Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia.
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.
Hool
Livia C
LC
Discipline of Physiology, The University of Western Australia, Crawley, WA, Australia. lhool@cyllene.uwa.edu.au
Di Maria
Carla A
CA
Viola
Helena M
HM
Arthur
Peter G
PG
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008689:Methacrylates / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000502:physiology*; D009861:Onium Compounds / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000032:analysis / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D007476:Ionophores; D008689:Methacrylates; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D013844:Thiazoles; D013481:Superoxides; D000804:Angiotensin II; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C007517:diphenyleneiodonium; C030517:myxothiazol; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases