Ciampani T, Fabbri A, Isidori A, Dufau M L
Section on Molecular Endocrinology, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Endocrinology. 1992 Dec;131(6):2785-92. doi: 10.1210/endo.131.6.1332849.
Rat GH-releasing hormone (GHRH), mainly contained in hypothalamic neurons, has also been identified in several extraneural tissues, including the gastrointestinal tract, placenta, ovary, and testis. In the testis, GHRH mRNA is ontogenically regulated, and GHRH immunoreactivity can be observed in interstitial cells and tubules, suggesting an intratesticular role for the peptide. Leydig cells in culture are able to produce hypothalamic releasing hormones, i.e. CRH, which acts as an autocrine negative regulator of Leydig cell function. In this study we investigated whether GHRH is present in Leydig cells and evaluated the role of the peptide in Leydig cell function. Adult Leydig cells in culture produced considerable amounts of immunoreactive GHRH [23.9 +/- 2.1 (+/- SE) pg/10(6) cells.30 min], and the release of the peptide was acutely stimulated by hCG. HPLC analysis of GHRH in media from basal and hCG-treated cultures showed the presence of a single peak eluting at the same retention time as that of hypothalamic rat GHRH. Radioligand binding and activation studies revealed a common receptor for vasoactive intestinal peptide (VIP) and rat GHRH in Leydig cell membrane. Specific binding of [125I]VIP to Leydig cell membranes showed the presence of a single site, with high affinity and low binding capacity. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP > rat GHRH > secretin > human GHRH. In cultured Leydig cells, GHRH and VIP stimulated cAMP production, consistent with coupling of the receptor to the adenylate cyclase system. VIP displayed a lower ED50 than GHRH in stimulating cAMP production (P < 0.01), comparable with the higher binding potency of this peptide. No additive effects of VIP- and GHRH-stimulated cAMP generation were observed, suggesting that both peptides compete for the same receptor protein. GHRH and VIP had no effect on basal steroidogenesis, indicating a lack of tonic actions and compartmentalization of the peptides' effect. On the other hand, GHRH acted as a potentiator of the acute gonadotropin stimulation of testosterone production and cAMP generation. [125I]hCG binding to the Leydig cells in culture showed that GHRH was unable to affect the number or affinity of binding sites for hCG, indicating that the GHRH-sensitizing effect on LH action is beyond the level of gonadotropin binding and possibly is through the facilitation of LH receptor coupling functions.(ABSTRACT TRUNCATED AT 400 WORDS)
大鼠生长激素释放激素（GHRH）主要存在于下丘脑神经元中，在包括胃肠道、胎盘、卵巢和睾丸在内的几种神经外组织中也已被鉴定出来。在睾丸中，GHRH mRNA受到个体发育调控，并且在间质细胞和小管中可观察到GHRH免疫反应性，这表明该肽在睾丸内具有作用。培养的睾丸间质细胞能够产生下丘脑释放激素，即促肾上腺皮质激素释放激素（CRH），它作为睾丸间质细胞功能的自分泌负调节因子。在本研究中，我们调查了睾丸间质细胞中是否存在GHRH，并评估了该肽在睾丸间质细胞功能中的作用。培养的成年睾丸间质细胞产生了相当数量的免疫反应性GHRH[23.9±2.1（±标准误）皮克/10⁶个细胞·30分钟]，并且该肽的释放受到人绒毛膜促性腺激素（hCG）急性刺激。对基础培养和hCG处理培养的培养基中GHRH进行高效液相色谱（HPLC）分析，结果显示存在一个单一峰，其洗脱保留时间与下丘脑大鼠GHRH相同。放射性配体结合和激活研究揭示了睾丸间质细胞膜上血管活性肠肽（VIP）和大鼠GHRH存在共同受体。[¹²⁵I]VIP与睾丸间质细胞膜的特异性结合显示存在一个单一位点，具有高亲和力和低结合容量。VIP相关肽对放射性配体结合抑制的相对效力为：VIP＞大鼠GHRH＞促胰液素＞人GHRH。在培养的睾丸间质细胞中，GHRH和VIP刺激环磷酸腺苷（cAMP）产生，这与受体与腺苷酸环化酶系统的偶联一致。在刺激cAMP产生方面，VIP的半数有效剂量（ED50）低于GHRH（P＜0.01），这与该肽较高的结合效力相当。未观察到VIP和GHRH刺激cAMP生成的相加效应，表明这两种肽竞争相同的受体蛋白。GHRH和VIP对基础类固醇生成无影响，表明这些肽缺乏紧张性作用且其作用具有区室化。另一方面，GHRH作为急性促性腺激素刺激睾酮产生和cAMP生成的增强剂。[¹²⁵I]hCG与培养的睾丸间质细胞结合显示，GHRH无法影响hCG结合位点的数量或亲和力，这表明GHRH对促黄体生成素（LH）作用的敏化效应超出了促性腺激素结合水平，可能是通过促进LH受体偶联功能实现的。（摘要截断于400字）
