The molecular basis of neurotrophic keratitis.

Endogenous proliferation of corneal epithelial cells is regulated by a bidirectional control process characterized by an adrenergic, cAMP-dependent 'off', and a cholinergic, muscarinic cGMP-dependent 'on' response. The adrenergic receptor(s) are located in the plasma membrane (microsomal fraction), whereas the novel feature of the system is a cholinergic receptor specific for acetylcholine (ACH) located in the nuclear membrane. Exogenous substances which raise intracellular cAMP levels such as isoproterenol or PGE1, shut off epithelial mitosis: and, carbamylcholine or ACH raise intranuclear cGMP levels and increase mitosis by specific, regulatory stimulation of RNA-polymerase II activity. We believe that this regulatory system explains the transitory mitotic suppression induced by superficial corneal wounding (interruption of adrenergic fibres, chalone-effect); and the marked, permanent depression of epithelial mitosis associated with decreased intracellular ACH levels which are produced by total corneal denervation, and which results in neurotrophic keratitis.

Activity of DNA and RNA polymerases in resurfacing rabbit corneal epithelium.

Activity of RNA polymerases I, II and III (distinguished using alpha-amanitin) and activity of DNA polymerases alpha and beta (distinguished using N-ethylmaleimide) were assayed for varying intervals and at varying substrate (UTP or dTTP) concentrations in the purified nuclear fraction from corneal epithelium of carbamylcholine-treated and control eyes of rabbits with resurfacing acid burn defects. Incorporation was linear with time for all enzymes up to 30 min. In 10 min assays at varying substrate concentrations, all polymerases from carbamylcholine-treated eyes had significantly elevated Vmax compared to corresponding control enzymes. The drug also increased apparent affinity of RNA polymerase II for UTP and apparent affinity of DNA polymerases alpha and beta for dTTP. Results are discussed in relation to potential mechanisms by which effects of carbamylcholine on polymerase activity may be mediated.

Subcellular localization of muscarinic effects on enzymes of cyclic nucleotide metabolism in cultured corneal epithelial cells of the rabbit.

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.

Effects of carbamylcholine on cyclic nucleotide-dependent protein kinase activity in corneal epithelium during resurfacing.

Nuclear and total cellular cyclic nucleotide-dependent protein kinase activity was assayed in corneal epithelium from carbamylcholine-treated and control eyes of rabbits with and without resurfacing acid burn defects. In epithelium from resurfacing corneas carbamylcholine significantly elevated both nuclear and total cellular cGMP-dependent protein kinase activity and reduced activity of cAMP-dependent protein kinase. The drug also increased the proportion of total cellular cGMP kinase activity represented by nuclear activity, suggesting that the drug may enhance nuclear translocation of cGMP-dependent protein kinase.

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)

Effects of topical carbamylcholine on corneal epithelial resurfacing.

Effects of topical carbamylcholine on resurfacing of epithelial defects produced in both eyes of New Zealand albino rabbits with 18 N H2SO4 were evaluated. One eye of each animal received carbamylcholine (50 microliter of a 1.5% solution in HBSS) t.i.d. (10 AM, 1 PM, and 4 PM) for 7 days, with treatment beginning the morning after defect production. The contralateral eye received 50 microliters HBSS and served as control. Resurfacing was monitored by fluorescein staining and photography. Rates of resurfacing were calculated by linear regression analysis of plots of defect surface area versus time. Data represent treated and control eyes of 19 animals in two experimental series. In these experiments, carbamylcholine significantly enhanced epithelial resurfacing, although the effect was not dramatic. Mean resurfacing rates were 11.9 +/- 1.4 mm2/24 hr for carbamylcholine-treated eyes versus 8.5 +/- 1.3 mm2/24 hr for control eyes (p less than .001). On each day after initiation of treatment, the mean percentage of original defect area remaining was significantly lower in treated than in control eyes. At the end of the 7-day treatment regimen, none of the defects were completely resurfaced (mean for treated eyes = 79.9 +/- 2.2%, mean for control eyes = 56.8 +/- 4.3%).

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

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.

Prostaglandins in ocular pathophysiology with special reference to diabetic retinopathy and glaucoma.

