Effect of the antianginal drug bepridil on intracellular Ca²⁺ release and extracellular Ca²⁺ influx in human neutrophils

To understand more fully the effects of bepridil, an antiarrhythmic and antianginal drug, on myocardial ischemia-reperfusion injury and systemic immune responses, its effect on intracellular Ca²⁺ levels ([Ca²⁺]_i) in human neutrophils was investigated by using fura-2 as a fluorescent probe. Bepridil (10-200 μM) increased [Ca²⁺]_i in a concentration-dependent fashion. This signal was partly inhibited by removal of extracellular Ca²⁺. In a Ca²⁺-free medium, pretreatment with bepridil (100 μM) abolished the Ca²⁺ release induced by thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, and by carbonylcyanide m-chlorophenylhydrazone (2 μM), a mitochondrial uncoupler. Pretreatment with carbonylcyanide m-chlorophenylhydrazone and thapsigargin, respectively, partly inhibited bepridil-induced Ca²⁺ release. Addition of Ca²⁺ (3 mM) increased [Ca²⁺]_i after pretreatment with bepridil (100 μM) in a Ca²⁺-free medium. Bepridil (100 μM)-induced Ca²⁺ release was not altered when phospholipase C was inhibited by U73122 (2 μM). Both Ca²⁺ release and Ca²⁺ entry induced by bepridil (100 μM) were augmented by activating protein kinase C with phorbol 12-myristate 13-acetate (10 nM), and were suppressed by inhibiting protein kinase C with GF 109203X (2 μM). Treatment with bepridil (10-20 μM) for 30 min increased the production of reactive oxygen intermediates (ROI) by more than 50%. Collectively, it was found that bepridil increased [Ca²⁺]_i concentration-dependently in human neutrophils by releasing Ca²⁺ from the endoplasmic reticulum, mitochondria and, possibly, other compartments in a phospholipase C-independent manner. Bepridil also activated Ca²⁺ influx. The activity of protein kinase C may regulate bepridil-induced Ca²⁺ release and Ca²⁺ entry.