期刊
PLACENTA
卷 32, 期 12, 页码 932-940出版社
W B SAUNDERS CO LTD
DOI: 10.1016/j.placenta.2011.09.003
关键词
Placenta; Arginase; Endothelium; Human; Nitric oxide; Hypoxia
资金
- Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT) [1080534, 1110977]
- Programa de Investigacion Interdisciplinario (PIA), Comision Nacional de Investigacion en Ciencia y Tecnologia (CONICYT) [Anillos ACT-73]
- CONICYT
- Apoyo a la Realizacion de la Tesis Doctoral, CONICYT [AT-24090200, 24100107]
Hypoxia relates with altered placental vasodilation, and in isolated endothelial cells, it reduces activity of the endothelial nitric oxide synthase (eNOS) and L-arginine transport. It has been reported that arginase-2 expression, an alternative pathway for L-arginine metabolism, is increased in adult endothelial cells exposed to hypoxia as well as in pre-eclamptic placentae. We studied in human umbilical vein endothelial cells (HUVEC) whether hypoxia-reduced NO synthesis results from increased arginase-mediated L-arginine metabolism and changes in subcellular localization of eNOS and arginase-2. In HUVEC exposed (24 h) to 5% (normoxia) or 2% (hypoxia) oxygen, L-arginine transport kinetics, arginase activity (urea assay), and NO synthase (NOS) activity (L-citrulline assay) were determined. Arginase-1, arginase-2 and eNOS expression were determined by RT-PCR and Western blot. Subcellular localization of arginase-2 and eNOS were studied using confocal microscopy and indirect immunofluorescence. Experiments were done in absence or presence of S-(2-boronoethyl)-L-cysteine-HCl (BEC, arginase inhibitor) or N-G-nitro-L-arginine methyl ester (L-NAME). Hypoxia-induced reduction in eNOS activity was associated with a reduction in eNOS phosphorylation at Serine-1177 and increased phosphorylation at Threonine-495. This was paralleled with an induction in arginase-2 expression and activity, and decreased L-arginine transport. In hypoxia the arginase inhibition, restored NO synthesis and L-arginine transport, without changes in the eNOS post-translational modification status. Hypoxia increased arginase-2/eNOS colocalization, and eNOS redistribution to the cell periphery. Altogether these data reinforce the thought that eNOS cell location, post-translational modification and substrate availability are important mechanisms regulating eNOS activity. If these mechanisms occur in pregnancy diseases where feto-placental oxygen levels are reduced remains to be clarified. (C) 2011 Elsevier Ltd. All rights reserved.
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