Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time- dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH₄) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines γ-interferon, interleukin-1β, and tumor necrosis factor-α increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate- limiting enzyme in BH₄ biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 ± 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH₄ through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production. The effect of DAHP on NO production was exacerbated in the presence of either methotrexate or aminopterin, inhibitors of dihydrofolate reductase and dihydropteridine reductase. Generation of NO from activated RPASM requires de novo biosynthesis of BH₄ and may serve as potential alternative pharmacological targets in modifying NO production.