Effects of the reactive oxygen species hydrogen peroxide and hypochlorite on endothelial nitric oxide production

Reactive oxygen species (ROS) hydrogen peroxide (H2O2) and hypochlorite (HO Cl) participate in the pathogenesis of ischemia/reperfusion injury, inflamm ation, and atherosclerosis. Both NO and ROS are important modulators of vas cular tone and architecture and of adhesive interactions between leukocytes , platelets, and vascular endothelium. We studied the effect of H2O2 and HO Cl on receptor-dependent (bradykinin [10(-6) mol/L] and ADP [10(-4) mol/L]) and receptor-independent mechanisms (calcium ionophore A23187 [10(-6) mol/ L]) of NO production by porcine aortic endothelial cells (ECs). Changes in the level of EC cGMP (the second messenger of NO) were used as a surrogate of NO production. EC cGMP increased 300% in response to bradykinin and A231 87 and 200% in response to ADP. Exposure of ECs to H2O2 (50 mu mol/L) for 3 0 minutes significantly impaired cGMP levels in response to ADP, bradykinin , and the receptor-independent NO agonist A23187. In contrast, preincubatio n with HOCl (50 mu mol/L) impaired cGMP production only in response to ADP and bradykinin but not A23187. These concentrations of H2O2 and HOCl did no t result in increased EC lethality as assessed by lactate dehydrogenase rel ease. Neither H2O2 nor HOCl affected EC cGMP production in response to NO d onor sodium nitroprusside, which suggests that guanylate cyclase is resista nt to these oxidants. We also demonstrated that neither H2O2 nor HOCl affec ts endothelial NO synthase (eNOS) catalytic activity as measured by convers ion Of L-arginine to L-citrulline in EC homogenates supplemented with eNOS cofactors. The present studies show that H2O2 impairs NO production in resp onse to both receptor-dependent and receptor-independent agonists and that these effects are due, at least in part, to inactivation of eNOS cofactors, whereas HOCl inhibits NO production by interfering with receptor-operated mechanisms at the level of the cell membrane. Concentrations of H2O2 and HO Cl used in the present studies have been shown to be generated in vivo duri ng inflammation and ischemia/reperfusion. Therefore, we infer that these ef fects of H2O2 and HOCl on EC NO production may contribute to disregulated v ascular tone and altered leukocyte-EC interactions that occur in vascular i njury as a result of those causes in which ROS generation is involved.