Acute hypoxic inhibition of the pore-forming alpha(1C) subunit of the L-type Ca2+ channel mediates hypoxic arterial vasodilatation, a physiological response which matches tissue O-2 demand and supply in the systemic vasculature. In numerous O-2-sensing cell types, reactive O-2 species (ROS) have been proposed as mediators linking lowered O-2 levels with the appropriate cellular response. In this study, we examined the roles of H2O2 and NADPH oxidase as mediators of hypoxic inhibition of recombinant alpha(1C) subunits. Human cardiac L-type Ca2+ channel alpha(1C) subunits were stably expressed in HEK 293 cells. Ca2+ currents were recorded using the whole-cell configuration of the patch-clamp technique. Bath application of 100 muM H2O2 significantly enhanced depolarisation-evoked Ca2+ currents in a voltage-dependent manner, while dialysis with 1000 U ml(-1) catalase reduced these currents. In the presence of catalase, hypoxic inhibition of Ca2+ currents was not significantly different compared to non-dialysed controls. The NADPH oxidase inhibitors diphenylene iodonium (10 muM) and phenylarsine oxide (5 muM) were without effect on either basal Ca2+ currents or responses to hypoxia. Thus, endogenous production of H2O2 regulates the alpha(1C) subunit. However, neither suppression of H2O2 levels nor inhibition of NADPH oxidase is involved in O-2-dependent regulation of the Ca2+ channel. (C) 2004 Elsevier Inc. All rights reserved.