Naor Z, Azrad A, Limor R, Zakut H, Lotan M
J Biol Chem. 1986 Sep 25;261(27):12506-12.
Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.
向预先用[32P]Pi或肌醇-[2-3H]肌醇标记的垂体细胞中添加促性腺激素释放激素（GnRH），导致[32P]磷脂酰肌醇4,5-二磷酸水平迅速下降（约10秒），[32P]磷脂酰肌醇4-磷酸水平下降（约1分钟），随后[32P]磷脂酰肌醇和[32P]磷脂酸的标记增加（1分钟）。在存在Li+（10 mM）的情况下，GnRH刺激[3H]肌醇1,4,5-三磷酸（10秒）、[3H]肌醇1,4-二磷酸（15秒）和[3H]肌醇1-磷酸（1分钟）的出现。单独的Li+刺激[3H]肌醇1-磷酸和[3H]肌醇1,4-二磷酸的积累，但不刺激[3H]肌醇1,4,5-三磷酸的积累，且对促黄体生成素释放无影响。GnRH对肌醇磷酸（Ins-P）产生的作用呈剂量依赖性（ED50 = 1 - 5 nM），并被强效拮抗剂[D-pGlu,pClPhe,D-Trp]GnRH阻断。分别通过离子霉素和A23187从细胞内或细胞外钙库升高胞质游离Ca2+水平（[Ca2+]i），对[3H]Ins-P产生无显著影响。GnRH诱导的[3H]Ins-P产生不依赖于细胞外Ca2+，并且在分别通过A23187或离子霉素动员细胞外或细胞内Ca2+后也能观察到。GnRH对[3H]Ins-P积累的作用不受肿瘤促进剂佛波酯12-O-十四酰佛波醇-13-乙酸酯或胰岛激活蛋白百日咳毒素预先处理细胞的影响。这些结果表明，GnRH刺激多磷酸肌醇的快速磷酸二酯水解。这种刺激作用不是通过胰岛激活蛋白-底物介导的，不依赖于[Ca2+]i的升高，也不受激活Ca2+/磷脂依赖性蛋白C激酶的12-O-十四酰佛波醇-13-乙酸酯的负调控。这些结果与以下假设一致，即GnRH诱导的磷脂酰肌醇周转负责Ca2+动员，随后导致促性腺激素释放。
