Carroll J A, Willard S T, Bruner B L, McArthur N H, Welsh T H
Department of Animal Science, Texas A&M University System, College Station 77843, USA.
Domest Anim Endocrinol. 1996 Jul;13(4):339-49. doi: 10.1016/0739-7240(96)00047-1.
Mifepristone (RU486), bovine corticotropin-releasing hormone (CRH), arginine vasopressin (VP), adrenocorticotropin (ACTH1-24), and protein kinase activators (forskolin, [FSK]; phorbol 12-myristate 13-acetate [PMA]) were used in vitro to investigate their direct effect on adrenocorticosteroidogenesis. Bovine adrenocortical fasciculata/reticularis cells (2 x 10(5) viable cells/well) were cultured for 3 d in medium supplemented with 10% fetal calf serum. After incubation for an additional 24 hr in serum-free medium, cells were treated with serum-free medium alone (Control) or various concentrations of ACTH, CRH, VP, FSK, PMA, RU486, and/or various concentrations for 1, 2, 4, or 24 hr. Medium content of cortisol and progesterone were determined by radioimmunoassays. ACTH, CRH, FSK, and PMA each stimulated (P < 0.05) secretion of cortisol in time- and dose-related manners. Although these agents stimulated (P < 0.05) secretion of progesterone in a dose-related manner, medium content of progesterone declined (P < 0.05) over time. The minimal effective doses of ACTH and CRH required to stimulate (P < 0.05) secretion of cortisol relative to the Control over a 4-hr culture period were 0.01 nM and 3 nM, respectively. Relative to observations at 1 hr posttreatment, 24-hr treatment with ACTH or CRH increased the medium content of cortisol by an additional 19.8- and 48-fold, respectively (whereas content of progesterone declined over that time period). VP-stimulated secretion of cortisol was time- (P < 0.05) but not dose-related. Specifically, by 24-hr posttreatment, the medium content of cortisol was increased (P < 0.05) 4.6-fold relative to the quantity of cortisol secreted by 1-hr postaddition of VP (0.01 to 1 microM). Co-treatment with RU486 (1 microM) decreased (p < 0.05) FSK-, ACTH- and CRH-stimulated secretion of cortisol by 77, 27, and 56%, respectively. Similarly, the stimulatory effects of ACTH and CRH on progesterone secretion were reduced (P < 0.05) by 40 and 22%, respectively, by co-addition of RU486. The inhibitory action of RU486 on production of cortisol was no longer apparent by 24 hr after treatment. These observations indicate that RU486 can act as a steroid agonist and as well as an antagonist. These data characterize time- and dose-related direct actions of ACTH, CRH, and RU486 on adrenocorticosteroidogenesis. This information will assist efforts to clarify complex intra-adrenal interactions of neurohormones, growth factors, and endogenous steroids.
米非司酮（RU486）、牛促肾上腺皮质激素释放激素（CRH）、精氨酸加压素（VP）、促肾上腺皮质激素（ACTH1 - 24）以及蛋白激酶激活剂（福斯高林，[FSK]；佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯[PMA]）被用于体外实验，以研究它们对肾上腺皮质类固醇生成的直接作用。牛肾上腺束状带/网状带细胞（2×10⁵个活细胞/孔）在补充有10%胎牛血清的培养基中培养3天。在无血清培养基中再孵育24小时后，细胞分别用单独的无血清培养基（对照）或不同浓度的促肾上腺皮质激素、促肾上腺皮质激素释放激素、精氨酸加压素、福斯高林、佛波酯、米非司酮和/或不同浓度处理1、2、4或24小时。通过放射免疫分析法测定培养基中皮质醇和孕酮的含量。促肾上腺皮质激素、促肾上腺皮质激素释放激素、福斯高林和佛波酯均以时间和剂量相关的方式刺激（P < 0.05）皮质醇的分泌。尽管这些试剂以剂量相关的方式刺激（P < 0.05）孕酮的分泌，但培养基中孕酮的含量随时间下降（P < 0.05）。在4小时培养期内，相对于对照，刺激（P < 0.05）皮质醇分泌所需的促肾上腺皮质激素和促肾上腺皮质激素释放激素的最小有效剂量分别为0.01 nM和3 nM。相对于处理后1小时的观察结果，促肾上腺皮质激素或促肾上腺皮质激素释放激素处理24小时后，培养基中皮质醇的含量分别增加了19.8倍和48倍（而在此时间段内孕酮的含量下降）。精氨酸加压素刺激的皮质醇分泌与时间相关（P < 0.05），但与剂量无关。具体而言，处理后24小时，相对于添加精氨酸加压素1小时后分泌的皮质醇量（0.01至1 microM），培养基中皮质醇的含量增加了（P < 0.05）4.6倍。与米非司酮（1 microM）共同处理分别使福斯高林、促肾上腺皮质激素和促肾上腺皮质激素释放激素刺激的皮质醇分泌减少了77%、27%和56%。同样，共同添加米非司酮分别使促肾上腺皮质激素和促肾上腺皮质激素释放激素对孕酮分泌的刺激作用降低了（P < 0.05）40%和22%。米非司酮对皮质醇产生的抑制作用在处理后24小时不再明显。这些观察结果表明，米非司酮既可以作为类固醇激动剂，也可以作为拮抗剂。这些数据描述了促肾上腺皮质激素、促肾上腺皮质激素释放激素和米非司酮对肾上腺皮质类固醇生成的时间和剂量相关的直接作用。这些信息将有助于阐明肾上腺内神经激素、生长因子和内源性类固醇之间复杂的相互作用。
