Cyclic nucleotides in muscarinic regulation of DNA and RNA polymerase activity in cultured corneal epithelial cells of the rabbit

Abstract

DNA and RNA polymerase activities in the purified nuclear fraction from cultured rabbit corneal epithelial cells were assayed over a range of substrate (labeled dTTP or UTP) concentrations using calf thymus DNA as template. Effects of carbamylcholine on polymerase activities were evaluated over a range of drug concentrations including those saturating muscarinic receptors. Carbamylcholine significantly (p less than 0.001) enhanced activity of both polymerases, both in nuclei incubated with the drug during assay and in nuclei from carbamylcholine-treated cells. Drug effects were blocked by atropine. Regression analysis of Hill plots for variation of polymerase activity with carbamylcholine concentration indicated half-maximal activity of both polymerases at approximately 1 microM carbamylcholine. Mechanisms by which carbamylcholine may alter polymerase activities are discussed in relation to effects of the drug on nuclear enzymes of cyclic nucleotide metabolism and on cyclic nucleotide-dependent protein phosphorylation.

The molecular basis of neurotrophic keratitis

“…Udenfriend et al 1972) The purity of the nuclear fraction was monitored microscopically and by using as fraction markers nuclear DNA, mitochondria1 isocitric dehydrogenase, plasma membrane S'nucleotidase and soluble cytoplasmic aldolase. (Colley et al 1985) The purified nuclear pellet was washed 3X in TMS and re-suspended in TMS-containing 10% glycerol for storage (up to two weeks at -80°C). Total protein was determined by the method of Bradford (1976) using reagents from BioRad.…”

“…The contralateral eye (right eye in half the animals in each series, left eye in the other half) received 50 pl HBSS. Re-surfacing was monitored by fluorescein staining and serial photography (Colley et al 1985;.…”

“…The 5'nucleotidase reaction was terminated by adding 500 @ of a 25% slurry of AG 1 X2 Ci/mmol, New England Nuclear), with calf thymus DNA (100 pg/ml template. (Colley et al 1985; to distinguish RNA polymerases I, I1 and 111, activity was assayed in the absence (total nuclear activity) and presence of a-amanitin at 1 pg/ml (inhibits polymerase I1 but not I or 111) or 250 pg/ml (inhibits polymerases II and 111 but not I) (1-7 DNA polymerase b was estimated as activity in the presence of 10 m N-ethylmaleimide; total activity in the absence of the inhibitor minus activity in its present served as an estimate of DNA polymerase a activity. Relative proportions of total nuclear DNA polymerase activity assayed as polymerases a and b in the presene and absence of N-ethylmaleimide were very similar to relative activities assayed using the polymerase a inhibitor aphidicolin (15 pg/ml) and using the polymerase b inhibitor dideoxyadenosine triphosphaft (100 pi).…”

“…Vmax and K, , or K, , , were calculated by linear regression analysis of Lineweaver-Burk plots of data at varying UTP or dTTP concentrations in 10 min incubations. (Colley et al 1985;.…”