Wiebe J P, Dhanvantari S, Watson P H, Huang Y
Hormonal Regulatory Mechanisms Laboratory, University of Western Ontario, London, Canada.
Endocrinology. 1994 Jan;134(1):377-82. doi: 10.1210/endo.134.1.8275953.
The gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. In a previous report, we showed that this suppression is achieved at least in part by an interaction at the plasma membrane level. We undertook to examine the possible interaction of 3 alpha HP at the level of intracellular Ca2+. Anterior pituitary cells from adult randomly cycling female rats were treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without protein kinase C activator (SC10), antagonist (H-7), intracellular Ca2+ chelator (TMB-8), and intracellular Ca2+ mobilizer (glutamate), and with or without EGTA and Ca2+ in the medium. FSH content in media and cells was determined by RIA. The protein kinase C (PKC) activator, SC10, increased basal levels of secreted FSH. 3 alpha HP suppressed (P < 0.05) SC10-stimulated basal FSH release. The PKC inhibitor, H7, decreased GnRH-induced FSH release; FSH was further suppressed (P < 0.05) by 3 alpha HP in the presence of H7. These results were interpreted to indicate that 3 alpha HP may act in part at the level of PKC and also at another site(s). The intracellular Ca2+ chelator, TMB-8, suppressed released and cellular GnRH-stimulated FSH to the same extent as 3 alpha HP; FSH was not further decreased by 3 alpha HP in the presence of TMB-8. 3 alpha HP suppressed glutamate-stimulated FSH release in Ca(2+)-free medium (P < 0.01). Moreover, GnRH-induced release of FSH was suppressed to the same degree by 10(-10) M 3 alpha HP as by 10(-4) M EGTA. In pituitary cell suspensions, the GnRH-induced [Ca2+]i elevations were significantly (P < 0.05) attenuated by 3 alpha HP. From these and previous results, a model is proposed for the action of 3 alpha HP. The model suggests that 3 alpha HP may interact with gonadotropes at the level of the PKC cell signaling pathway and intracellular Ca2+ mobilization, in addition to the plasma membrane/calcium channel. The interaction effects a decrease in intracellular Ca2+, leading to decreases in FSH release from those pituitary gonadotropes that are responsible for FSH. The consistent decrease in total FSH (released plus cellular content) by 3 alpha HP suggests that this neurosteroid may also suppress FSH synthesis.
性腺类固醇和神经类固醇3α-羟基-4-孕烯-20-酮（3αHP）可抑制垂体前叶细胞培养物中促卵泡激素（FSH）的释放。在先前的一份报告中，我们表明这种抑制至少部分是通过质膜水平的相互作用实现的。我们着手研究3αHP在细胞内Ca2+水平上可能的相互作用。将成年随机发情雌性大鼠的垂体前叶细胞用10 nM促性腺激素释放激素（GnRH）和0.1 nM 3αHP处理4小时，同时添加或不添加蛋白激酶C激活剂（SC10）、拮抗剂（H-7）、细胞内Ca2+螯合剂（TMB-8）和细胞内Ca2+动员剂（谷氨酸），并且在培养基中添加或不添加乙二醇双乙醚二胺四乙酸（EGTA）和Ca2+。通过放射免疫分析法（RIA）测定培养基和细胞中的FSH含量。蛋白激酶C（PKC）激活剂SC10增加了分泌型FSH的基础水平。3αHP抑制（P<0.05）SC10刺激的基础FSH释放。PKC抑制剂H7降低了GnRH诱导的FSH释放；在存在H7的情况下，3αHP进一步抑制（P<0.05）FSH释放。这些结果被解释为表明3αHP可能部分作用于PKC水平，也作用于其他位点。细胞内Ca2+螯合剂TMB-8与3αHP一样程度地抑制释放型和细胞内GnRH刺激的FSH；在存在TMB-8的情况下，3αHP不会进一步降低FSH。3αHP在无Ca2+培养基中抑制谷氨酸刺激的FSH释放（P<0.01）。此外，10-10 M 3αHP与10-4 M EGTA一样程度地抑制GnRH诱导的FSH释放。在垂体细胞悬液中，3αHP显著（P<0.05）减弱GnRH诱导的细胞内Ca2+升高。根据这些以及先前的结果，提出了一个3αHP作用的模型。该模型表明，除了质膜/钙通道外，3αHP可能在PKC细胞信号通路和细胞内Ca2+动员水平上与促性腺激素细胞相互作用。这种相互作用导致细胞内Ca2+减少，从而导致负责FSH的垂体促性腺激素细胞释放的FSH减少。3αHP使总FSH（释放型加上细胞内含量）持续减少，这表明这种神经类固醇也可能抑制FSH合成。
