Chang J P, Stojilković S S, Graeter J S, Catt K J
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Endocrinology. 1988 Jul;123(1):87-97. doi: 10.1210/endo-123-1-87.
The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.
在静态培养中暴露于促性腺激素释放激素（GnRH）3小时或在柱灌流期间暴露2分钟的培养大鼠垂体细胞中，研究了促黄体生成素（LH）对GnRH的反应对细胞外钙的依赖性。在正常培养基中进行静态培养时，GnRH以0.3 nM的半数有效浓度（ED50）刺激LH释放，钾离子（K+）以32 mM的ED50刺激LH释放。在缺钙（未添加钙）或无钙培养基（含有100 μM乙二醇双四乙酸（EGTA））中孵育，显著降低但并未消除LH对10和100 nM GnRH的反应，而在缺钙培养基中，K+诱导的LH释放几乎完全被消除。钙离子通道激动剂（BK 8644）和拮抗剂（硝苯地平、尼卡地平、维拉帕米和钴离子（Co2+））分别增强或降低了LH对GnRH和K+的反应。然而，钙拮抗剂完全消除了LH对K+去极化的反应，但仅部分抑制了LH对GnRH的反应，证实了GnRH作用存在一个不依赖于细胞外钙的重要成分。在灌流的垂体细胞中，暴露于缺钙培养基或含有5 mM EGTA或5 mM乙二胺四乙酸（EDTA）的正常培养基中，降低了对10 nM GnRH 2分钟脉冲的初始快速LH反应，并消除了LH释放的第二阶段。在GnRH脉冲结束时重新引入含钙培养基导致LH分泌第二阶段的恢复，表明细胞外钙的流入对于LH对GnRH反应的早期阶段不是必需的，而是似乎对其延长至关重要。对花生四烯酸的LH释放反应与GnRH分泌作用机制有关，完全独立于细胞外钙，并且不受添加10 nM BK 8644的影响。这些观察结果表明，对GnRH分泌反应的启动在很大程度上独立于钙的进入，而促性腺激素分泌的延长由钙流入维持，部分通过电压敏感性钙通道。花生四烯酸代谢产物在GnRH作用中的作用可能与GnRH诱导的LH分泌的钙非依赖性成分有关。由于GnRH是间歇性且短时间分泌的，其对脉冲性促性腺激素释放的许多生理作用可能独立于细胞外液中的钙流入。
