SALIVARY SECRETION-ROLE OF CALCIUM

拟议研究的总体目标是阐明 钙离子调节分泌过程的机制。 证据 这表明钙和环磷酸腺苷信号 转导途径以协调的方式起作用以调节分泌。 在小鼠腮腺毒蕈碱受体相互作用导致 磷酸肌醇（PI）周转，以及刺激和抑制 β-肾上腺素能诱导的cAMP蓄积。 待检验的假设 这个建议是毒蕈碱亚型与不同的 功能反应和这些调节的机制 功能反应是不同的。 具体目标是：（1）确定 并在分离的小鼠腮腺腺泡中鉴定毒蕈碱亚型， 放射性配体结合; 2）将毒蕈碱受体亚型与 通过比较选择性拮抗剂与受体的结合 拮抗剂对PI周转和cAMP代谢的影响，以及3） 以确定调节功能的潜在机制， a）检查毒蕈碱和β-肾上腺素能的相互作用 激动剂对受体结合和PI转换的作用;和通过B）检查 cAMP代谢的细胞内介质。 细胞循环水平 GMP、钙和蛋白激酶C（PKC）活性将与 腺苷酸环化酶和磷酸二酯酶活性的影响 细胞和/或无细胞制剂。 游离钙 将使用fura-2测量浓度（Cai）;将使用环核苷酸测量浓度（Cai）。 通过放射免疫测定法测定，并通过 酶测定 拟议的研究应提供一个重要的 了解参与相互作用的分子机制， 唾液腺调节中的两条信号转导通路 分泌，并提供了一个框架，检查细胞的生化 囊性纤维化等疾病的缺陷。

The overall objective of the proposed research is to elucidate the mechanisms by which calcium ions regulate the secretory process. Evidence has accumulated which suggests that the calcium and cyclic AMP signal transduction pathways act in a coordinated manner to regulate secretion. In mouse parotid gland muscarinic receptor interaction leads to phosphoinositide (PI) turnover, and to both stimulation and inhibition of beta-adrenergic-induced cAMP accumulation. The hypothesis to be tested in this proposal is that muscarinic subtypes couple to the different functional responses and the mechanisms underlying regulation of these functional responses are different. Specific aims are 1) to characterize and identify muscarinic subtypes in isolated mouse parotid acini by radioligand binding; 2) to correlate muscarinic receptor subtypes with functional responses by comparing selective antagonist binding to receptors with the effects of antagonists on PI turnover and cAMP metabolism and 3) to determine the mechanisms underlying the regulation of functional responses by a) examining the interaction of muscarinic and beta-adrenergic agonists on receptor binding and PI turnover; and by b) examining the role of intracellular mediators on cAMP metabolism. Cellular levels of Cyclic GMP, calcium and protein kinase C (PKC) activity will be correlated with effects on adenylate cyclase and phosphodiesterase activities in intact cells and/or in cell-free preparations. The free intracellular calcium concentration (Cai) will be measured using fura-2; cyclic nucleotides will be determined by radioimmunoassay and PKC activity will be measured by enzyme assay. The proposed studies should provide a significant understanding of the molecular mechanisms involved in the interaction of the two signal transduction pathways in the regulation of salivary gland secretion, and provide a framework for examining the cellular biochemical defects in diseases such as Cystic Fibrosis.