title: Subcellular localization of muscarinic effects on enzymes of cyclic nucleotide metabolism in cultured corneal epithelial cells of the rabbit.
abstract: Activities of adenylate and guanylate cyclases and cAMP and cGMP phosphodiesterases (cAPDE, cGPDE) were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine on enzyme activities in each fraction were evaluated. Activity of cyclases and phosphodiesterases was detectable in control incubations of homogenates, nuclei, the mitochondrial/lysosomal fraction, microsomes, and cytosol, although microsomal guanylate cyclase represented a very small proportion of the total cellular activity. In homogenates, carbamylcholine significantly elevated guanylate cyclase and cAPDE and reduced cGPDE activity. In mitochondria/lysosomes, guanylate cyclase was elevated and cGPDE reduced, but the drug did not alter cAPDE activity. In microsomes, carbamylcholine enhanced cAPDE but did not alter guanylate cyclase of cGPDE activity. In the soluble cytoplasmic fraction the drug reduced guanylate cyclase activity. The purified nuclear fraction exhibited substantial activity of cyclases and phosphodiesterases. Carbamylcholine significantly elevated activity of nuclear guanylate cyclase and cAPDE and significantly reduced nuclear cGPDE activity. The drug did not significantly alter adenylate cyclase in homogenates or in any cell fraction. The presence of activity of enzymes of cyclic nucleotide metabolism in the cell nucleus and the sensitivity of nuclear guanylate cyclase, cAPDE and cGPDE to carbamylcholine, which in the same concentration range enhances activity of DNA and RNA polymerases, suggested the hypothesis that effects on cyclic nucleotide-dependent phosphorylation of nuclear proteins might be among regulatory mechanisms by which the drug alters rates of replication and transcription in corneal epithelial cells.
authors.affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322.
authors.forename: A M
authors.initials: AM
authors.lastname: Colley
authors.forename: H D
authors.initials: HD
authors.lastname: Cavanagh
authors.forename: M L
authors.initials: ML
authors.lastname: Law
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis; D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects* / Q000201:enzymology; D004847:Epithelial Cells; D006162:Guanylate Cyclase / Q000032:analysis; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D009712:Nucleotides, Cyclic; D002217:Carbachol; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase