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.
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