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

描述（由申请人提供）：唾液的正常分泌对口腔健康很重要。了解控制唾液腺液体和蛋白质分泌的具体机制是唾液腺功能障碍治疗策略发展的核心。唾液液体成分的产生主要受胞质钙（[Ca2+]i）通过激活空间分离的离子电导值而增加的控制。虽然蛋白质的胞吐是通过Ca2+和cAMP介导的效应物发生的，但要验证的中心假设是，液体和蛋白质分泌的动力学是由小鼠腮腺腺泡细胞中引发的[Ca2+]i信号的独特特征控制的。在Ca2+刺激胞吐的背景下，腮腺腺泡细胞中负责蛋白质分泌的机制将被研究，特异性目的旨在阐明负责启动、快速发展到全球Ca2+i信号以及随后有效终止信号的机制。利用指示染料结合闪光光解肌醇1,4,5-三磷酸（InsP3）和色氨酸受体（RyR）激动剂的数字成像，将确定InsP3受体和RyR对Ca2+信号起始的相对贡献。通过施加高细胞内Ca2+缓冲和笼内分子的局部光解作用，腮腺细胞中协调一致的全局Ca +信号的机制将被定义。不同钙负荷下Ca2+清除机制对Ca2+信号终止的相对贡献将通过选择性药理干预来确定。时间分辨膜电容测量和光学方法将被用于监测单个细胞的胞吐。使用这种技术的Ca2+敏感性的胞吐将被定义。此外，通过探索cAMP和Ca2+刺激胞吐之间协同作用的机制，将建立cAMP和Ca2+在颗粒募集和融合过程中的相对作用。预计腮腺分泌液体和蛋白质的具体机制的知识将在阐明唾液腺功能障碍的新疗法中发挥重要作用。

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.