L‐type Ca2+ Current Increase by Rotenone, Stimulant of Mitochondrial Superoxide Release, in A7r5 Arterial Smooth Muscle Cells. Effect of Dithiothreitol, Hydrogen Peroxide, Tempol and Staurosporine

Abstract

L‐type Ca current (ICaL) is primary Ca2+ current to initiate contraction in arterial smooth muscle cells. Mitochondrial respiratory chain (MRC) generates and releases superoxide (O2−) in energy metabolism. O2− is converted to H2O2 by superoxide dismutase (SOD). Rotenone (Rot), phytogenic toxin, inhibits complex I of MRC and stimulates the release of O2−. We recently found that Rot increases ICaL in A7r5 cultured aortic smooth muscle cells. Here, we performed further pharmacological study of the increase by applying several redox‐related agents. Rot (0.01 mM) and other drugs were introduced during repetitive depolarization at 1/20s or recording I–V relationship with 2 min interval. Rot increased ICaL by 25% in 1–2 minute. It increased macroscopic conductance and shifted V0.5 of activation curve to the hyperpolarizing direction. Dithiothreitol (DTT, 2 mM), thiol‐reducing agent, increased ICaL by 20% without producing the negative shift and, in the presence of DTT, Rot increased ICaL with the negative shift. H2O2 (0.3 mM) inhibited ICaL by 13% in 5 min without shifting I–V and, in the presence of H2O2, Rot induced 17% increase of ICaL with negative shift. Pretreatment by Tempol, SOD mimetic scavenger of O2−, markedly depressed Rot‐induced increase of ICaL. Staurosporine (100 nM), broad‐spectrum protein‐kinase inhibitor, inhibited ICaL with slow time course and completely suppressed Rot‐induced increase of ICaL. Rotenone increases ICaL by increasing the release of O2− from mitochondrial complex I and the increase is independent of thiol‐oxidation but is mediated by phosphorylation of proteins that regulates ICaL.