Apocynin inhibits and its removal augments L‐type Ca2+ currents in coronary artery smooth muscle cells and ventricular myocytes

Abstract

Exogenously applied H2O2 augments L‐type Ca2+ current (ICa,L). NADPH oxidase (NOX) initiates sequential reactions to generate reactive oxygen species (ROS) including H2O2. We studied the regulatory role of NOX‐generated ROS on ICa,L by applying apocynin (APO), a NOX inhibitor, and diphenyleneiodonium (DPI), a flavoprotein inhibitor. Whole cell ICa,L was recorded from bovine coronary arterial smooth muscle (BCASMC), rat ventricular myocytes (RVMC) and HEK 293 cells transiently expressing human cardiac CaV1.2. APO (0.1–10 mM) rapidly and dose‐dependently inhibited ICa,L: 10‐mM APO decreased ICa,L to 27, 34 and 16% in BCASMC, RVMC and 293 cells, respectively, while DPI (10 μM) inhibited more slowly and less intensively (to 64% in RVMC). The inhibitory effects of both inhibitors were mainly additive. Surprisingly, washout of high concentrations of APO rapidly recovered ICa,L to slightly above control in BCASMC and increased to 150% of control with prolonged current decay in RVMC. ROS levels measured simultaneously with ICa,L by increase rate of CM‐H2DCF fluorescence were decreased by APO and markedly increased by its removal in BCAVSM and RVMC. Possible accumulation of NADPH during NOX inhibition by APO explains the increased generation of ROS. We conclude that decrease of ROS inhibits and its increase augments cardiovascular ICa,L and NOX is involved in the regulation of ICa,L. This study is supported by NIH grant #R01HL85352