Acceleration of oxidative stress-induced endothelial cell death by nitric oxide synthase dysfunction accompanied with decrease in tetrahydrobiopterin content.
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.
Ishii
M
M
Department of Clinical Pharmacy and Pharmacology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan.
Shimizu
S
S
Yamamoto
T
T
Momose
K
K
Kuroiwa
Y
Y
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
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