Fundamental to developing more informed pharmacologic approaches to the promotion of healing of painful, often blinding injuries and diseases of the ocular surface is an understanding of mechanisms regulating replication, transcription, and translation during corneal epithelial growth, and of mechanisms by which drugs may alter rates of macromolecular synthesis during wound repair. Our findings to date in cultured corneal epithelial cells of the rabbit indicate (a) cGMP-mediated growth enhancement (increased nucleic acid and protein precursor incorporation) by binding of cholinergic agonists to muscarinic receptors; (b) cAMP-mediated Beta-adrenergic growth inhibition by catecholamines; and (c) potentiation of adrenergic effects by PGE1.
The specific aims of the proposed research are (1) to elucidate molecular mechanisms by which receptor-mediated alterations in cyclic nucleotide levels lead to altered replicative, transcriptional, or translational activity, and (2) to assess whether mechanisms of adrenergic and cholinergic growth regulation in cultured cells are operative during corneal wound repair in vivo. Investigative approaches include (1) subcellular localization of receptors and enzymes of cyclic nucleotide metabolism and function, and (2) evaluation of potential roles of drugs and cyclic nucleotide-dependent protein phosphorylation (a) in regulating DNA polymerase activities; (b) in transcriptional regulation of gene expression via phosphorylation of histones, nonhistone nuclear proteins, or RNA polymerases; and (c) in posttranscriptional regulation (e.g., via phosphorylation of ribosomal proteins). Influences of carbamylcholine (cholinergic agonist) alone or with propranolol (Beta-adrenergic antagonist) or indomethacin (PG synthesis inhibitor) on receptor number and ligand affinity and on cyclase, phosphodiesterase, and kinase activities will be assessed in subcellular fractions from drug-treated and control cells and from tissue of drug-treated and control eyes in various stages of defect resurfacing in vivo. Polymerase activities and nuclear and ribosomal protein phosphorylation will be assayed after appropriate purification. The proposed research represents the first in-depth effort to explore regulatory aspects of the molecular genetics of the corneal epithelium. As such, these evaluations should elucidate mechanisms by which a variety of drugs and endogenous mediators may influence healing via direct or in direct effects on components of the replicative, transcriptional, and translational machinery of corneal epithelial cells.
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