Apocynin Reversibly Inhibits L-type Ca2+ Channel Current Involvement of Reactive Oxygen Species

Abstract

L-type Ca2+ channel current (ICa,L) is a major Ca2+ entry mechanism and contraction of arterial smooth muscle (ASM) is regulated by reactive oxygen species (ROS). We utilized apocynin (APO) as a tool to clarify the contribution of ROS in the regulation of ICa,L. APO, a natural organic compound contained in a variety of plants, is widely used as inhibitor of NADPH oxidase (NOX) that reduces oxygen to superoxide in the presence of NADPH to generate ROS including H2O2. We recorded whole cell ICa,L with Ba2+ as charge carrier from isolated bovine coronary ASM (BCASM) and HEK293 cells transiently expressing human cardiac CaV1.2. APO was introduced and washed out during continuous application of depolarization step to 0 mV from HP −80 mV at 1/min. We found that APO inhibited ICa,L in a dose-dependent manner between 0.1 and 10 mM decreasing its amplitude to ∼20% of control. APO also accelerated ICa,L decay during depolarization. Surprisingly, washout of the high concentration of APO caused rapid recovery of ICa,L. It could even produce a rebound increase of ICa,L with its peak at ∼3 min in BCASM. We performed fluorometric analysis of APO-induced change of cellular ROS by loading cells with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Oxidation of CM-H2DCF to CM-DCF by ROS induces an increase of fluorescence. APO markedly inhibited the fluorescence increase, while washout of APO caused more intensive increase of fluorescence than control. The results are explained by the inhibition of NOX by APO which results in decrease of ROS and overproduction of ROS during washout utilizing accumulated NADPH produced by prior NOX inhibition. ROS which changes dynamically in situ, e.g. hypoxia and reoxygenation, seems to be vital to sustain ICa,L.