This report reviews evidence for influences of prostaglandins (PGs) on intraocular pressure in glaucoma and on the progressive vascular and blood cell dysfunction in diabetes mellitus, of which retinopathy is a manifestation, and includes a brief outline of evidence for roles of PGs in ocular inflammation. Sequences in pathways of biosynthesis and conversion of PGs are summarized, and PG assay methods are evaluated briefly.

Binding of [3H]dihydroalprenolol and [3H]quinuclidinyl benzilate to intact cells of cultured corneal epithelium.

In intact cultured rabbit corneal epithelial cells we have identified [3H]dihydroalprenolol ( [3H]DHA) and [3H]quinuclidinyl benzilate ( [3H]QNB) binding activities which meet criteria for beta-adrenergic and muscarinic cholinergic receptors. For saturable, propranolol-sensitive [3H]DHA binding, Bmax = 0.374 +/- 0.063 fmol/microgram protein; KDHA = 12.5 +/- 2.4 nM from Scatchard analysis. For saturable, atropine-sensitive [3H]QNB binding, Bmax = 0.403 +/- 0.053 fmol/microgram protein; KQNB = 15.4 +/- 0.7 nM. The order of potency of unlabeled adrenergic agonists in competition for [3H]DHA sites was isoproterenol greater than epinephrine greater than norepinephrine. For unlabeled cholinergic agonists competing for [3H]QNB sites, the order was oxotremorine greater than acetylcholine greater than or equal to carbamylcholine. Acetylcholine did not inhibit [3H]DHA binding, nor did isoproterenol or choline inhibit [3H]QNB binding. Effectiveness of drugs in stimulating cAMP or cGMP accumulation closely paralleled efficacy in competition for [3H]DHA or [3H]QNB sites. Results confirm the presence in intact cultured corneal epithelial cells of beta-adrenergic receptors (demonstrated by others in corneal membrane suspensions), identify in intact cells muscarinic cholinergic receptors (not previously detected in broken cell preparations), and supply evidence for receptor-mediated regulation of cyclic nucleotide levels in these cells, further supporting our hypothesis of bidirectional influence by cAMP-mediated beta-adrenergic and cGMP-mediated cholinergic "first messengers" on proliferation during healing of corneal epithelial defects.

Cholinergic, adrenergic, and PGE1 effects on cyclic nucleotides and growth in cultured corneal epithelium.

Effects of adrenergic and cholinergic drugs and prostaglandin E1 on cyclic nucleotide accumulation and parameters of growth and basement membrane synthesis were examined in corneal epithelial cell cultures. 8-bromo-cGMP significantly (p less than 0.05) enhanced incorporation of labeled thymidine and leucine, as did acetylcholine and carbamylcholine, which elevated cGMP and decreased cAMP/cGMP ratio. Responses to acetylcholine were abolished by atropine and alpha-bungarotoxin. Precursor incorporation was inhibited by dibutyryl cAMP and adenosine 5'-monophosphate and by norepinephrine, epinephrine, prostaglandin E1, and theophylline, which significantly elevated cAMP levels and cAMP/cGMP ratio. Propranolol, but not phenoxybenzamine, blocked responses to effective concentrations of norepinephrine. Norepinephrine, PGE1, and dibutyryl cAMP also significantly elevated uptake of labeled glucosamine and incorporation of labeled proline into collagenase-sensitive protein or the hydroxyproline fraction of protein hydrolysates, while acetylcholine had no effect on parameters of basement membrane synthesis. Propranolol blocked responses to norepinephrine. Results were consistent with a cGMP-mediated stimulatory role of the cholinergic transmitter in corneal epithelial growth regulation, cAMP-mediated beta-adrenergic suppression of regrowth and increased basement membrane production after initial injury to the corneal epithelium, and potentiation of the adrenergic effect by prostaglandins.

Metabolic approaches to studies on diabetic microangiopathy.

The chief purposes of this report are (a) to focus attention on various metabolic and pathophysiologic parameters relating prostaglandins (PGs) and thromboxanes to the slow but inexorable progression of vascular and blood cell dysfunction in diabetes mellitus and (b) to suggest areas of investigation that may be of fundamental importance for expanded areas of diabetes research. The prime thrust of these investigations would be to correlate these metabolic and pathophysiologic parameters with the vasculopathy of diabetes mellitus.