Effects of mibefradil on intracellular Ca²⁺ release in cultured rat cardiac fibroblasts and human platelets

Abstract

The Ca²⁺ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca²⁺ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K⁺ depolarization and Ca²⁺ channel block by verapamil or other Ca²⁺ antagonists. At concentrations above 10 μM, mibefradil elevated substantially cytosolic [Ca²⁺] without affecting the peak level of agonist-induced Ca²⁺ transients. These Ca²⁺-mobilizing actions of 10 or 100 μM mibefradil stand in contrast to the Ca²⁺ antagonism and relaxation of vascular muscle at 1μM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca²⁺ was independent of extracellular Ca²⁺, and in order to define better the mechanism of Ca²⁺ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 μM activated dose-dependent Ca²⁺ release from intracellular Ca²⁺ stores. Verapamil had no effect at concentrations of up to 100 μM. Mibefradil-induced Ca²⁺ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca²⁺- nor disulfide reagent-induced Ca²⁺ release were involved and that mibefradil did not release Ca²⁺ by inhibition of the Ca²⁺-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca²⁺ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca²⁺ release. Depletion of Ca²⁺ stores of permeabilized cells using inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca²⁺ release, and depletion of Ca²⁺ stores by mibefradil prevented further Ca ²⁺ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (≥/10 μM) thus mobilized Ca²⁺ from an inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool in cultured rat cardiac fibroblasts and human platelets.