Pecori Giraldi F, Cavagnini F
Ospedale San Luca, IRCCS, Istituto Auxologico Italiano, 2nd Chair of Endocrinology, University of Milan, Milan, 20149 Italy.
J Clin Invest. 1998 Jun 1;101(11):2478-84. doi: 10.1172/JCI443.
Anterior pituitary hormone secretion is mainly regulated by hypothalamic releasing factors, which reach the pituitary via portal vessels. It has been demonstrated recently that these peptides can also be produced by the pituitary itself, thus possibly modulating hormone secretion in a paracrine/autocrine fashion. The object of this study was to seek evidence for the synthesis and secretion of corticotropin-releasing hormone (CRH) within the anterior pituitary and to ascertain its biological relevance. Messenger RNA from adult rat anterior pituitary fragments and cell cultures was reverse transcribed and subjected to PCR amplification using primers specific to the rat CRH gene. As in the hypothalamus, a single 232-bp band was obtained. The correspondence of the amplified fragment to the sequence of the CRH gene was confirmed by Southern blotting and restriction enzyme digestion. Combined in situ reverse transcription-PCR amplification/immunocytochemistry demonstrated the presence of CRH mRNA in corticotropes. Medium from anterior pituitary primary cultures contained approximately 7 pg/microg protein of CRH immunoreactivity which presented the same chromatographic profile on HPLC as the mature CRH peptide. Incubation of anterior pituitary cells with an antibody directed against CRH markedly reduced basal ACTH secretion compared with serum-treated control wells (0.89+/-0.11 vs. 1.74+/-0.14 ng/200,000 cells in control wells after 1 h, P < 0.05; 1.17+/-0.10 vs. 2.16+/-0. 39 ng/200,000 cells after 2 h, P < 0.05; 1.45+/-0.12 vs. 3.12+/-0.61 ng/200,000 cells after 3 h, P < 0.05). Further, the ACTH response to potassium and to forskolin was markedly blunted by the CRH antiserum as well as by the CRH antagonist, alpha-helical CRH(9-41). In conclusion, this study demonstrates the presence of CRH mRNA in normal rat corticotropes and the secretion of the mature peptide by the anterior pituitary, pointing to the production of CRH at the site of its target cells. In addition, intrapituitary CRH contributes in a paracrine/autocrine fashion to ACTH secretion.
垂体前叶激素的分泌主要受下丘脑释放因子的调节，这些因子通过门静脉到达垂体。最近有研究表明，这些肽也可由垂体自身产生，从而可能以旁分泌/自分泌方式调节激素分泌。本研究的目的是寻找垂体前叶内促肾上腺皮质激素释放激素（CRH）合成和分泌的证据，并确定其生物学意义。从成年大鼠垂体前叶片段和细胞培养物中提取的信使核糖核酸进行逆转录，并用大鼠CRH基因特异性引物进行聚合酶链反应（PCR）扩增。与下丘脑一样，获得了一条单一的232碱基对的条带。通过Southern印迹法和限制性内切酶消化证实了扩增片段与CRH基因序列的对应性。原位逆转录-PCR扩增/免疫细胞化学联合检测显示促肾上腺皮质激素细胞中存在CRH信使核糖核酸。垂体前叶原代培养物的培养基中含有约7皮克/微克蛋白质的CRH免疫反应性物质，其在高效液相色谱（HPLC）上的色谱图谱与成熟的CRH肽相同。与血清处理的对照孔相比，用抗CRH抗体孵育垂体前叶细胞可显著降低基础促肾上腺皮质激素（ACTH）分泌（1小时后，对照孔中为1.74±0.14纳克/200,000个细胞，处理孔中为0.89±0.11纳克/200,000个细胞，P<0.05；2小时后，分别为2.16±0.39纳克/200,000个细胞和1.17±0.10纳克/200,000个细胞，P<0.05；3小时后，分别为3.12±0.61纳克/200,000个细胞和1.45±0.12纳克/200,000个细胞，P<0.05）。此外，CRH抗血清以及CRH拮抗剂α-螺旋CRH（9-41）可显著减弱ACTH对钾和福司可林的反应。总之，本研究证明正常大鼠促肾上腺皮质激素细胞中存在CRH信使核糖核酸，且垂体前叶可分泌成熟肽，表明其靶细胞部位可产生CRH。此外，垂体内CRH以旁分泌/自分泌方式促进ACTH分泌。
