Hydrogen Peroxide Promotes Endothelial Dysfunction by Stimulating Multiple Sources of Superoxide Anion Radical Production and Decreasing Nitric Oxide Bioavailability

Abstract

Hydrogen peroxide (H₂O₂) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H₂O₂. Exposure of aortic rings to 25 or 50 µM, but not 10 µM, H₂O₂ for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide (^·NO) donor sodium nitroprusside. Treatment of PAEC with 50 µM H₂O₂ significantly decreased ACh-induced accumulation of ^·NO, as measured with a ^·NO-selective electrode, yet such treatment increased nitric oxide synthase activity ∼3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased ^·NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O₂^–·), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O₂^–· production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H₂O₂ decreases the bioavailability of agonist-induced ^·NO as a result of increased production of O₂^–· likely derived from xanthine oxidase, NADPH-oxidasse and mitochondria. These processes could contribute to H₂O₂-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.