calcium currents could also contribute to this action.

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Introduction:
1,4-dihydropyridine derivatives are well known Ca2+ channel blockers and are used in clinics since long for the treatment of several cardiovascular disorders (Nayler, 1988). These compounds bind with high affinity to the a1 subunit of L-type Ca2+ channels and this binding is state-dependent so binding affinity increased when Ca2+ channels were in the inactivated state (Glossmann and Striessnig, 1990). There is still, however, a need for more vascular selective compounds due to the potentially life-threatening effects of their cardiac depressant action (Waters, 1997). Recently, two new 1,4-dihydropyridines, elgodipine and oxodipine, with high vascular selectivity have been developed.
Elgodipine possess strong vasodilator action attributed to the depression of Ca2+ entry through potential-operated channels in smooth muscle (Chulia et al., 1995). The potent coronary relaxing properties of elgodipine without significant depression of cardiac contraction could be the basis for its beneficial effects in patients with coronary artery disease. Elgodipine inhibits both T- and L-type Ca2+ currents (ICaT and ICaL respectively) in portal vein myocytes although it is more potent on ICaL (Lepretre et al., 1994). Oxodipine has been reported to strongly depress contraction in aorta and mesenteric resistance vessels (Tejerina et al., 1992) at concentrations significantly lower than those required to induce cardiodepressant effects. In rat portal vein myocytes, Baron et al., (1994) showed that oxodipine depressed the high-threshold Ca2+ current ICaL in a voltage-dependent manner and was without effect on the low-threshold Ca2+ current ICaT .

Objectives:
The present study was undertaken to compare the pharmacological properties of oxodipine and elgodipine on vascular and cardiac preparations and to characterize their mechanisms of blockade of Ca2+ channels (T- and L-types) in isolated cardiomyocytes.

Methods:
Abdominal aortic rings were dissected from rabbits (New Zealand) under pentobarbital anaesthesia, fixed to a force transducer and placed in bath chamber continuously perfused with a Tyrode solution. Contraction was induced by replacing all NaCl by KCl (140 mM) or by norepinephrine (10 µM). Standard microelectrode techniques were used to record action potentials from small right ventricular strips dissected out from neonatal (3 - 4 days) and adult rat hearts (Wistar). In adult rat ventricular strips contractions were simultaneously recorded. In all cases, strips were continuously perfused with normal Tyrode solution and stimulated at a frequency of 1.25 Hz. All experiments were conducted at 35 °C. T- and L-type Ca2+ currents (ICaT and ICaL respectively) were recorded in rat neonatal cardiomyocytes in primary culture (ICaT and ICaL) and in isolated cardiomyocytes from adult rat hearts (ICaL only). The "whole cell" variant of the patch-clamp method was used with intra- and extracellular solutions designed to isolate the Ca2+ currents. Currents were evoked by 200-ms voltage-clamp pulses to -40 mV (ICaT) or to +10 mV (ICaL) from a holding potential of -90 mV at a frequency of 0.125 Hz. Results were analysed by the students' "t"-test and are expressed as means and standard deviations. The criterion for significance was p < 0.05.


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Results:
Both elgodipine and oxodipine exerted a concentration-dependent depression of the KCl-induced contraction of rabbit aortic rings. The results are summarized in Table 1. The estimated vascular selectivity indexes (IC50-heart/IC50-aorta) for elgodipine and oxodipine were »680 and »72 respectively. Oxodipine but not elgodipine (10 µM) significantly depressed the norepinephrine-induced contraction of rabbit aorta by 56.5 ± 2.9 % (n = 5).
When applying 10 µM elgodipine or oxodipine on neonatal and adult rat ventricular strips, the only electrical parameter affected was action potential duration. Elgodipine was more effective in decreasing action potential duration at the level of 0 mV in adult ventricle while oxodipine was more effective in neonatal ventricle (Table 2). Both drugs were less effective in decreasing action potential duration at -60 mV repolarization level.
Elgodipine and oxodipine inhibited ICaT and ICaL in cultured neonatal and adult cardiomyocytes. Table 3 summarizes IC50 values of both dihydropyridines on these currents. Elgodipine was five times less potent on ICaT than oxodipine. Both compounds were less effective on adult ICaL than on neonatal ICaL. Elgodipine and oxodipine at concentrations near their respective IC50s values also decreased the inactivation time constants of ICaT and ICaL.
We studied the frequency-dependent effects of elgodipine and oxodipine on ICaT and ICaL using voltage-clamp protocols (holding potential = -90 mV) at high stimulation rates (2 Hz), under control conditions (no drug) and in the presence of elgodipine or oxodipine. Tonic block was estimated as the difference between peak control Ca2+ current (T or L) and peak Ca2+ current at the first pulse after drug superfusion. Use-dependent blockade was considered to be the difference between peak Ca2+ current for the first and the 20th (or 30th) pulses after drug exposure. An example of the frequency-dependent effects of elgodipine and oxodipine (10 µM) on ICaL is illustrated in Figures 1A and 1B (neonatal and adult cardiomyocytes respectively). As can be seen elgodipine exhibits both tonic and use- or frequency-dependent blocks while oxodipine behaves as a "classic" dihydropyridine exerting a mostly tonic block. Figures 2A and 2B show the same frequency-dependent effects of both drugs on ICaT of neonatal cardiomyocytes. Use-dependent blocks of ICaT and ICaL by elgodipine amounted to 78.6 ± 5.3% and 59.2 ± 3.7% of the respective total blocks.
As expected from compounds that bind with high affinity to the inactivated state of the Ca2+ channels, in well polarized cells (holding potential = -90 mV) elgodipine and oxodipine shifted to more negative potentials the availability curves of ICaL by 10 to 12 mV. These voltage-dependent effects were also evident as an enhanced inhibition of ICaL when cardiomyocytes were clamped from less negative holding potentials (-50 mV). At 10 µM elgodipine and oxodipine inhibited ICaL by 53.0 ± 17.0% and 63.5 ± 5.7% respectively from a holding potential of -90 mV while from a holding potential of -50 mV ICaL was inhibited by 77.0 ± 13.0% and 90.0 ± 3.4% respectively. Voltage-dependent effects of both drugs on ICaT were less pronounced. Availability curves of ICaT were only shifted by » 5 mV by elgodipine (10 µM) and oxodipine (1 µM) and ICaT inhibition was only enhanced by » 10% at a reduced holding potential of -70 mV.

