The lacrimal gland is a major source of tear proteins that are important for the health of the corneal surface. In dry-eye patients, tear protein deficiencies result in an inadequate physiologic response to ocular surface disease or trauma, leading to corneal dysfunction. Thus, as one approach to the development of treatment that may enhance secretion in a physiologically compromised state, the long-term objective of this work is to define the cellular mechanisms of control of regulated secretion of lacrimal tear proteins.
The specific aims of this proposal are designed to investigate the role of guanine-nucleotide binding proteins (G proteins) in stimulus-secretion coupling in the lacrimal gland. Within the models of signal transduction of the IP3-Ca2+ and cAMP pathways, coupling of specific seven transmembrane receptors to phospholipase C or adenylyl cyclase via GTPdependent mechanisms has been proposed. This work will identify and characterize the specific G proteins that link receptors to the generation of intracellular second messengers and regulation of the lacrimal secretory response. The identification of G proteins present in the gland will be accomplished by Western blotting of membrane preparations of acinar cells and detection with antisera directed against peptide sequences of the alpha subunits of both toxin- sensitive and toxin-insensitive G proteins. Introduction of anti-G antisera into permeabilized cells and the measurement of secretion in response to specific receptor activation will allow characterization of the physiologic significance of the G proteins present in the gland. Because G proteins also are involved in intracellular events associated with exocytosis that are distal to second messenger generation, secretion by cells that have been permeabilized and exposed to antisera will be measured in response to agents that by-pass G protein-linked effector activation of the lacrimal response. The coupling of receptor activation to the generation of the second messengers 1P3 and cAMP, important in the control of lacrimal secretion, by specific G proteins will also be assessed. Membrane preparations of acini will be used to measure phospholipase C activity in the presence of muscarinic agonists and antagonists and anti-Gsalpha antisera will be used to investigate coupling of receptor activation to phospholipase C activity by G proteins. The characterization of G protein coupling of receptor activation that results in stimulation (VIP) or inhibition (enkephalin) of lacrimal adenylyl cyclase also will be determined. This will be accomplished by exposure of lacrimal tissues to anti-Galpha antisera and the measurement of adenylyl cyclase in the presence of VIP or enkephalin analogs.
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