title: H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules.
abstract: Although elevated levels of H(2)O(2) have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H(2)O(2) on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H(2)O(2)-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca(2+)](i) was measured on fura-2-loaded vessels. Perfusion of H(2)O(2) (10 μM) caused a delayed and progressively increased endothelial [Ca(2+)](i) and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H(2)O(2) induced two phases of NO production followed by caspase activation, intracellular Ca(2+) accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser(1177) phosphorylation in the absence of elevated endothelial [Ca(2+)](i), whereas the second phase of NO depended on increased [Ca(2+)](i) and was associated with Thr(495) dephosphorylation without increased Ser(1177) phosphorylation. Inhibition of NOS prevented H(2)O(2)-induced caspase activation, cell apoptosis, and increases in endothelial [Ca(2+)](i) and Lp. Our results indicate that H(2)O(2) at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca(2+) accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases.
authors.affiliation: Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, WV 26505, USA.
authors.forename: Xueping
authors.initials: X
authors.lastname: Zhou
authors.forename: Dong
authors.initials: D
authors.lastname: Yuan
authors.forename: Mingxia
authors.initials: M
authors.lastname: Wang
authors.forename: Pingnian
authors.initials: P
authors.lastname: He
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019793:Fluorescein; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008643:Mesentery / Q000098:blood supply*; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010477:Perfusion; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D013912:Threonine; D013997:Time Factors; D014699:Venules / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
chemical_list: C414689:4,5-diaminofluorescein diacetate; D002147:Calmodulin; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D016877:Oxidants; C049925:fura-2-am; D013912:Threonine; D009569:Nitric Oxide; D012694:Serine; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; C505410:Casp3 protein, rat; D053148:Caspase 3; D053179:Caspase 7; D002118:Calcium; D019793:Fluorescein; D016257:Fura-2