A decrease in lacrimal gland secretin is a primary cause of ocular surface problems occurring in keratoconjunctivitis sicca (KCS), aging, and contact lens wear. The long-term objective of this proposal is to investigate the cellular mechanisms normally used by lacrimal gland cells to secrete the proteins, electrolytes, and water that make up lacrimal gland fluid. The proposed experiments will address the following steps in the control of secretion: (1) determine whether guanine nucleotide- binding regulatory proteins (G proteins) couple the stimulus-induced activation of receptors to the production of second messenger; (2) assess the role of the second messengers inositol phosphates, Ca2+, and diacylglycerol (DAG) in stimulation of secretion; (3) define the role of specific protein kinases activated by second messengers in the control of secretion and the interaction of different cellular pathways used for secretion; (4) characterize the role that enzymes and transport proteins located on secretory granules play in the control of protein and electrolyte/water secretion. Acini, which secrete protein, electrolytes, and water, will be isolated from rat exorbital lacrimal glands. The role of G proteins will be determined by using nonhydrolyzable GTP analogs in permeabilized acini and specific toxins to G proteins in acini cultured overnight. The role of inositol phosphates will be measured by anion exchange chromatography and high-performance liquid chromatography. The role of Ca2+ will be investigated by using fluorescent calcium dyes and DAG by phosphorylation of exogenous substrates by the DAG-activated protein kinase. The role of protein kinases will be determined by measuring phosphorylation of endogenous substrates by specific protein kinases. Secretory granules will be isolated by density gradient centrifugation and characterized by use of specific toxins to G proteins, measurement of phosphorylation by endogenous kinases, and characterization of ion transport proteins in the granule membranes. Knowledge of the normal mechanism of lacrimal gland fluid secretion will provide a basis for determining the steps in the secretory process affected in KCS and a rationale for developing drugs to treat the deficiency of lacrimal secretion or to stimulate the remaining functional lacrimal gland tissue.
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