A decrease in lacrimal gland secretion is a primary cause of the ocular surface problems that occur in diseases of tear film deficienCy such as keratoconjunctivitis sicca and Sjogren's syndrome; in aging; and in contact lens wear. The long-term objective of the present proposal is to determine specific and offer unique steps in the signal transduction pathways that neurotransmitters and peptides use to activate the lacrimal gland to secrete proteins, electrolytes, and water. Knowledge of the normal mechanism of lacrimal gland secretion has led to be the development of a possible topical treatment for diseases of tear deficiency. Identification of the specific components in each signal transduction pathway could allow development of improved treatments with increased efficacy and decreased systemic side effects. Cholinergic agonists, alpha1-adrenergic agonists, and VIP are the major stimuli of lacrimal gland secretion. The present proposal will focus on cholinergic and alpha1-adrenergic agonists. The proposed experiments will investigate the following steps in the signal transduction pathway activated by these agonists in the lacrimal gland. (1) determine which stimuli of secretion activate which specific guanine-nucleotide-binding proteins (G proteins); (2) determine which stimuli of secretion activate phospholipase D and A2 activity, characterize this stimulation, and determine which phospholipase C isozymes are activated by cholinergic agonists; (3) determine which stimuli of secretion activate which protein kinase C isozymes and determine the role each of these isozymes plays in stimulating secretion. Acini, which secrete proteins, electrolytes, and water, will be isolated from rat exorbital lacrimal glands. Specific G proteins activated by cholinergic, alpha1-adrenergic, and VIP receptors will be covalently attached to [alpha32P]GTP to allow identification. Function-blocking antibodies to G proteins will also be used. Which phospholipases D and A2 as well as the isozymes of PLC are activated by cholinergic and alpha1- adrenergic agonists will be determined by thin-layer chromatography and function-blocking antibodies. The specific isozymes of PKC used by cholinergic and alpha1-adrenergic agonists will be determined using antibodies, synthetic substrates, inhibitory pseudosubstrates, and permeable myristoylated pseudosubstrates. The interaction of cholinergic agonists and VIP causes synergistic secretion. To determine if activators of protein kinase C or if specific protein kinase C isozymes cause the synergism, antibodies and permeable myristoylated pseudosubstrates of protein kinase C will be used.
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