Understanding molecular mechanisms regulating RNA synthesis during growth and differentiation of corneal epithelial cells is important in developing more effective treatment of defects arising from corneal injury or disease. The research in progress represents the first in-depth effort to characterize nuclear enzymes of transcription in mammalian corneal epithelium and to determine mechanisms by which their activity is regulated. Our previous studies have provided evidence for cyclic nucleotide-mediated, muscarinic cholinergic regulation of RNA polymerase activity in rabbit corneal epithelial cells.
The specific aims of the proposed research are as follows: (1) To characterize RNA polymerase I, II and III of rabbit corneal epithelium (including electrophoretic and further biochemical/kinetic characterization of chromatographically purified enzymes). (2) To determine regulatory mechanisms by which the cholinergic agonist carbamylcholine enhances activity of RNA polymerase in rabbit corneal epithelium. Hypotheses to be tested concerning mechanisms mediating observed enhancement of RNA polymerase activity by carbamylcholine include the following potential effects of the drug, each of which is tentatively consistent with our kinetic data for one or more RNA polymerase studies of crude enzymes from isolated, purified nuclei: (a) Effects on posttranslational phosphorylation of RNA polymerase by cyclic nucleotide-dependent (cGMP-dependent) protein kinases (""""""""targeted hypothesis""""""""); (b) Effects on phosphorylation of other chromatin associated proteins (histones, HMG nonhistone proteins) influencing accessibility of DNA template to RNA polymerase; (c) Effects on RNA polymerase synthesis; (d) Effects on relative proportions of kinetically and electrophoretically distinct RNA polymerase isozymes with different substrate affinities. Experimental approaches include electrophoretic analysis of phosphorylation and synthesis of nuclear proteins in drug-treated and control cells, in conjunction with chromatographic purification and electrophoretic characterization of RNA polymerase I, II and III, histones and HMG proteins. Long-range objectives of this research include (1) elucidating molecular regulatory mechanisms by which cholinergic agents may influence gene expression at the transcriptional level during corneal epithelial cell growth and differentiation and (2) determining the suitability of cholinergic agents, alone or in combination with other drugs, for management of various types of injury and disease of the ocular surface.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY004604-04A4
Application #
3259056
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1989-07-01
Project End
1992-09-29
Budget Start
1989-09-30
Budget End
1990-09-29
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Georgetown University
Department
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Lind, G J; Cavanagh, H D (1995) Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells. Invest Ophthalmol Vis Sci 36:1492-507
Lind, G J; Cavanagh, H D (1993) Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit. Invest Ophthalmol Vis Sci 34:2943-52
Cavanagh, H D; Colley, A M (1989) The molecular basis of neurotrophic keratitis. Acta Ophthalmol Suppl 192:115-34
Colley, A M; Law, M L; Drake, L A et al. (1987) Activity of DNA and RNA polymerases in resurfacing rabbit corneal epithelium. Curr Eye Res 6:477-87
Colley, A M; Cavanagh, H D; Drake, L A et al. (1985) Cyclic nucleotides in muscarinic regulation of DNA and RNA polymerase activity in cultured corneal epithelial cells of the rabbit. Curr Eye Res 4:941-50