Protective effects of tetrahydrobiopterin against nitric oxide-induced endothelial cell death

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, attenuates NO-induced endothelial cell death. S-Nitroso-N-acetyl-DL-penicillamine (SNAP) was used as a NO donor. Endothelial cell death was assessed by the leakage of intracellular lactate dehydrogenase (LDH). Addition of SNAP to endothelial cells time- and concentration-dependently induced endothelial cell death. The SNAP-induced endothelial cell death was strongly reduced by the treatment with carboxy-PTIO, a NO scavenger, or catalase, but not with superoxide dismutase (SOD). Moreover, pretreatment with sepiapterin, a precursor of BH4, increased intracellular BH4 content, and strongly reduced the SNAP-induced endothelial cell death. Both the increase in BH4 content and the protective effects of sepiapterin were prevented by co-pretreatment with N-acetylserotonin (NAS), an inhibitor of BH4 synthesis. These findings suggest that the cytotoxicity of NO released from SNAP involves H2O2 production, and increase in intracellular BH4 content attenuates NO-induced endothelial cell death. Scavenging of H2O2 by BH4 may be at least one of the mechanisms by which BH4 reduces NO-induced endothelial cell death.