[Ca2+]i and Secretory Dynamics in Parotid Acinar Cells

描述(申请人提供)：唾液的正常分泌对口腔健康很重要。了解控制唾液腺分泌液体和蛋白质的具体机制对于制定治疗唾液腺功能障碍的策略至关重要。唾液中液体成分的产生主要受细胞内钙([Ca~(2+)]i)的增加的控制，这种增加是通过激活空间分离的离子电导值来实现的。虽然蛋白质的胞吐是通过钙离子和cAMP介导的效应器发生的，但需要检验的中心假设是，液体和蛋白质的分泌动力学由小鼠腮腺腺泡细胞中诱发的[钙]i信号的独特特征控制。负责腮腺腺泡细胞蛋白质分泌的机制将在钙离子刺激的胞吐作用的背景下进行研究，其特定目的旨在阐明负责启动、快速发展为全球钙离子i信号以及随后有效终止该信号的机制。利用指示染料的数字成像结合1，4，5-三磷酸肌醇(InsP3)和兰尼定受体(RyR)激动剂的闪光光解，可以确定Insp3受体和RyR对启动钙信号的相对贡献。通过施加高的细胞内钙缓冲和笼子分子的局部光解，腮腺细胞中协调的、始终如一的全局钙信号的机制将被确定。不同钙负荷下的钙清除机制对钙信号终止的相对贡献将通过选择性药物干预来确定。时间分辨膜电容测量和光学方法将被用来监测单个细胞的胞吐作用。利用这项技术，将定义胞吐作用的钙敏感性。此外，通过探讨cAMP和Ca~(2+)刺激胞吐之间协同作用的机制，将建立cAMP和Ca~(2+)在颗粒募集和融合过程中的相对作用。可以预见，对腮腺负责液体和蛋白质分泌的特定机制的了解将对阐明唾液腺功能障碍的新疗法非常重要。

DESCRIPTION (provided by applicant): The normal production of saliva is important for oral health. Understanding the specific mechanisms which govern fluid and protein secretion from salivary glands is central to the development of strategies for the treatment of salivary gland dysfunction. The production of the fluid component of saliva is primarily controlled by an increase in cytosolic calcium ([Ca2+]i) through activation of spatially separated ionic conductance values. While the exocytosis of proteins occurs through both Ca2+ and cAMP mediated effectors, the central hypothesis to be tested is that the dynamics of both fluid and protein secretion are controlled by the unique characteristics of the [Ca2+]i signals elicited in mouse parotid acinar cells. The mechanisms responsible for protein secretion in parotid acinar cells will be studied in the context of Ca2+ stimulated exocytosis, and the Specific Aims are designed to elucidate the mechanisms responsible for the initiation, rapid progression to a global Ca2+i signal, and subsequent efficient termination of the signal. Using digital imaging of indicator dyes combined with flash photolysis of inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptor (RyR) agonists the relative contribution of InsP 3 receptors and RyR to the initiation of Ca2+ signals will be determined. By imposing high intracellular Ca2+ buffering and by local photolysis of caged molecules the mechanism underlying the coordinated, invariably global Ca + signal in parotid cells will be defined. The relative contribution of Ca2+ clearance mechanisms at different Ca loads to the termination of Ca2+ signals will be determined by selective pharmacological intervention. Time-resolved membrane capacitance measurements and optical methods will be utilized to monitor exocytosis from single cells. Using this technique the Ca2+ sensitivity of exocytosis will be defined. In addition, by probing the mechanism which is responsible for the synergism exhibited between cAMP and Ca2+ stimulated exocytosis the relative roles of cAMP and Ca2+ in the processes of granule recruitment and fusion will be established. It is envisioned that the knowledge gained into the parotid specific mechanisms responsible for fluid and protein secretion will be important in elucidating new therapy for salivary gland dysfunction.