Effects of carbamylcholine on nuclear cyclic nucleotide-dependent protein kinase activity in cultured corneal epithelial cells of the rabbit.
Cyclic nucleotide-dependent protein kinase activity and cyclic nucleotide binding were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine (1 mM) on activity in each fraction were evaluated. In cell homogenates and in nuclei, carbamylcholine significantly elevated cGMP-dependent kinase activity and [3H]cGMP binding, and reduced cAMP-dependent kinase activity and [3H]cAMP binding. In the cytosol, the drug significantly reduced cAMP kinase and cAMP binding but did not alter cGMP binding or kinase activity. In both mitochondria/lysosomes and microsomes, cGMP binding was significantly enhanced and cAMP binding reduced, but differences in protein kinase activity were not significant. The drug did not alter cyclic nucleotide-independent protein kinase activity. All observed effects were blocked by atropine. Kinase activity in the purified nuclear fraction also was assayed over a range of carbamylcholine, substrate, and cyclic nucleotide concentrations. Vmax for cGMP kinase in control nuclei was 295 +/- 18 pmol/mg protein/min (KM = 367 +/- 70 micrograms/ml histone) vs. Vmax = 846 +/- 6 pmol/mg/min (KM = 131 +/- 13 mu/ml histone) in carbamylcholine-treated nuclei. Vmax for cAMP kinase in control nuclei was 282 +/- 12 pmol/mg/min (KM = 172 +/- 7 micrograms/ml histone); 1 mM carbamylcholine abolished nuclear cAMP-dependent kinase activity. Cyclic nucleotide concentrations at half-maximal binding were 5.4 +/- 0.9 nM cGMP and 9.3 +/- 0.4 nM cAMP, as compared to 15.4 +/- 5.0 nM cGMP and 36.2 +/- 6.2 nM cAMP for half-maximal protein kinase activity. Regression analysis of Hill plots for variation of nuclear cyclic nucleotide binding and kinase activity as a function of carbamylcholine concentration indicated half-maximal drug effects on activity of both enzymes at approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Colley
A M
AM
Department of Ophthalmology, Emory University School of Medicine, Emory Eye Center, Atlanta, GA 30322.
Law
M L
ML
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects* / Q000201:enzymology; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004847:Epithelial Cells; D007700:Kinetics; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions
D002217:Carbachol; D000242:Cyclic AMP; D011494:Protein Kinases; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D006152:Cyclic GMP