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.