SIN-1-induced cytotoxicity in cultured endothelial cells involves reactive oxygen species and nitric oxide: protective effect of sepiapterin.
The purpose of this study was to examine whether tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, attenuates endothelial cell death induced by 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), which is known to produce both superoxide and NO. Endothelial cell death was assessed by the release of intracellular lactate dehydrogenase (LDH). Addition of SIN-1 (500, 1,000 microM) to endothelial cells induced cell death from 6 h after its addition. The SIN-1-induced endothelial cell death was strongly reduced by treatment with carboxy-PTIO, a NO scavenger, or superoxide dismutase (SOD). Iron chelators and hydroxyl radical scavengers also reduced the SIN-1-induced endothelial cell death. Interestingly, the SIN-1-induced endothelial cell death was also reduced by treatment with catalase. Thus NO, superoxide, hydroxyl radical, and hydrogen peroxide are likely to be implicated in SIN-1-induced endothelial cell death. Moreover, pretreatment with sepiapterin, a precursor of BH4 synthesis, reduced the SIN-1-induced endothelial cell death and increased the intracellular BH4 content. Both the protective effect of sepiapterin and the increase in intracellular BH4 content were prevented by co-pretreatment with N-acetylserotonin (NAS), an inhibitor of BH4 synthesis. The protective effect of sepiapterin also was observed when up-take of trypan blue was used as another marker of cell death. These findings suggest that BH4 has a protective effect against endothelial cell death caused by the presence of NO and superoxide. The protective effect of BH4 may at least partly involve scavenging of superoxide or hydrogen peroxide or both, because we and other groups previously found that BH4 has a scavenging activity for reactive oxygen species.
Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
M
M
Ishii
S
S
Shimizu
K
K
Momose
T
T
Yamamoto
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta / Q000187:drug effects; D001565:Benzoates / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000633:toxicity; D009569:Nitric Oxide / Q000378:metabolism; D010936:Plant Extracts / Q000494:pharmacology; D011621:Pteridines / Q000378:metabolism / Q000494:pharmacology*; D011622:Pterins; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D014343:Trypan Blue / Q000493:pharmacokinetics
D000975:Antioxidants; D001565:Benzoates; D016166:Free Radical Scavengers; D007093:Imidazoles; D007502:Iron Chelating Agents; D010936:Plant Extracts; D011621:Pteridines; D011622:Pterins; D017382:Reactive Oxygen Species; C079393:1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole; D001708:Biopterins; D009569:Nitric Oxide; C002385:linsidomine; C015378:nas; C016727:sepiapterin; D008981:Molsidomine; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D013482:Superoxide Dismutase; C003402:sapropterin; D014343:Trypan Blue