Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle.
Since controversy exists on how hypoxia influences vascular reactive oxygen species (ROS) generation, and our previous work provided evidence that it relaxes endothelium-denuded bovine coronary arteries (BCA) in a ROS-independent manner by promoting cytosolic NADPH oxidation, we examined how hypoxia alters relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in BCA. Methods were developed to image and interpret the effects of hypoxia on NAD(P)H redox based on its autofluorescence in the cytosolic, mitochondrial, and nuclear regions of smooth muscle cells isolated from BCA. Aspects of anaerobic glycolysis and cytosolic NADH redox in BCA were assessed from measurements of lactate and pyruvate. Imaging changes in mitosox and dehydroethidium fluorescence were used to detect changes in mitochondrial and cytosolic-nuclear superoxide, respectively. Hypoxia appeared to increase mitochondrial and decrease cytosolic-nuclear superoxide under conditions associated with increased cytosolic NADH (lactate/pyruvate), mitochondrial NAD(P)H, and hyperpolarization of mitochondria detected by tetramethylrhodamine methyl-ester perchlorate fluorescence. Rotenone appeared to increase mitochondrial NAD(P)H and superoxide, suggesting hypoxia could increase superoxide generation by complex I. However, hypoxia decreased mitochondrial superoxide in the presence of contraction to 30 mM KCl, associated with decreased mitochondrial NAD(P)H. Thus, while hypoxia augments NAD(P)H redox associated with increased mitochondrial superoxide, contraction with KCl reverses these effects of hypoxia on mitochondrial superoxide, suggesting mitochondrial ROS increases do not mediate hypoxic relaxation in BCA. Since hypoxia lowers pyruvate, and pyruvate inhibits hypoxia-elicited relaxation and NADPH oxidation in BCA, mitochondrial control of pyruvate metabolism associated with cytosolic NADPH redox regulation could contribute to sensing hypoxia.
Department of Physiology, New York Medical College, Valhalla, NY 10595, USA.
Qun
Q
Gao
Michael S
MS
Wolin
D000818:Animals; D002417:Cattle; D003331:Coronary Vessels / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D005456:Fluorescent Dyes; D000860:Hypoxia / Q000201:enzymology*; D066298:In Vitro Techniques; D019344:Lactic Acid / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009126:Muscle Relaxation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D017382:Reactive Oxygen Species; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D013481:Superoxides; D019344:Lactic Acid; D019289:Pyruvic Acid; D009247:NADH, NADPH Oxidoreductases