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.

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Background: Muscle degradation caused by severe burn releases myoglobin (Mb), which accumulates in the kidney (termed myoglobinuria). Mb is a pro-oxidant. Aim: To demonstrate that Mb promotes oxidative stress and dysfunction in cultured Madin-Darby canine kidney type II (MDCK II) cells. Methods: The glutathione redox ratio was used to monitor oxidative stress. Regulation of antioxidant response genes was determined with RT-PCR. Propidium iodide and annexin V staining were markers of necrosis and apoptosis, respectively. Mitochondrial function was assessed by monitoring mitochondrial depolarisation. Endocytosis was determined with immune fluorescence microscopy, and monolayer permeability was monitored with labelled inulin. Results: Kidney epithelial cells exposed to (0–100 µM) Mb showed a dose-dependent decrease in the glutathione redox ratio indicative of enhanced oxidative stress. In parallel, the expression of antioxidant genes for superoxide dismutase (SOD)-1/2, inducible haemoxygenase (HO-1) and catalase (CAT) increased in MDCK II cells, coupled with increases in corresponding activity. Notably, apoptosis and necrosis remained unaffected. However, transferrin endocytosis and monolayer permeability decreased significantly, while clathrin distribution and mitochondrial function were unaffected. Conclusion: Low concentrations of Mb promote oxidative stress in kidney epithelial cells that manifest as subtle changes to function without decreasing viability. Whether this impairs kidney function in burns patients is not clear.

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