Stojilković S S, Chang J P, Izumi S, Tasaka K, Catt K J
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20982.
J Biol Chem. 1988 Nov 25;263(33):17301-6.
The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of gonadotropin releasing hormone (GnRH) and phorbol esters in cultured pituitary cells. During incubation in normal medium, GnRH stimulated LH release with an ED50 of 0.35 nM. Incubation in Ca2+-deficient medium (Ca2+-free, 10 microM) substantially decreased but did not abolish the LH responses to GnRH. The extracellular Ca2+-dependent component of GnRH action could be mimicked by high K+ concentrations, consistent with the presence of voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs. Ca2+ channel agonist (Bay K 8644) and antagonist (nifedipine) analogs, respectively, enhanced or partially inhibited LH responses to GnRH and also to K+, the latter confirming the participation of two types of VSCC (dihydropyridine-sensitive and -insensitive) in K+-induced secretion. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), 4 beta-phorbol-12,13-dibenzoate, and 4 beta-phorbol-12,13-diacetate, stimulated LH release with ED50s of 5, 10, and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca2+]e in the incubation medium, and the residual LH response was identical with that elicited by GnRH in Ca2+-deficient medium. TPA increased [Ca2+]i to a peak after 20 s in normal medium but not in the absence of extracellular Ca2+, indicating that protein kinase C (Ca2+/phospholipid-dependent enzyme) promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca2+]i. The extracellular Ca2+-dependent action of TPA on LH release was blocked by Co2+. However, nifedipine did not alter TPA action on [Ca2+]i and LH release. These observations indicate that protein kinase C can participate in GnRH-induced LH release that is independent of Ca2+ entry, but also promotes the influx of extracellular Ca2+ through dihydropyridine-insensitive Ca2+-channels.
在对促性腺激素释放激素（GnRH）和佛波酯在培养的垂体细胞中的作用进行的研究中，分析了蛋白激酶C在促黄体生成素（LH）释放中的作用。在正常培养基中孵育期间，GnRH以0.35 nM的半数有效浓度（ED50）刺激LH释放。在缺钙培养基（无钙，10 microM）中孵育可显著降低但并未消除LH对GnRH的反应。GnRH作用的细胞外钙依赖性成分可被高钾浓度模拟，这与垂体促性腺细胞中存在电压敏感性钙通道（VSCC）一致。钙通道激动剂（Bay K 8644）和拮抗剂（硝苯地平）类似物分别增强或部分抑制LH对GnRH以及对钾的反应，后者证实了两种类型的VSCC（对二氢吡啶敏感和不敏感）参与钾诱导的分泌。佛波酯，包括12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）、4β - 佛波醇 - 12,13 - 二苯甲酸酯和4β - 佛波醇 - 12,13 - 二乙酸酯，分别以5、10和1000 nM的ED50刺激LH释放，且刺激效果约为GnRH的70%。随着孵育培养基中细胞外钙（[Ca2 + ]e）浓度逐渐降低，佛波酯刺激的LH分泌减少但未被消除，并且剩余的LH反应与在缺钙培养基中GnRH引发的反应相同。在正常培养基中，TPA在20秒后使细胞内钙（[Ca2 + ]i）升高至峰值，但在无细胞外钙的情况下则不会，这表明蛋白激酶C（钙/磷脂依赖性酶）促进钙内流，但也可在钙内流和[Ca2 + ]i无变化的情况下介导分泌反应。TPA对LH释放的细胞外钙依赖性作用被钴离子阻断。然而，硝苯地平并未改变TPA对[Ca2 + ]i和LH释放的作用。这些观察结果表明，蛋白激酶C可参与GnRH诱导的LH释放，这种释放独立于钙内流，但也通过对二氢吡啶不敏感的钙通道促进细胞外钙的内流。
