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Induction of Guanosine Triphosphate-Cyclohydrolase by Follicle-Stimulating Hormone Enhances Interleukin-1ß-Stimulated Nitric Oxide Synthase Activity in Granulosa Cells¹

Department of Biochemistry Molecular Biology and Physiology, University of Las Palmas School of Medicine, Las Palmas, Spain

Address all correspondence and requests for reprints to: Dr. Carlos M. Ruiz de Galarreta, Department of Biochemistry Molecular Biology and Physiology, University of Las Palmas School of Medicine, Las Palmas 35016, Spain.

In cultured granulosa cells, interleukin-1ß (IL-1ß) induced a time-dependent (16–72 h) and dose-related (0.3–30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [³H]arginine to [³H]citrulline and NO₂^- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ml) or the cell-permeant cAMP analog (Bu)₂cAMP (0.5 mM) markedly enhanced IL-1ß-induced NO generation in cultured granulosa cells. The effect of IL-1ß on citrulline biosynthesis and NO₂^- accumulation was abrogated by the NOS inhibitor N^G-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-, or tumor necrosis factor-, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [³H]citrulline formation or NO₂^- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1ß-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH₄) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1ß and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1ß-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH₄ biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH₄ is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH₄ is essential for full iNOS biosynthetic capacity in IL-1ß-stimulated granulosa cells.