VASCULAR FREE-RADICAL RELEASE - EX-VIVO AND IN-VIVO EVIDENCE FOR A FLOW-DEPENDENT ENDOTHELIAL MECHANISM

Mechanisms underlying production of vascular free radicals are unclear . We hypothesized that changes in blood flow might serve as a physiolo gical stimulus for endothelial free radical release. Intact isolated a ortas from 45 rabbits were perfused with the spin trap alpha-phenyl-N- tert-butylnitrone (PBN, 20 mmol/L) and formed radical adducts detected by electron paramagnetic resonance spectroscopy (EPR). Sequential per fusion at 2, 7.5, and 12 mL/min changed cumulative vascular PBN radica l adduct yields, respectively, from 3.2+/-0.9 to 4.1+/-0.7 (P=.05) and 7.0+/-1.5 (P<.005) pmol/mg with endothelium and from 3.6+/-1.6 to 3.8 +/-1.4 and 2.2+/-0.8 pmol/mg without endothelium (P=NS). In endothelia lized aortas, superoxide dismutase (SOD) completely blocked flow-induc ed free radical production, whereas inactivated SOD, indomethacin, and the nitric oxide synthetase antagonist nitro-L-arginine methyl ester (L-NAME) had no effect; relaxations to acetylcholine remained unchange d with higher flows. To assess the role of flow on in vivo radical pro duction, femoral arterial plasma levels of the ascorbyl radical, a sta ble ascorbate oxidation product, were measured by direct EPR in 56 oth er rabbits. Ascorbyl levels were assessed at baseline (30.2+/-0.7 nmol /L) and at peak-induced iliac flow changes. Flow increases from 25% to 100% due to saline injections through an extracorporeal aortic loop i nduced significant dose-dependent increases in ascorbyl levels (n=5). In addition, after papaverine bolus injections, flow increased by 114/-8% versus baseline, and ascorbyl levels increased by 5.4+/-0.7 nmol/ L (n=31, P<.001); similar results occurred with adenosine, isoproteren ol, or hyperemia after 30-second occlusions (P<.05, n=4 or 5 in each g roup). Active SOD completely blocked papaverine-induced ascorbyl radic al increase, despite preserved flow response (DELTAascorbyl=0.02+/-1.6 nmol/L, P=NS); inactivated SOD, catalase, indomethacin, and L-NAME ha d no effect. Blood flow decreases of 65% to 100% due to phenylephrine or 60-second balloon occlusions were accompanied by an average decreas e of 4.4 nmol/L (P<.05) in ascorbyl levels. No change in ascorbyl sign al was observed when rabbit blood alone was submitted to in vitro flow increases through a tubing circuit. Thus, increases in blood flow tri gger vascular free radical generation; such a response seems to involv e endothelium-derived superoxide radicals unrelated to cyclooxygenase or nitric oxide synthetase activities. This mechanism may contribute t o explain vascular free radical generation in physiological or patholo gical circumstances.