A decrease in lacrimal gland secretion is a primary cause of the ocular surface problems that occur in tear-deficient dry eye including Sjogren's Syndrome, contact lens wear, and aging. The long term objective of the present proposal is to characterize the specific and different signaling proteins that are used by cholinergic and a1-adrenergic agonist to stimulate lacrimal gland protein, electrolyte, and water secretion. A second objective is to determine if these signal transduction pathways are altered in a mouse model of Sjogren's Syndrome. Once an alteration in these pathways is described, new treatment for tear-deficient dry eye could be developed, treatments that bypass the defect in secretion. To obtain these goals, the following Specific Aims a1-adrenergic agonist activation of lacrimal gland secretion; 2) correlate location of protein kinase C agonists; and 3) determine if cholinergic and a1-adrenergic agonist-induced increases in Ca2+ or activation of protein kinase C isozymes is altered in a mouse model of tear-deficient dry eye. Acini or pieces of normal rat, cholinergic and a1-adrenergic agonists on phospholipase and phosphoinositol 3-kinase activity will be measured by thin layer chromatography. The effect of cholinergic and a1-adrenergic agonists on protein kinase C isozyme location will be determined by conventional and confocal immunofluorescence microscopy. Presence of protein kinase C isozymes will be measured in purified subcellular fractions by Western blotting. The effect of cholinergic and a1-adrenergic agonists on [Ca2+] and protein kinase C activity in diseased and control mouse lacrimal glands will be determined using: 1) [Ca2+] and protein kinase C activity in diseased and control mouse lacrimal glands will be determined using: 1) Ca2+ sensitive fluorescent dyes with florescence spectrophometry and 2) Western blot and immunofluorescent microscopy for protein kinase C isozymes.
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