INDUCTION OF GUANOSINE TRIPHOSPHATE-CYCLOHYDROLASE BY FOLLICLE-STIMULATING-HORMONE ENHANCES INTERLEUKIN-1-BETA-STIMULATED NITRIC-OXIDE SYNTHASE ACTIVITY IN GRANULOSA-CELLS

In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation o f nitric oxide (NO) synthase (NOS) activity, as determined by the cata lytic conversion of [H-3]arginine to [H-3]citrulline and NO2 accumulat ion in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ml) or t he cell-permeant cAMP analog (Bu)2(c)AMP analog (Bu)(2)cAMP (0.5 mM) m arkedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2 acc umulation was abrogated by the NOS inhibitor N-G-methyl-L-arginine of the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha , which are well known inducers of inducible NOS (iNOS) in a variety o f immunocompetent and nonimmunocompetent cell types, failed to increas e [H-3]citrulline formation or NO2 accumulation in untreated or FSH-st imulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependen t increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GT PCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) bio synthesis. Treatment with FSH augmented only GTPCH messenger RNA expre ssion, and a more than additive GTPCH signal was observed when cells w ere simultaneously challenged with IL-1 beta and FSH. Treatment with t he GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta -induced NOS activity in untreated or FSH-stimulated cells, and this i nhibition was completely reversed by sepiaterin, a substrate for BH1 b iosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is essential for full iNOS biosynthetic capacity i n IL-1 beta-stimulated granulosa cells.