Tinajero J C, Fabbri A, Dufau M L
Section on Molecular Endocrinology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Endocrinology. 1992 Apr;130(4):1780-8. doi: 10.1210/endo.130.4.1312425.
CRF is produced in the Leydig cells and acts as a negative autocrine regulator of Leydig cell function. To clarify the hormonal control of CRF secretion by Leydig cells, we evaluated the participation of serotonin (5HT) and serotonin agonists in the release of CRF from Leydig cells and their effects on hCG-induced cAMP generation and steroidogenesis. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosterone production (ID50, 1 nM). The inhibitory action of 5HT was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The selective 5HT2 receptor agonist (+-)1-[2,5-dimethoxy-4-iodophyryl]2-amino propane hydrochloride (DOI) also stimulated CRF secretion and inhibited hCG-stimulated cAMP generation and testosterone production to control levels (ID50, 7 microM). Serotonergic 5HT1A, 5HT1B/1C, 5HT1D, and 5HT3/5HT2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the 5HT3 receptor had no effect. [125I]DOI binding studies in Leydig cells demonstrated two sets of receptors with Kd values in the nanomolar and micromolar range, with low and high capacities, respectively. The low affinity site differed from that of brain receptors (Kd, 4.2 nM) and displayed higher binding capacity (50-fold). The selective 5HT2 receptor antagonist ketanserin prevented CRF stimulation and blocked the inhibitory actions of 5HT and DOI, while the alpha 1-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sensitivity to hCG and raised maximal cAMP and testosterone production. 5HT was a more effective stimulus than hCG in stimulating CRF secretion, and gonadotropin-induced CRF release was inhibited by ketanserin. Inhibitory effects of exogenous CRF were demonstrable after blockade of 5HT action by ketanserin. The inhibitory actions of 5HT were unaffected by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that 5HT acts on 5HT2 receptors in Leydig cells that are distinct from those in the brain to stimulate CRF secretion through a pertussis toxin-insensitive G-protein. This action of 5HT is predominantly mediated by the low affinity 5HT2-binding site and requires full occupancy for maximal CRF stimulation, indicating the absence of spare receptors. 5HT-stimulated CRF inhibits basal and hCG-induced cAMP generation and steroidogenesis. Furthermore, 5HT mediates the stimulatory action of LH/hCG on CRF secretion from Leydig cells and, thus, participates in a negative autoregulatory loop to limit the testosterone response to the gonadotropic stimulus.
促肾上腺皮质激素释放因子（CRF）由睾丸间质细胞产生，作为睾丸间质细胞功能的负性自分泌调节因子。为阐明睾丸间质细胞对CRF分泌的激素调控，我们评估了血清素（5HT）和血清素激动剂在睾丸间质细胞释放CRF中的作用，以及它们对人绒毛膜促性腺激素（hCG）诱导的环磷酸腺苷（cAMP）生成和类固醇生成的影响。血清素刺激CRF分泌，使其比基础水平高出4倍，并抑制基础和hCG刺激的cAMP生成及睾酮产生（半数抑制浓度，1 nM）。CRF抗体和α-螺旋CRF-（9 - 41）拮抗剂可阻止5HT的抑制作用。选择性5HT2受体激动剂（±）1 - [2,5 - 二甲氧基 - 4 - 碘苯基] - 2 - 氨基丙烷盐酸盐（DOI）也刺激CRF分泌，并将hCG刺激的cAMP生成和睾酮产生抑制至对照水平（半数抑制浓度，7 μM）。血清素能的5HT1A、5HT1B/1C、5HT1D和5HT3/5HT2激动剂对hCG刺激的cAMP和睾酮产生的抑制作用较弱，而5HT3受体激动剂则无作用。睾丸间质细胞中的[125I]DOI结合研究显示有两组受体，解离常数（Kd）值分别在纳摩尔和微摩尔范围内，亲和力低和高。低亲和力位点与脑受体不同（Kd，4.2 nM），且结合能力更高（50倍）。选择性5HT2受体拮抗剂酮色林可阻止CRF刺激，并阻断5HT和DOI的抑制作用，而α1 - 肾上腺素能拮抗剂哌唑嗪则无作用。此外，用酮色林处理细胞可增加对hCG的敏感性，并提高cAMP和睾酮的最大生成量。在刺激CRF分泌方面，5HT比hCG更有效，且酮色林可抑制促性腺激素诱导的CRF释放。在用酮色林阻断5HT作用后，可证明外源性CRF的抑制作用。5HT的抑制作用不受百日咳毒素和霍乱毒素影响，添加8 - 溴 - cAMP可使其逆转。这些结果表明，5HT作用于睾丸间质细胞中的5HT2受体，该受体与脑内的不同，通过对百日咳毒素不敏感的G蛋白刺激CRF分泌。5HT的这一作用主要由低亲和力的5HT2结合位点介导，且需要完全占据才能实现最大的CRF刺激，表明不存在备用受体。5HT刺激的CRF抑制基础和hCG诱导的cAMP生成及类固醇生成。此外，5HT介导促黄体生成素/人绒毛膜促性腺激素（LH/hCG）对睾丸间质细胞CRF分泌的刺激作用，从而参与负性自调节环路，以限制睾酮对促性腺激素刺激的反应。
