The mucus layer, secreted primarily by the conjunctival goblet cells, is a critical protective layer for the ocular surface, shielding it from pathogenic and environmental challenges. To defend the ocular surface, goblet cells must be able to respond to the external challenges. These external challenges activate sensory nerves in the cornea to stimulate parasympathetic and sympathetic nerves that surround the goblet cells. These nerves release neurotransmitters that interact with receptors in the goblet cell membranes and activate intracellular signaling pathways to induce goblet cell mucin secretion. The long term objective of this research is to identify, characterize the function of, and determine the activity of the specific cellular biochemical processes in the signaling pathways activated by parasympathetic and sympathetic neurotransmitters and to stimulate goblet cell mucin secretion and goblet cell proliferation. The focus of the present proposal will be cholinergic agonists (parasympathetic pathway), one of the major stimulatory pathways in the conjunctiva. It will be determined if cholinergic agonists: (1) activate a Ca2+/calmodulin-dependent pathway to stimulate goblet cell mucin secretion; (2) activate a PKC-dependent pathway to stimulate goblet cell mucin secretion; (3) activate the MAP kinase pathway and the stress responsive transcription factors NF-kappaB and AP-1 to induce goblet cell proliferation, and (4) mediate neurally-induced goblet cell proliferation. Rat conjunctiva will be incubated in vitro and an enzyme-linked lectin assay used to measure secretion of goblet cell mucin. This method will be combined with immunoprecipitation, Western blotting, confocal immunofluorescence microscopy, ELISA, and electrophoretic mobility shift assay to determine identify, location, and activity of the individual components of the signaling pathways and bromo-2 deoxyuridine labeling to measure cell proliferation. Mucin production by the goblet cells is tightly regulated as either an increase or decrease in mucin secretion is associated with ocular surface disease. Neural stimulation of goblet cell secretion and proliferation is especially important. In diseases of mucin overproduction, such as giant papillary conjunctivitis, a constant irritative stimulus would activate sensory nerves in the cornea to stimulate efferent nerves in the conjunctiva to increase goblet cell secretion and proliferation. In diseases of mucin deficiency, such as anesthetic cornea and herpetic keratitis, sensory nerves in the cornea are rendered dysfunctional preventing activation of the efferent pathway and blocking goblet cell secretion and proliferation. Study of the cellular components of the signaling pathways that stimulate goblet cell secretion and proliferation will lead to the design of goblet cell-specific therapies for diseases of both mucus overproduction and deficiency.
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