Dhanvantari S, Wiebe J P
Hormonal Regulatory Mechanisms Laboratory, University of Western Ontario, London, Canada.
Endocrinology. 1994 Jan;134(1):371-6. doi: 10.1210/endo.134.1.8275952.
We have previously shown that the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. We undertook exploration of the mechanisms of this suppression by examining the possible sites of 3 alpha HP action in isolated anterior pituitary cells of rats. The specific objective of this study was to determine if 3 alpha HP suppresses FSH by action at the level of the gonadotrope membrane and/or calcium channels. Pituitary cells from adult randomly cycling female rats were precultured for 72 h and then treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without Ca2+ channel agonists or antagonist. In other experiments, cells were treated with BSA-conjugated 3 alpha HP, progesterone, or 3 beta HP (the stereoisomer of 3 alpha HP). Levels of FSH were determined by RIA in media and cells. GnRH-stimulated FSH release and the total FSH (released plus cellular) were significantly suppressed by 3 alpha HP. The Ca2+ ionophore A23187 induced FSH release and 3 alpha HP significantly suppressed both released and total FSH in its presence. In combination with a high dose (100 microM) of the dihydropyridine-sensitive Ca2+ channel antagonist nifedipine, 3 alpha HP suppressed FSH secretion to a greater extent than the antagonist alone. Cellular content of FSH was also decreased by nifedipine (100 microM) and was further suppressed in the presence of 3 alpha HP. The phenylalkylamine-sensitive Ca2+ channel antagonist methoxyverapamil (D600) suppressed GnRH-induced FSH release, and 3 alpha HP significantly potentiated the suppression. Released and cellular FSH were increased by the dihydropyridine-sensitive agonist BAYK 8644, whereas 0.1 nM 3 alpha HP suppressed this agonist-induced FSH to a greater extent than the maximum dose (100 microM) of nifedipine. In order to test for direct action at the level of the gonadotrope membrane, 3 alpha HP was conjugated to BSA (3 alpha HP-BSA) and administered to cultured pituitary cells. The 3 alpha HP-BSA conjugate (but not progesterone-BSA or 3 beta HP-BSA) significantly suppressed release of FSH. The results of the study suggest that 3 alpha HP may be interacting with the Ca2+ channel component of the GnRH signal transduction mechanism; in addition, 3 alpha HP may also suppress FSH release (and possibly synthesis) through direct action at the level of the gonadotrope membrane.
我们之前已经表明，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。我们通过研究3αHP在大鼠离体垂体前叶细胞中的可能作用位点，对这种抑制作用的机制进行了探索。本研究的具体目的是确定3αHP是否通过作用于促性腺激素细胞的膜水平和/或钙通道来抑制FSH。将成年随机发情的雌性大鼠的垂体细胞预培养72小时，然后用10 nM促性腺激素释放激素（GnRH）和0.1 nM 3αHP处理4小时，同时添加或不添加钙通道激动剂或拮抗剂。在其他实验中，细胞用牛血清白蛋白（BSA）偶联的3αHP、孕酮或3βHP（3αHP的立体异构体）处理。通过放射免疫分析法（RIA）测定培养基和细胞中的FSH水平。3αHP显著抑制了GnRH刺激的FSH释放以及总FSH（释放的加上细胞内的）。钙离子载体A23187诱导FSH释放，3αHP在其存在时显著抑制了释放的和总的FSH。与高剂量（100 μM）的对二氢吡啶敏感的钙通道拮抗剂硝苯地平联合使用时，3αHP比单独使用拮抗剂更能抑制FSH分泌。硝苯地平（100 μM）也降低了细胞内FSH的含量，并且在3αHP存在时进一步受到抑制。对苯烷基胺敏感的钙通道拮抗剂甲氧基维拉帕米（D600）抑制GnRH诱导的FSH释放，3αHP显著增强了这种抑制作用。对二氢吡啶敏感的激动剂BAYK 8644增加了释放的和细胞内的FSH，而0.1 nM 3αHP比最大剂量（100 μM）的硝苯地平更能抑制这种激动剂诱导的FSH。为了测试在促性腺激素细胞膜水平的直接作用，将3αHP与BSA偶联（3αHP-BSA）并施用于培养的垂体细胞。3αHP-BSA偶联物（但不是孕酮-BSA或3βHP-BSA）显著抑制了FSH的释放。研究结果表明，3αHP可能与GnRH信号转导机制的钙通道成分相互作用；此外，3αHP也可能通过直接作用于促性腺激素细胞膜水平来抑制FSH释放（可能还有合成）。
