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

描述（由申请人提供）：唾液的正常产生对口腔健康很重要。了解唾液腺分泌液体和蛋白质的具体机制是制定治疗唾液腺功能障碍策略的关键。唾液的流体组分的产生主要由通过激活空间分离的离子电导值的胞质钙（[Ca 2 +]i）的增加来控制。虽然蛋白质的胞吐作用通过Ca 2+和cAMP介导的效应器发生，但待测试的中心假设是液体和蛋白质分泌的动力学受小鼠腮腺腺泡细胞中引发的[Ca 2 +]i信号的独特特征控制。将在Ca 2+刺激的胞吐作用的背景下研究腮腺腺泡细胞中负责蛋白分泌的机制，具体目的旨在阐明负责启动、快速进展为全局Ca 2 +i信号以及随后有效终止信号的机制。使用指示剂染料的数字成像结合肌醇1，4，5-三磷酸（InsP 3）和兰尼碱受体（RyR）激动剂的闪光光解，将确定InsP 3受体和RyR对Ca 2+信号起始的相对贡献。通过施加高细胞内Ca 2+缓冲和局部光解的笼状分子的协调，总是全球的钙信号在腮腺细胞的机制将被定义。将通过选择性药理学干预来确定不同Ca负荷下Ca 2+清除机制对Ca 2+信号终止的相对贡献。将利用时间分辨膜电容测量和光学方法来监测单细胞的胞吐作用。使用这种技术，胞吐的Ca 2+敏感性将被定义。此外，通过探讨cAMP和Ca ~（2+）刺激的胞吐之间的协同作用机制，将建立cAMP和Ca ~（2+）在颗粒募集和融合过程中的相对作用。据设想，腮腺的具体机制，负责液体和蛋白质分泌的知识将是重要的，在阐明新的治疗唾液腺功能障碍。

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.