The importance of tear proteins for maintenance of the integrity of the corneal surface as well as responses to ocular surface disease or trauma is evident in dry-eye patients in whom tear protein deficiencies result in corneal dysfunction. As one approach towards the development of therapeutic treatment of pathologies resulting from aqueous tear film deficiencies in dry-eye states, the long-term objective of this work is to define the cellular mechanisms of control of regulated secretion of tear proteins. Because the lacrimal gland is the major source of the protein components of the aqueous tear film, identification and characterization of a multiple system of modulators of lacrimal secretion is an important step in this process.
The specific aims of this proposal are designed to investigate the modulation of intracellular pathways by endogenous neurotransmitters and neuropeptides known to be negative regulators in stimulus-secretion coupling associated with exocytosis of lacrimal proteins. Within the model of transmembrane signalling, it is proposed that activation of receptors that are negatively coupled through Gi to adenylyl cyclase results in inhibition of lacrimal secretion. the physiologic importance of opioid, muscarinic cholinergic and alpha2- adrenergic receptor activation to lacrimal protein secretion will be assessed by in vitro measures of lacrimal function. The characterization of opioid inhibition of lacrimal secretion will be continued by investigation of the control of lacrimal acinar protein secretion by beta- endorphin. In addition, receptor subtypes associated with opioid inhibition will be identified by the measurement of secreted protein in the presence of specific opiate receptor ligands. The assay of secreted proteins will also be used to determine if alpha2-adrenergic agonists inhibit lacrimal acinar secretion, alone, or in combination with opioids resulting in a synergistic inhibitory effect. In order to assess the intracellular mechanism of negative control, adenylyl cyclase activity will be measured by a protein kinase binding assay in the presence of ligands that result in inhibition of secretion. Included in these studies will be the investigation of the effect of muscarinic cholinergic agonists, known to enhance secretion, but also reported to decrease intracellular cAMP accumulation in lacrimal acini. Because significant interactions occur between the Ca2+-IP3/DAG pathway and the cAMP pathway resulting in either synergistic stimulation or inhibition of secretion, cross-over effects between the two signal transduction pathways will be defined. This will be accomplished by the protein binding assay of 1,4,5-inositol trisphosphate (IP3) production in isolated acini in the presence of inhibitory agonists. The intracellular steps at which the pathway interact will also be determined. Because calcium ionophores and phorbol esters by-pass receptor activation and second messenger production, these agents will be used to determine if the cAMP-mediated inhibition of cholinergic stimulation of secretion can be overcome by increasing intracellular Ca2+ or by activation of protein kinase C.
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