Hydroxylamine is a vasorelaxant and a possible intermediate in the oxidative conversion of L-arginine to nitric oxide

Our objective was to determine whether hydroxylamine is a possible intermediate in the oxidative conversion of L-arginine to nitric oxide. Vasorelaxation by hydroxylamine is known to be mediated by nitric oxide. The vasorelaxant properties of hydroxylamine were examined using rat aortic rings and an isolated rat lung perfusion model. Hydroxylamine and acetylcholine were equally effective in relaxing norepinephrine-contracted intact aortic rings, whereas only hydroxylamine relaxed aortic rings with endothelium removed. This endothelium-independent vasorelaxation by hydroxylamine indicated that the hydroxylamine-converting enzyme is not localized solely within endothelial cells. Catalase, an enzyme known to oxidize hydroxylamine to nitric oxide, was present in homogenates of intact and endothelium-denuded rings. Cyanamide, another catalase substrate and a known precursor of nitroxyl (HNO), was not a vasorelaxant of aortic rings or of isolated, hypoxia-constricted lungs. These results suggest that free nitroxyl is not an intermediate in the oxidation of hydroxylamine to nitric oxide. An overall pathway for the oxidative conversion of L-arginine through an hydroxylamine intermediate to nitric oxide is proposed

Induction of Endothelial Cell Injury by Cigarette Smoke

Cigarette smoke contains different populations of free radicals which may be responsible for endothelial cell (EC) injury of smokers. The purpose of this study was to examine the effects of gas-phase cigarette smoke on EC endothelium-derived relaxing factor (EDRF)/NO-guanylate cyclase (GC)-cGMP pathway and on EC detatchment-type injury after incubation with smoke. Furthermore, we examined whether different kind of antioxidants can prevent smoke-caused EC injury. We measured cGMP pathway using direct (sodium nitroprusside, SNP) and indirect (A23187, the calcium ionophore and bradykinin, BK) activators of GC. Directly and indirectly stimulated EC cGMP production dose-dependently decreased and EC detatchment increased after incubation with smoke. Externally added thiols (glutathione, GSH; D-Penicillamine, DP; N-acetylcysteine, NAC) protected EC from damage of cGMP production and cell detatchment. Other antioxidants (catalase, deferoxamine and superoxide dismutase) were ineffective. These results suggest that the thiol containing GC in EC is destroyed or inactivated or thiol like species responsible for activation of GC is incomplete in EC after incubation with smoke. It is also possible that externally added thiols bind an unknown component of smoke and this way, EC is protected. EC injury may contribute to vascular diseases associated with cigarette smoking

Stable Compounds of Cigarette Smoke Induce Endothelial Superoxide Anion Production via NADPH Oxidase Activation

Objective— Endothelial dysfunction is an early manifestation of cigarette smoke (CS) toxicity. We have previously demonstrated that CS impairs nitric oxide (NO)-mediated endothelial function via increased generation of superoxide anion (O 2 ). In these studies, we investigated whether stable compounds present in CS activate specific pathways responsible for the increased endothelial O OV0254; 2 production. Methods and Results— Short exposure of bovine pulmonary artery endothelial cells (BPAECs), human pulmonary artery endothelial cells, and rat pulmonary arteries to CS extracts (CSEs) resulted in a large increase in O OV0254; 2 production (20-fold, 3-fold, and 2-fold increase, respectively; P <0.05 versus control), which was inhibited by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodinium, apocynin, and gp91 docking sequence-tat peptide but not by oxypurinol, the NO synthase inhibitor N G -nitro- l -arginine methyl ester, or the mitochondrial respiration inhibitor rotenone. Exposure of BPAECs to acrolein, a stable thiol-reactive agent found in CS, increased O OV0254; 2 production 5-fold, which was prevented by prior inhibition of NADPH oxidase. Conclusions— These studies demonstrate that thiol-reactive stable compounds in CS can activate NADPH oxidase and increase endothelial O OV0254; 2 production, thereby reducing NO bioactivity and resulting in endothelial dysfunction. Clinically, these studies may contribute to the development of agents able to mitigate CS-mediated vascular toxicity. Exposure of pulmonary artery endothelial cells and pulmonary arteries to CSEs increased O 2 production that was prevented by NADPH oxidase inhibition. Exposure to acrolein, a thiol-reactive agent found in CS, also increased endothelial O 2 production. We conclude that stable thiol-reactive compounds in CS activate endothelial NADPH oxidase