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 beta-adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (I(Ca-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 I(Ca-L) to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of I(Ca-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 I(Ca-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 I(Ca-L) during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.
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 beta-adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (I(Ca-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 I(Ca-L) to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of I(Ca-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 I(Ca-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 I(Ca-L) during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.