Serotonergic effects of dotarizine in coronary artery and in oocytes expressing 5-HT2 receptors

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Abstract

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37°C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 μM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 μM, respectively. Flunarizine inhibited both types of contractions with IC50 of 1.7 μM for 5-HT and 2.4 μM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2+-activated Cl channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 μM dotarizine and blocked around 62% by 10 μM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 μM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.

Introduction

This study was aimed at characterizing the differential effects of dotarizine on 5-HT2 receptor subtypes and on vascular voltage-dependent Ca2+ channels. Dotarizine, a novel piperazine derivative structurally related to flunarizine (Fig. 1), is currently being evaluated in clinical trials for its antimigraine and antivertigo effects. Its parent piperazine compound, flunarizine, has been used for the prophylactic treatment of migraine crisis (Todd and Benfield, 1989) and its clinical efficacy has been related with its Ca2+ channel antagonist properties, since disorders of voltage-dependent Ca2+ and Na+ channels have been implicated in the pathogenesis of migraine. Like flunarizine, dotarizine exhibits Ca2+ channel antagonist properties. Thus in rabbit aortic smooth muscle, dotarizine inhibits 45Ca2+ uptake and vessel contractility (Tejerina et al., 1993). Furthermore, in bovine adrenal chromaffin cells dotarizine blocks whole-cell Ca2+ and Ba2+ currents generated by depolarizing test pulses in voltage-clamped cells, and also inhibits the 45Ca2+ uptake, the transient rise of cytosolic Ca2+ concentrations as well as the release of catecholamines triggered by K+ depolarization (Villarroya et al., 1995).

Additionally, dotarizine exhibits potent antiserotonergic activity, both in vitro and in vivo (Brasó et al., 1989, Brasó et al., 1994; Cartheuser et al., 1994). Since various drugs with 5-HT2 receptor antagonist properties have been used in migraine prophylaxis, for example, pizotifen, methysergide or the β-adrenoceptor antagonist, propranolol (Welch, 1993; Goadsby and Olesen, 1996), the 5-HT receptor blocking effect of dotarizine, in addition its being a Ca2+ channel antagonist, should increase its possible therapeutic potential. In this study we compared the vascular Ca2+ channel antagonist properties of dotarizine with its antiserotoninergic effects. In addition, by using Xenopus oocytes as a system to express specific receptors, we analyzed the possible selectivity of dotarizine for a given 5-HT2 receptor subtype. The presence of an intracellular site of action for dotarizine at inositol 1,4,5-trisphosphate (IP3) receptor level was also explored.

Section snippets

Preparation of the coronary artery and experimental protocols

Hearts from Large White pigs of both sexes were obtained from a local slaughterhouse within 10 min of slaughter, and were transferred to the laboratory in ice-cold Krebs-bicarbonate solution with the following composition (in mM): NaCl 119; KCl 4.7; MgSO4 1.2; CaCl2 1.5; KH2PO4 1.2; NaHCO3 25 and glucose 11. The first 3 cm of the left circumflex coronary artery was dissected out and the surrounding adipose and connective tissue was removed. Arteries were cut into helicoidal strips 15 mm long

Effects of dotarizine on the contractile response of pig coronary strips stimulated with 5-HT or high K+

Initial testing of increasing concentrations of 5-HT applied cumulatively to pig coronary strips showed that the EC50 value for the contractile response was 2.6×10−7 M. Therefore, repeated applications of 5-HT, separated by 30 min washout, at a concentration double the EC50 (5×10−7 M) were selected to induce substantial contractions in these vessels. Usually, after five or six initial challenges, the responses to 5-HT were stable and reproducible for at least ten further challenges (not shown).

Discussion

Though of similar chemical structure, the two piperazine derivatives, dotarizine and flunarizine, have important pharmacological differences (Table 1). Thus dotarizine blocked the phasic contractions induced by 5-HT in pig coronary arteries with a 17-fold greater potency than the K+-induced responses. On the contrary, flunarizine inhibited the 5-HT and K+ responses with similar IC50 values. From previous experiments with rabbit basilar and aorta smooth muscle and in bovine chromaffin cells, it

Acknowledgements

Supported by a grant from Fundación Ferrer (Barcelona, Spain) and by Janssen España, (Madrid, Spain). Also supported by grants from DGICYT No. PB94-0185 to CM and Fundación Ramón Areces and DGICYT No. PB94-0150 to AGG. We would like to thank the EC Human Capital and Mobility scheme for support to RBL. cDNAs of the 5-HT2A and 5-HT2C receptors were kindly provided by Dr. David Julius (University of California, San Francisco, USA). Dotarizine, flunarizine and elgodipine were generous gifts from

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