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

Abstract

Mibefradil is a tetralol derivative that inhibits cloned and native T-type voltage-operated calcium channels (VOCCs) at an IC50 of 1 microM and with an apparent 15-fold selectivity for T- over L-type VOCCs. Recent electrophysiological studies in Xenopus oocytes and inhibition of noradrenaline release studies in human isolated right atria concluded that mibefradil (0.3-3 microM) can block N-type VOCCs like omega-conotoxin GVIA. We tested this hypothesis in rat and guinea pig isolated driven left atria and rat vas deferens in response to sympathetic nerve stimulation. Mibefradil (3 microM) did not inhibit the inotropic responses to sympathetic electrical field stimulation in atria. In contrast, these responses were blocked by GVIA (10 nM) and tetrodotoxin (TTX 0.1 microM). In rat vas deferens, pretreated with benextramine (BNX 10 microM), contractions considered to be due to transmitter ATP evoked by electrical field stimulation were blocked by: (a) TTX 0.1 microM, (b) P2x receptor desensitisation (alphabeta-MeATP 100 microM), and (c) GVIA (pIC50 9.04+/-0.15, n=5). In contrast, mibefradil (0.3-30 microM) had no effect on these N-type VOCC-sensitive sympathetic nerve responses. Potassium (62 mM K+) and alphabeta-MeATP-induced contractions were unaffected by GVIA (10 nM), but mibefradil inhibited the potassium-induced contractions with a pIC50 6.02+/-0.08 (n=5), consistent with a T- and L-type VOCC blocking action on the postjunctional smooth muscle. We conclude that mibefradil up to 30 microM does not block N-type VOCCs in these isolated intact tissue assays of sympathetic nerve-muscle transmission. Therefore, at least in these tissues, mibefradil can be used to define T- and L-type VOCC activities at submicromolar and micromolar concentrations in sympathetic nerve-mediated responses.

Involvement of Different Calcium Channels in the Depolarization‐Evoked Release of Noradrenaline from Sympathetic Neurones in Rabbit Carotid Artery*

“…Our findings ( fig. 6 and 7) are in line with the conclusion that mibefradil is not a blocking agent of N-type Ca 2π channels (Xi & Angus 2001;Xi et al 2002).…”

Voltage‐dependent calcium channels of dog basilar artery

“…One or possibly two types of channels mediate current at a low voltage range of activation. This analysis also readily shows that low concentrations (0.01 μ m ) of nimodipine and mibefradil increased I Ba at voltages below –20 mV, another characteristic of T‐type Ca 2+ channels (Oike et al 1990; Avdonin et al 2000; Xi & Angus, 2001). It does not, however, exclude the possibility of opening of non‐selective cation channels, similar to leak channels in skeletal muscle that are activated by nifedipine (Alderton & Steinhardt, 2000).…”

Involvement of T‐type calcium channels in excitatory junction potentials in rat resistance mesenteric arteries

“…For each experiment, the n value was equal to the number of rats from which the arteries were obtained. Some data for this figure is re‐plotted from a previously published paper from our laboratory (Xi et al ., 2001)…”

“…In contrast both the Na + ‐channel blocker TTX (0.1 μ M ) and the N‐channel blocker ω‐conotoxin GVIA (10 n M ) essentially abolished the EJPs. We have previously established that twitch responses in the rat vas deferens preparation can be used as a highly sensitive functional assay of N‐channel function (Xi & Angus, 2001). Mibefradil in concentrations up to 30 μ M did not show any inhibitory effects in the rat vas deferens preparation but ω‐conotoxin GVIA (10 n M ) and TTX (0.1 μ M ) caused full inhibition of the twitch responses (Xi & Angus, 2001).…”

“…We have previously established that twitch responses in the rat vas deferens preparation can be used as a highly sensitive functional assay of N‐channel function (Xi & Angus, 2001). Mibefradil in concentrations up to 30 μ M did not show any inhibitory effects in the rat vas deferens preparation but ω‐conotoxin GVIA (10 n M ) and TTX (0.1 μ M ) caused full inhibition of the twitch responses (Xi & Angus, 2001). Thus, in our functional assays, we have no evidence to suggest a prejunctional action of mibefradil on either N‐channels or the Na + ‐channels.…”