Data_Sheet_9.PDF

The neurohypophyseal hormone oxytocin (Oxt) has been shown to stimulate prolactin (Prl) synthesis and release from the adenohypophysis in rats. However, little is known about the functional roles of Oxt-like neuropeptides in the adenohypophysis of non-mammalian vertebrates. In this study, cDNAs encoding ricefield eel oxytocin-like receptors (Oxtlr), namely isotocin (Ist) receptor 1 (Istr1) and 2 (Istr2), were isolated and specific antisera were generated, respectively. RT-PCR and Western blot analysis detected the presence of both Istr1 and Istr2 in the brain and pituitary, but differential expression in some peripheral tissues, including the liver and kidney, where only Istr1 was detected. In the pituitary, immunoreactive Istr1 and Istr2 were differentially distributed, with the former mainly in adenohypophyseal cell layers adjacent to the neurohypophysis, whereas the latter in peripheral areas of the adenohypophysis. Double immunofluorescent images showed that immunostaining of Istr1, but not Istr2 was localized to growth hormone (Gh) cells, but neither of them was expressed in Prl cells. Ist inhibited Gh release in primary pituitary cells of ricefield eels and increased Gh contents in the pituitary gland of ricefield eels at 6 h after in vivo administration. Ist inhibition of Gh release is probably mediated by cAMP, PKC/DAG, and IP3/Ca2+ pathways. In contrast, Ist did not affect either prl gene expression or Prl contents in primary pituitary cells. Results of this study demonstrated that Ist may not be involved in the regulation of Prl, but inhibit Gh release via Istr1 rather than Istr2 in ricefield eels, and provided evidence for the direct regulation of Gh cells by oxytocin-like neuropeptides in the pituitary of non-mammalian vertebrates.神经垂体激素（neurohypophyseal hormone）催产素（oxytocin, Oxt）已被证实可刺激大鼠腺垂体（adenohypophysis）合成并释放催乳素（prolactin, Prl）。然而，目前对于非哺乳类脊椎动物（non-mammalian vertebrates）腺垂体内催产素样神经肽的功能作用仍知之甚少。本研究成功克隆了黄鳝催产素样受体（oxytocin-like receptors, Oxtlr）的互补DNA（cDNA），即异催产素（isotocin, Ist）受体1（Istr1）与受体2（Istr2），并分别制备了特异性抗血清（antisera）。通过逆转录聚合酶链反应（RT-PCR）与蛋白质印迹（Western blot）分析，研究人员在脑与垂体（pituitary）中均检测到Istr1与Istr2的表达，但在肝脏、肾脏等部分外周组织（peripheral tissues）中仅能检测到Istr1，二者呈现差异化表达模式。在垂体内，免疫反应性Istr1与Istr2的分布存在显著差异：前者主要定位于与神经垂体（neurohypophysis）相邻的腺垂体细胞层，而后者则分布于腺垂体的外周区域。双重免疫荧光成像结果显示，Istr1的免疫染色可定位于生长激素（growth hormone, Gh）细胞，而Istr2则无此定位特征；且二者均未在Prl细胞中表达。异催产素可抑制黄鳝原代垂体细胞的生长激素释放，并在体内给药6小时后提升黄鳝垂体中的生长激素含量。异催产素对生长激素释放的抑制作用可能通过环磷酸腺苷（cAMP）、蛋白激酶C/二酰甘油（PKC/DAG）以及肌醇三磷酸/钙离子（IP3/Ca²+）通路介导。与之相反，异催产素对原代垂体细胞的催乳素基因表达与催乳素含量均无显著影响。本研究结果表明，异催产素可能并未参与催乳素的调控过程，而是通过Istr1（而非Istr2）抑制生长激素的释放，这为非哺乳类脊椎动物垂体内催产素样神经肽直接调控生长激素细胞提供了直接的实验证据。