REGULATION OF PANCREATIC MUSCARINIC RECEPTORS IN VITRO

胰腺毒蕈碱受体的体外调节

• 批准号: 3239852
• 负责人: SETH R HOOTMAN
• 金额: $ 7.22万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-15 至 1991-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3239852
• 关键词: O glycosidase; acetylcholine; cell membrane; chemical structure function; cystic fibrosis; epithelium; exo alpha sialidase; gel electrophoresis; guinea pigs; hormone regulation /control mechanism; laboratory rat; lacrimal apparatus; muscarinic receptor; neurotransmitter receptor; organelles; pancreas; pancreatitis; parotid gland; peptidases; phosphorylation; protein kinase C; secretion; tritium

The proposed research will probe the molecular structure of muscarinic acetylcholine receptors in the pancreas and lacrimal and parotid glands and define the mechanisms involved in modulation of receptor densities and affinity for agonists. These studies will enlarge understanding of the cellular processes responsible for control of neurotransmitter receptors in epithelia a goal of particular importance as dysfunction in these mechanisms is implicated in the pathology of pancreatitis and cystic fibrosis. To investigate structural characteristics of muscinic receptors, acini prepared from these organs will be labeled with (3H) propylbenzilycholine mustard (PrBCM), an irreversible cholinergic antagonist. After labelling, plasma membranes will be prepared and treated with neuraminidase, endoglycosidase F, and proteases and then solubilized and electrophoresed. Shifts in the electrophoretic mobility of labelled receptor protein induced by these enzymes will reveal the contribution of sialic acid and total glycosyl residues to the apparent molecular size of the muscarinic receptor as well as the size of the receptor domains on the extracellular and cytoplasmic sides of the membrane bilayer. Regulation of muscarinic receptor population size and affinity for agonists also will be examined by investigation of the effects of chronic exposure of cultured acini to acetyl-choline analogues. The subcellular pathways and organelles involved in receptor biosynthesis and degradation will be probed using the reversible muscarinic antagonists, (3H)-N- methylscopolamine and (3H)-quinuclidinyl benzilate and specific inhibitors such as methylamine, monensine, swainsonine, cycloheximide, and tunicamycin. The possible involvement of protein phosphorylation in muscarinic receptor desensitization and down regulation also will be examined through the use of compounds such as phorbol esters, which directly activate protein kinase in the acinar cell. Effects of phosphorylating conditions on the electrophoretic mobility and isoelectric point of solubilized muscarinic receptors also will be examined, utilizing (3H) PrBCM as a tag for the receptor molecule. Additionally, the ability to manipulate the size of the acinar muscarinic receptor population in culture will allow the direct determination of the stoichiometric relationship between the numbers of functional receptors and secretory responsiveness. The proposed studies will address fundamental questions regarding the mechanisms by which exocrine gland secretory cells adjust to varying intensity and duration of neurohumoral stimulation.

这项拟议的研究将探索 胰腺和泪腺的M胆碱能受体 和腮腺，并定义了参与的机制 受体密度和激动剂亲和力的调节。这些 研究将扩大对细胞过程的理解 负责控制上皮细胞中的神经递质受体 一个特别重要的目标是这些功能障碍 机制与胰腺炎和胰腺炎的病理有关 囊性纤维化。调查…的结构特征 从这些器官中制备的粘液素受体、腺泡蛋白将 标记(~3H)丙基苄基胆碱芥末(PrBCM)，以及 不可逆转的胆碱能拮抗剂。贴标签后，血浆 膜将被制备并用神经氨酸酶处理， 内切糖苷酶F和蛋白酶，然后溶解和 电泳法。标记物的凝胶迁移率变化 这些酶诱导的受体蛋白将揭示 唾液酸和总糖基残基对蛋白质的贡献 M受体的表观分子大小以及 胞外和胞浆受体结构域的大小 膜双层的两侧。M受体的调节 对激动剂的种群规模和亲和力也将进行检查 养殖腺泡长期暴露效应的研究 到乙酰胆碱类似物。亚细胞通路和 参与受体生物合成和降解的细胞器将 使用可逆性毒扁豆碱拮抗剂，(~3H)-N- 甲基东莨菪碱和(~3H)-奎宁基苯甲酸酯及其专一性 甲胺、莫能辛、苦马豆素等抑制剂， 环己亚胺和衣霉素。可能参与其中的 M受体脱敏和蛋白磷酸化 下调监管也将通过使用 直接激活蛋白质的化合物，如佛波醇酯 腺泡细胞中的激酶。磷化条件对脱脂效果的影响 溶液的电泳率和等电点 还将利用(~3H)PrBCM检测毒鼠强受体 作为受体分子的标签。此外，能够 操纵腺泡M受体种群的大小 将允许直接确定文化中 官能团数目之间的化学计量关系 受体和分泌反应性。拟议的研究将 解决与以下机制有关的基本问题 外分泌腺分泌细胞调节不同的强度和 神经体液刺激持续时间。