SALIVARY SECRETION--ROLE OF CALCIUM

拟议研究的长期目标是阐明 钙离子调节分泌过程的机制。 是 越来越清楚的是，测量电离或自由 胞浆钙（Ca-1）是获得确定性证据的必要条件， 细胞内钙在分泌中的调节作用。 也是 很明显，钙离子和其他离子之间存在强烈的相互关系， 定义了细胞内信使。 对这项提议特别感兴趣的是 研究Ca ~（2+）对cAMP代谢的调节作用， 钙，环GMP和钠离子在这种调节中的相互关系。 小鼠腮腺腺泡将通过涉及酶促的程序分离， 消化、二价阳离子耗尽和机械剪切。 免费 完整细胞中的胞质钙水平将通过使用高浓度的 钙荧光指示剂Quin-2及促分泌素的作用 测定Ca-1和淀粉酶释放。 通过高电压“透化”的细胞 也将用于评估各种Ca-1对淀粉酶的影响 释放;（Ca-1）浓度将通过使用Ca-EGTA测定 缓冲器。 Ca ~（2+）对环核苷酸（cAMP）代谢的调节将是一个新的研究方向。 通过检查腺苷酸和磷酸二酯酶活性测定。 为 涉及腺苷酸环化酶分离腮腺膜的研究将 制备和钙调节的腺苷酸环化酶活性测定， 钠离子的存在和不存在。 相互作用的部位 将检查钠和钙离子。 抑制剂的存在 鸟嘌呤核苷酸结合位点和其通过钠的调节也将被 测定 对于涉及磷酸二酯酶的研究， 腮腺组织中存在的磷酸二酯酶将通过 DEAE柱层析和抗体研究。 酶的调节 将确定胆碱能激动剂的活性， cGMP评估。 与细胞内生化事件相关的知识， 分泌过程可以提供有价值的洞察疾病状态， 存在这种细胞内事件的缺陷。

The long range goal of the proposed research is to elucidate the mechanism(s) by which calcium ions regulate the secretory process. It is becoming increasingly clear that the measurement of ionized or free cytosolic calcium (Ca-i) is essential for obtaining definitive evidence for the regulatory role of intracellular calcium in secretion. It is also clear that strong interrelationships exist between calcium ions and other defined intracellular messengers. Of particular interest in this proposal is to examine the regulation of cyclic AMP metabolism by Ca-i and the interrelationship of calcium, cyclic GMP and sodium ions in such regulation. Mouse parotid acini will be isolated by procedures involving enzymatic digestion, divalent cation depletion and mechanical shearing. Free cytosolic calcium levels in intact cells will be measured by using a highly fluorescent calcium indicator (Quin-2) and the effects of secretogogues on Ca-i and amylase release determined. Cells "permeabilized" by high voltage will also be used to assess the effects of a wide range of Ca-i on amylase release; (Ca-i) concentrations will be determined by use of Ca-EGTA buffers. Regulation of cyclic nucleotide (cAMP) metabolism by Ca-i will be determined by examining adenylate and phosphodiesterase activities. For studies involving adenylate cyclase isolated parotid membranes will be prepared and calcium-regulated adenylate cyclase activity measured in the absence and presence of sodium ions. The site of interaction between sodium and calcium ions will be examined. The presence of an inhibitory guanine nucleotide binding site and its modulation by sodium will also be determined. For studies involving phosphodiesterases, the forms of phosphodiesterase present in parotid tissue will be identified by DEAE-column chromatography and antibody studies. Regulation of enzyme activity by cholinergic agonists will be determined and the role of calcium and cGMP assessed. Knowledge of the intracellular biochemical events associated with the secretory process may provide valuable insight into disease states where defects in such intracellular events exist.