Tabraue C, Diaz Peñate R, Gallardo G, Hernandez I, Quintana J, Lopez Blanco F, Gonzalez Reyes J, Fanjul L F, Ruiz de Galarreta C M
Department of Biochemistry Molecular Biology and Physiology, University of Las Palmas School of Medicine, Spain.
Endocrinology. 1997 Jan;138(1):162-8. doi: 10.1210/endo.138.1.4854.
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 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-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or 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 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-1 beta-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-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-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-1 beta-stimulated granulosa cells.
在培养的颗粒细胞中，白细胞介素-1β（IL-1β）可诱导一氧化氮（NO）合酶（NOS）活性呈时间依赖性（16 - 72小时）和剂量相关性（0.3 - 30 ng/ml）的刺激，这是通过[3H]精氨酸催化转化为[3H]瓜氨酸以及培养基中NO2-积累来确定的。尽管单独使用促卵泡激素（FSH）未能刺激NOS活性，但与促性腺激素（200 ng/ml）或细胞可渗透的环磷酸腺苷类似物（Bu）2cAMP（0.5 mM）同时处理时，可显著增强培养的颗粒细胞中IL-1β诱导的NO生成。NOS抑制剂NG-甲基-L-精氨酸或IL-1受体拮抗剂蛋白可消除IL-1β对瓜氨酸生物合成和NO2-积累的影响。相比之下，细菌内毒素（脂多糖）、干扰素-γ或肿瘤坏死因子-α，这些在多种免疫活性和非免疫活性细胞类型中众所周知的诱导型NOS（iNOS）诱导剂，在未处理或FSH刺激的细胞中未能增加[3H]瓜氨酸的形成或NO2-积累。逆转录聚合酶链反应（RT-PCR）分析表明，IL-1β刺激的NO生成伴随着iNOS和GTP-环水解酶（GTPCH）信使RNA水平的时间依赖性增加，GTPCH是从头合成四氢生物蝶呤（BH4）的限速步骤。FSH处理仅增加GTPCH信使RNA表达，当细胞同时受到IL-1β和FSH刺激时，观察到GTPCH信号有超过相加的效应。用GTPCH抑制剂2,4-二氨基-6-羟基嘧啶处理可阻止未处理或FSH刺激的细胞中IL-1β诱导的NOS活性，并且这种抑制可被sepiapterin完全逆转，sepiapterin是BH4生物合成的底物，通过许多细胞类型中存在的替代蝶呤挽救途径起作用。由于BH4是NOS催化活性的必需辅因子，这些观察结果强烈表明FSH诱导的内源性BH4生物合成对于IL-1β刺激的颗粒细胞中完整的iNOS生物合成能力至关重要。