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Discussion:
The present results indicate that elgodipine and oxodipine behave as classical Ca2+ channel antagonists blocking the contraction of cardiac and vascular smooth muscle. However, both drugs, and particularly elgodipine, are more potent inhibitors of smooth muscle contraction. The vascular selectivity indexes (680 for elgodipine and 72 for oxodipine) are in the same order of magnitude of those reported for other second generation dihydropyridines (e.g. nitrendipine, felodipine, niludipine; see Nayler, 1988). Both drugs decrease the cardiac action potential duration an effect which is related to the inhibition of the T- and L-type Ca2+ currents. Elgodipine and oxodipine decreased ICaL in neonatal and adult cardiomyocytes but they are at least one order of magnitude more potent on ICaL in neonatal cardiomyocytes. Both compounds inhibit ICaT with IC50 in the micromolar range. There was a good correlation between the IC50 for inhibition of cardiac contraction and ICaL by oxodipine. However, the IC50 for the inhibition of ICaL by elgodipine was »20 times greater than the IC50 for the inhibition of cardiac contraction a difference that can be partially explained by the use-dependent action of elgodipine.
The major finding of the present study is the voltage-dependent block of ICaT and ICaL by elgodipine and oxodipine. Blockade of both currents was enhanced when currents were evoked from reduced holding potentials. This voltage-dependent inhibition of Ca2+ channels could be the basis of the high vascular selectivity of elgodipine and oxodipine. In vascular smooth muscle, resting potentials are lower than in cardiac muscle and the fraction of Ca2+ channels in the inactivated state is greater. Thus, interaction of these dihydropyridines with inactivated Ca2+ channels is favoured in smooth muscle. One major difference between elgodipine and oxodipine is the marked use-dependent effect of elgodipine on Ca2+ channels. It could be possible that the use-dependent inhibition of ICaT and ICaL by elgodipine contribute to enhance its inhibitory action on the vascular smooth muscle contraction.

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Conclusions:
1.- Elgodipine and oxodipine are potent blockers of both L-type and T-type Ca2+ channels in rat cardiomyocytes.
2.- The mechanisms of action of the two drugs on Ca2+ channels are different since oxodipine behaves essentially as a tonic blocker of ICaT and ICaL, while elgodipine exhibits a smaller tonic block and a prominent use-dependent block.
3.- Their vascular selectivity could be attributable to the voltage-dependent action on Ca2+ channels. Furthermore, the use-dependent inhibition of Ca2+ currents by elgodipine could also contribute to confer this molecule a higher vascular selective action.

References:
Baron A; Rakotoarisoa L; Leprętre N; Mironneau J.: Eur. J. Pharmacol. 269: 105-113, 1994.
Chulia T; González P; del Río M; Tejerina T.: Eur. J. Pharmacol. 285: 115-122, 1995.
Glossmann H; Striessnig J.: Rev. Physiol. Biochem. Pharmacol. 114: 1-105, 1990.
Leprętre N; Arnaudeau S, Mironneau J; et al.: J. Pharmacol. Exp. Therap. 271: 1209-1215, 1994.
Nayler WG.: Calcium antagonists. London, Academic Press, 1988.
Tejerina T; Cauvin C; Tamargo J.: Europ. J. Pharmacol. 219: 279-284, 1992.
Waters D: Can. J. Cardiol. 13: 757-766, 1997.

 

Questions, contributions and commentaries to the Authors: send an e-mail message (up to 15 lines, without attachments) to pharma-pcvc@pcvc.sminter.com.ar , written either in English, Spanish, or Portuguese.

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