title: Acceleration of oxidative stress-induced endothelial cell death by nitric oxide synthase dysfunction accompanied with decrease in tetrahydrobiopterin content.
abstract: The purpose of this study was to examine whether nitric oxide (NO) synthase dysfunction accompanied with decrease in tetrahydrobiopterin (BH4) content increases H2O2-induced endothelial cell death. Endothelial cell death was measured by the release of intracellular lactate dehydrogenase (LDH). Intracellular BH4 content was changed by pretreatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, or pretreatment with sepiapterin, a substrate for the salvage pathway of BH4 synthesis, and the intracellular content was measured by high performance liquid chromatography equipped with a fluorescence detector. Moreover, production of superoxide was detected by a chemiluminescence technique using MCLA, a Cypridina luciferin analogue, for the superoxide-sensitive probe. Pretreatment with DAHP (10 mM) for 24 h decreased intracellular BH4 content to 14% and increased H2O2-induced cell death. The toxic effect of DAHP was reduced by co-pretreatment with sepiapterin (100 microM) or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM), an inhibitor of NO synthase, but not by N(G)-methyl-L-arginine (L-NMA, 1 mM), the other inhibitor of NO synthase. Moreover, production of superoxide in endothelial cells induced by Ca2+-ionophore ionomycin (1 microM) increased by the pretreatment with DAHP, and the increase in superoxide production was blocked by L-NAME (1 mM) but not L-NMA (1 mM). Co-pretreatment with sepiapterin decreased the production of superoxide. These findings suggested that dysfunction of NO synthase with a decrease in BH4 content in endothelial cells produced superoxide instead of NO and increased the oxidative stress-induced endothelial cell death.
authors.affiliation: Department of Clinical Pharmacy and Pharmacology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ishii
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Momose
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroiwa
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D002417:Cattle; D016923:Cell Death / Q000187:drug effects*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007042:Hypoxanthines / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018384:Oxidative Stress; D011621:Pteridines / Q000494:pharmacology; D011622:Pterins; D013481:Superoxides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007042:Hypoxanthines; D011621:Pteridines; D011622:Pterins; D013481:Superoxides; D001708:Biopterins; D006861:Hydrogen Peroxide; C016727:sepiapterin; D019001:Nitric Oxide Synthase; C017202:2,4-diaminohypoxanthine; C003402:sapropterin