TETRAHYDROBIOPTERIN SYNTHESIS AND INDUCIBLE NITRIC-OXIDE PRODUCTION IN PULMONARY-ARTERY SMOOTH-MUSCLE

We recently reported (Am. J. Respir. Cell Mel. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a t ime-dependent increase in mRNA and protein expression of inducible nit ric oxide synthase (iNOS) in cultured rat pulmonary artery smooth musc le cells (RPASM). In the current study we extend observations on regul ation of iNOS in RPASM by showing that de novo synthesis of tetrahydro biopterin (BH4) is critical for LPS and cytokine-induced NO production . A mixture of LPS and the cytokines gamma-interferon, interleukin-1 b eta, and tumor necrosis factor-or increased steady-state levels of mRN A of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 bi osynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with fu rther increases at 24 h by Northern blot analysis and reverse-transcri ptase 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-i nduced NO production, determined by nitrite concentrations in the medi um, was decreased in a concentration-dependent manner by the GTP-CH in hibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inh ibited completely the LPS- and cytokine-induced accumulation of intrac ellular 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 wa s exacerbated in the presence of either methotrexate or aminopterin, i nhibitors of dihydrofolate reductase and dihydropteridine reductase. G eneration of NO from activated RPASM requires de novo biosynthesis of BH4 and may serve as potential alternative pharmacological targets in modifying NO production.