Van Strien F J, Roubos E W, Vaudry H, Jenks B G
Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, Subfaculty of Biology, University of Nijmegen, The Netherlands.
Endocrinology. 1996 Oct;137(10):4298-307. doi: 10.1210/endo.137.10.8828489.
The release of POMC-derived peptides from the melanotrope cells in the pituitary pars intermedia of Xenopus laevis is regulated by multiple inhibitory and stimulatory neurochemical messengers. In this study we examined the role of acetylcholine (ACh) in regulating melanotrope cell activity. The origin of ACh was studied in in vitro biosynthesis experiments, where it was demonstrated that dissociated melanotropes can synthesize ACh. In addition, immunocytochemical studies showed the presence of the ACh-synthesizing enzyme choline acetyltransferase in the cytoplasm of melanotrope cells. Oscillations in the concentration of intracellular free calcium ions are thought to be the driving force for the secretion of POMC-derived peptides from Xenopus melanotrope cells. A technique was developed to study simultaneously calcium oscillations and peptide secretion from the same population of single melanotrope cells in vitro. ACh concentration-dependently raises the intracellular free calcium concentration by increasing both the frequency and amplitude of the calcium oscillations as well as the release of POMC-derived peptides. The actions of ACh on the melanotrope cell were mimicked by muscarine, but not by nicotine, indicating the exclusive presence of a muscarinic cholinergic receptor. Furthermore, selective muscarinic receptor antagonists showed that the actions of ACh are mediated through an M1-subtype muscarinic receptor. Immunofluorescence cytochemistry in combination with confocal laser scanning microscopy visualized muscarinic receptors on the surface of melanotrope cells. It is concluded that ACh stimulates the release of POMC-derived peptides from the Xenopus melanotrope cell in an autocrine way, acting via a muscarinic M1 receptor on the intracellular free calcium concentration.
非洲爪蟾脑垂体中间叶黑素细胞中源自阿黑皮素原（POMC）的肽类释放受多种抑制性和刺激性神经化学信使调节。在本研究中，我们检测了乙酰胆碱（ACh）在调节黑素细胞活性中的作用。在体外生物合成实验中研究了ACh的来源，实验表明解离的黑素细胞能够合成ACh。此外，免疫细胞化学研究显示黑素细胞胞质中存在合成ACh的酶——胆碱乙酰转移酶。细胞内游离钙离子浓度的振荡被认为是非洲爪蟾黑素细胞分泌源自POMC的肽类的驱动力。我们开发了一种技术，用于在体外同时研究同一群单个黑素细胞的钙振荡和肽分泌。ACh浓度依赖性地提高细胞内游离钙浓度，这是通过增加钙振荡的频率和幅度以及源自POMC的肽类的释放来实现的。毒蕈碱模拟了ACh对黑素细胞的作用，而烟碱则不能，这表明仅存在毒蕈碱型胆碱能受体。此外，选择性毒蕈碱受体拮抗剂表明，ACh的作用是通过M1亚型毒蕈碱受体介导的。免疫荧光细胞化学结合共聚焦激光扫描显微镜观察到黑素细胞表面存在毒蕈碱受体。得出的结论是，ACh以自分泌方式刺激非洲爪蟾黑素细胞释放源自POMC的肽类，通过毒蕈碱M1受体作用于细胞内游离钙浓度。
