REGULATION OF PANCREATIC MUSCARINIC RECEPTORS IN VITRO

拟议的研究将探测分子结构， 胰腺和泪腺中的毒蕈碱型乙酰胆碱受体 和腮腺，并确定参与的机制， 调节受体密度和对激动剂的亲和力。 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， 机制与胰腺炎的病理学有关， 囊性纤维化 为了研究 muscinic受体，从这些器官制备的腺泡将被 用（3 H）丙基苄胆碱芥子气（PrCl 3）标记， 不可逆胆碱能拮抗剂。 标记后，血浆 制备膜并用神经氨酸酶处理， 内切糖苷酶F和蛋白酶，然后溶解， 我很抱歉。 标记蛋白电泳迁移率的变化 这些酶诱导的受体蛋白将揭示 唾液酸和总糖基残基对 毒蕈碱受体的表观分子大小以及 细胞外和细胞质上受体结构域的大小 膜双层的两侧。 毒蕈碱受体的调节 群体大小和对激动剂的亲和力也将通过 培养腺泡慢性暴露效应的研究 乙酰胆碱类似物。 亚细胞途径和 参与受体生物合成和降解的细胞器将 使用可逆的毒蕈碱拮抗剂（3 H）-N- 甲基东莨菪碱和（3 H）-奎宁环二苯乙酸酯和特异性 抑制剂如甲胺，莫能辛，苦马豆素， 放线菌酮和衣霉素。 可能参与的 毒蕈碱受体脱敏中的蛋白磷酸化， 下调也将通过使用 直接激活蛋白质的化合物，如佛波醇酯， 腺泡细胞中的激酶。 磷酸化条件对 增溶的电泳迁移率和等电点 还将利用（3 H）Pre来检查毒蕈碱受体 作为受体分子的标签。 此外，能够 操纵腺泡毒蕈碱受体群体的大小 将允许直接确定 官能团数之间的化学计量关系 受体和分泌反应。 拟议的研究将 解决有关机制的基本问题， 外分泌腺分泌细胞调节不同的强度， 神经体液刺激的持续时间。

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.