Carboxy-PTIO increases the tetrahydrobiopterin level in mouse brain microvascular endothelial cells

The aim of the present study was to characterize the increase in tetrahydro biopterin (BH4), which is a cofactor for nitric oxide synthase (NOS), by ca rboxy-PTIO, a scavenger of nitric oxide (NO), in vascular endothelial cells . BH4 level was determined by oxidation under acidic conditions as biopteri n. Addition of lipopolysaccharide (LPS) to endothelial cells increased mRNA levels of inducible NOS (iNOS) and GTP-cyclohydrolase I (GTPCH), which is a rate-limiting enzyme for BH4 synthesis, and the biopterin level. NOS inhi bitors, NO-donors and L-arginine, a substrate of NOS, did not affect the in crease in the biopterin level induced by LPS, suggesting that BH4 synthesis is unlikely to be modulated by NO produced by iNOS during LPS treatment. H owever, carboxy-PTIO increased the biopterin level in the absence and the p resence of LPS. Carboxy-PTIO did not affect the expression of GTPCH mRNA le vel. Moreover, 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, inhi bited only about 30% of the carboxy-PTIO-induced increase in the biopterin level. Whereas, N-acetylserotonin, an inhibitor of sepiapterin reductase, s trongly inhibited the increase in biopterin level. Carboxy-PTIO inhibited t he accumulation of pterin, a decomposition product of BH4 in endothelial ce lls. These findings suggest that carboxy-PTIO accumulates BH4 under basal a nd LPS-treated conditions in vascular endothelial cells due to both inhibit ion of the decomposition of BH4 to pterin and activation of the salvage pat hway of BH4 synthesis via sepiapterin reductase.