title: Hypoxia-reduced nitric oxide synthase activity is partially explained by higher arginase-2 activity and cellular redistribution in human umbilical vein endothelium.
abstract: 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.
authors.affiliation: Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile.
authors.forename: C P
authors.initials: CP
authors.lastname: Prieto
authors.forename: B J
authors.initials: BJ
authors.lastname: Krause
authors.forename: C
authors.initials: C
authors.lastname: Quezada
authors.forename: R
authors.initials: R
authors.lastname: San Martin
authors.forename: L
authors.initials: L
authors.lastname: Sobrevia
authors.forename: P
authors.initials: P
authors.lastname: Casanello
mesh_terms: D000328:Adult; D001119:Arginase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D001120:Arginine / Q000378:metabolism; D001897:Boronic Acids / Q000494:pharmacology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D018613:Microscopy, Confocal; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis*; D010766:Phosphorylation; D011247:Pregnancy; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: C425062:(2-boronoethyl)-cysteine; D001897:Boronic Acids; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C477631:ARG2 protein, human; D001119:Arginase; D019331:NG-Nitroarginine Methyl Ester