Lactate and P o 2 Modulate Superoxide Anion Production in Bovine Cardiac Myocytes

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Abstract
Background Lactate increases lucigenin chemiluminescence (CL)–detectable superoxide anion (O 2 • − ) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by P o 2 and cytosolic NAD(H) redox appears to be the detected source of O 2 • − production. Little is known about the importance of this O 2 • − -producing system in cardiac myocytes. Methods and Results In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O 2 • − levels to ≈1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O 2 • − . A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b 558 , was a major source of O 2 • − production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O 2 • − production. However, mitochondria released H 2 O 2 in the presence of 5 mmol/L succinate and 30 μmol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)–elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O 2 • − that was potentiated by lactate and inhibited by DPI. Conclusions NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O 2 • − production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in P o 2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.