Inhibitory effect of mibefradil on contractions induced by sympathetic neurotransmitter release in the rat tail artery

Abstract

This study tested whether mibefradil exerts a stronger inhibitory effect than verapamil on sympathetic neurotransmitter release provoked by electrical field stimulation. Tail arteries (diameter 620+/-9 microm) were obtained from male Wistar rats. Ring segments of 2 mm length were mounted in an isometric wire myograph. After an appropriate period of equilibration and a priming procedure the vessels were either subjected to electrical field stimulation (EFS; frequency 0.25-4 Hz for 30 s) or a concentration-response curve was generated with either noradrenaline (concentration range 0.03-3 microM) or ATP (concentration 0.3 mM) which served as baseline parameters. EFS-induced contractions were stable and reproducible and were blocked by tetrodotoxin (1 microM), guanethidine (3 microM), and the combination of suramin (0.5 mM) and prazosin (3 microM). EFS-induced contractions (1 Hz) were almost completely inhibited by 10 microM mibefradil (97%) but only partly by 10 microM verapamil (73%). Log IC50 values were -5.6 for mibefradil and -6.6 for verapamil. Calcium antagonists were equipotent in inhibiting noradrenaline (maximum inhibition by mibefradil and verapamil by 70% and 75%, respectively; log IC50: -6.5 and -6.7, respectively) and ATP-mediated contractions (maximum inhibition by mibefradil and verapamil by 92% and 97%, respectively; log IC50: -6.5 and -7.0, respectively). Consequently mibefradil displays an additional effect on contractions provoked by EFS-induced sympathetic noradrenaline release which cannot be explained by L-type calcium channel blockade. Probably this effect of mibefradil is mediated by the blockade of prejunctional N-type calcium channels, thereby inhibiting sympathetic noradrenaline release. Since activation of the sympathetic nervous system in hypertension is both common and undesirable, a calcium antagonist displaying both L- and N-type calcium channel blocking activities, would have major advantages over calcium antagonists lacking N-type calcium channel blocking activities.

The evaluation of the N‐type channel blocking properties of cilnidipine and other voltage‐dependent calcium antagonists

“…Electrical stimulation has been shown to result in pronounced and reproducible increases of smooth muscle contraction [19] (Figure 2), which is sensitive to tetrodotoxin, guanethidine and prazosine, thus confirming the essential involvement of sympathetic nerves in the induction of this mechanical response. We used this particular preparation as it is densely innervated by sympathetic neurones.…”

“…In a previous study [19], we demonstrated that the contractile force evoked by EFS could be inhibited (>90%) by prazosin (0.1 lM) and abolished by tetrodotoxin (1 lM). Consequently, the contractions appear to be predominantly caused by stimulation-induced release of noradrenaline from the sympathetic nerve terminals.…”

“…As expected, the selective N-type calcium-channel inhibitor x-conotoxin GVIA abolished the stimulation-induced sympathetic outflow, thus confirming that N-type Ca 2+ -channels predominate in the control of sympathetic transmission in the thoracic aorta ( Figure 1). Mibefradil has been shown to exert N-type Ca 2+ -channel blocking properties in various models [19,22], besides its blockade of L-and T-type channels. Accordingly, it is assumed that cilnidipine inhibits stimulation-induced sympathetic neurotransmission by reducing Ca 2+ -influx via N-type calcium-channels.…”

“…In order to verify whether the stimulation-induced contractions were indeed neuronally mediated, control experiments with tetrodotoxin (1 lM), guanethidine (3 lM) and prazosin (0.1 mM) have been carried out as described previously [19].…”

Evidence against an action of mibefradil at N-type voltage-operated calcium channels

“…Recently, a series of papers, using functional and electrophysiological approaches, have concluded that mibefradil may also block N-type VOCCs (Bezprozvanny and Tsien 1995;Göthert and Molderings 1997;Molderings et al 2000;van der Lee et al 2000). In addition, in some human and rat CNS and rat spinal motoneurone preparations, mibefradil may inhibit other VOCCs including N, P…”