Objective: The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca2+ channel during acute hypoxia was examined in adult ventricular myocytes from guinea pig.Methods: The fluorescent indicator dihydroethidium (DHE) was used to detect superoxide and the response of the L-type Ca2+ channel to adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (ICa-L) to isoproterenol (Iso).Results: Hypoxia caused a 41.2 +/- 5.2% decrease in the rate of the DHE signal (n=21; p < 0.01). Of the classical NAD(P)H oxidase inhibitors, DPI but not apocynin mimicked the effect of hypoxia on the sensitivity Of ICa-L to Iso. However, the potent NAD(P)H oxidase agonist angiotensin 11 had no effect on cellular superoxide or the sensitivity Of ICa-L to Iso. Although DPI inhibits NAD(P)H oxidase, it also decreased superoxide in isolated mitochondria in a concentration-dependent manner. Partial inhibition of mitochondrial function with nanomolar concentrations of FCCP or myxothiazol mimicked the effect of hypoxia on cellular superoxide and the sensitivity Of ICa-L to Iso. In addition, hypoxia caused a 69.3 +/- 0.8% decrease in superoxide in isolated mitochondria (n=4; p < 0.01), providing direct evidence for a role for the mitochondria.Conclusions: Our data suggest that mitochondria appear to be involved in oxygen sensing, regulation of cellular ROS, and the function of ICa-L during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.