Objective: The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca 2+ 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 Ca 2+ channel to hadrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca 2+ channel (I Ca-L ) to isoproterenol (Iso). Results: Hypoxia caused a 41.2 T 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 T 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.

Changes in the microdialysis outflow-to-inflow (O/I) ratio for [(14)C]ethanol and (3)H(2)O were determined in the perfused rat hindlimb after increases and decreases in nutritive flow mediated by the vasoconstrictors norepinephrine (NE) and serotonin (5-HT), respectively. Microdialysis probes (containing 10 mM [(14)C]ethanol and (3)H(2)O pumped at 1 or 2 microl/min) were inserted through the calf of the rat. Hindlimb perfusion flow rate was varied from 6 to 56 ml x min(-1) x 100 g(-1) in the presence of NE, 5-HT, or saline vehicle. The O/I ratios for both tracers were determined at each perfusion flow rate, as was perfusion pressure, oxygen uptake (a surrogate indicator of nutritive flow), and lactate release. Both tracers showed a decreased O/I ratio as hindlimb perfusion flow was increased, with [(14)C]ethanol being higher than (3)H(2)O. NE decreased the O/I ratio compared with vehicle, and 5-HT increased it for both tracers and both microdialysis flow rates. We conclude that the microdialysis O/I ratio, while able to detect changes in total flow, is also sensitive to changes in nutritive and nonnutritive flow, where the latter still extracts tracer, but less than the former.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.

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