AN NADPH OXIDASE SUPEROXIDE-GENERATING SYSTEM IN THE RABBIT AORTA

Superoxide anion can modulate vascular smooth muscle tone and potentially affect the growth response in vascular disease. The present studies were undertaken to characterize the source of superoxide in rabbit aorta. Rings of aorta (5 mm) were incubated in physiological salt solution (PSS) for 30 min at 37 degrees C in the presence of 10 mM diethyldithiocarbamate (DDC) with or without inhibitors of superoxide-generating systems. Rings were then placed in PSS containing 250 mu M lucigenin at 37 degrees C in the presence or absence of inhibitors, and changes in amounts of superoxide were determined by measuring chemiluminescence (units). The inhibitors of xanthine oxidase, oxypurinol (300 mu M), and of mitochondrial NADH dehydrogenase, rotenone (50 mu M), had no significant effect on superoxide levels. An inhibitor of NADPH oxidase, iodonium thiophen, caused a concentration-dependent inhibition of superoxide anion (12.49 +/- 1.48 vs. 5.27 +/- 1.81 and 2.30 +/- 0.36 units, control vs. 7 mu M and 70 mu M iodonium thiopen, respectively). A structurally related iodonium compound, diphenyleneiodonium (20 mu M), caused a 78% reduction in basal and DDC-evoked superoxide levels. In the presence or absence of DDC, exogenous administration of NADPH (10 mu M(-1) mM), but not NADP (1 mM), elicited a concentration-dependent rise in superoxide levels that was inhibited by iodonium thiophen. Particulate fractions of whole aortic tissue exhibited NADPH-dependent superoxide production that was inhibited by 1 mu M diphenyleneiodonium. Particulate fractions of whole aortic tissue and isolated medial cells contained similar NADPH oxidase superoxide-generating activity (3.6 +/- 0.41 and 2.9 +/- 1.4 nmol superoxide . min(-1). mg(-1) protein, respectively), whereas particulate fractions prepared from the adventitia had higher activity (12 +/- 6 nmol . min(-1). mg(-1) protein). NADH does not appear to be a suitable substrate for superoxide generation. These studies suggest that a major source of supero;dde anion in the rabbit aorta is an NADPH oxidase located in medial smooth muscle and adventitia.