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.     

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.     

Specific receptor sites for PAF in iris and ciliary body of the rabbit eye

The existence of specific binding sites by using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF) was investigated on iris and ciliary body tissue of pigmented rabbit eyes. The binding was saturable, specific, time-dependent and reversible. Scatchard's analysis indicated the presence of two types of binding sites with a Kd1 4.90 +/- 0.47 nM, a Kd2 11.60 +/- 0.33 nM, a Bmax1 3.17 +/- 0.50 pmol/mg protein and a Bmax2 12.45 +/- 2.30 pmol/mg protein for iris tissue, and a Kd1 5.71 +/- 0.09 nM, a K2d 24.40 +/- 0.91 nM, a Bmax1 3.41 +/- 1.00 pmol/mg protein and a Bmax2 16.60 +/- 0.51 pmol/mg protein for ciliary body. The binding was fully displaced by unlabelled PAF in both iris and ciliary body preparations, and partially inhibited by the PAF antagonist BN 52021 in iris tissue.     

Intraocular extracellular cyclic nucleotide concentrations: the influence of vitreous surgery

Levels of cAMP (23.1 pmol/ml) and cGMP (22.6 pmol/ml) are nearly equal in normal rabbit vitreous, although aqueous CAMP levels (29.9 pmol/ml) are 10 times higher than aqueous cGMP levels (2.6 pmol/ml) (P<0.01). Intravitreal cAMP values decrease slightly 1 week after vitrectomy and lensectomy and return to normal whether the retina is attached, detached, or surgically removed. In contrast, intravitreal cGMP levels are profoundly depressed at 7 days (approximating normal aqueous levels) and are more than 50% lower than normal 30–42 days after surgery. The presence of these cyclic nucleotides in intraocular fluid and their responsiveness after surgery may signify a potential regulatory role in the physiologic responses of the eye to retinal detachment and its repair.This study was funded in part by NIH grants EY-02934, EY-05230, and EY-06520 and a grant from the William Beaumont Hospital Research Institute, Royal Oak, Michigan     

Cyclic nucleotide-dependent phosphorylation of proteins in rabbit ciliary processes

Cyclic nucleotide-dependent protein phosphorylation in albino rabbit ciliary processes was studied in particulate and soluble fractions of the tissue by the technique of SDS-polyacrylamide gel electrophoresis and autoradiography. In the presence of gamma-32P-ATP, the soluble fraction showed increased phosphorylation of proteins of 200, 32 and 16 kDa molecular weight when 10 microM cAMP was added. Protein phosphorylation increased with time up to 5 min. No significant augmentation of phosphorylation was observed in the presence of 10 microM cGMP compared to control. In the particulate fraction, proteins with molecular weights of 200, 160, 105, 72, 58, 32 and 16 kDa showed increased phosphorylation in the presence of 10 microM cAMP. Phosphorylation caused by the addition of cAMP was maximal between 30 sec and 1 min for the particulate membrane fraction, but with longer incubation times the incorporation of phosphate residues decreased. The same molecular weight proteins of the membrane fraction that were phosphorylated in a cAMP-dependent manner were phosphorylated in the absence of exogenous cAMP by addition of either the catalytic subunit of cAMP-dependent protein kinase or activators of membrane-bound adenylate cyclase such as l-isoproterenol, vasoactive intestinal peptide, aluminum fluoride or forskolin. A cAMP-dependent dephosphorylation of a 56 kDa protein was observed in the membrane fraction. Cyclic GMP did not cause observable changes in the pattern of protein phosphorylation in the particulate fraction of rabbit ciliary processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide content and phosphodiesterase activity in the rds mouse (020/A) retina

Cyclic nucleotide metabolism was examined in the retina and in the retinal pigment epithelium (RPE)-choroid complex of the rds mouse (020/A), a mutant in which discrete photoreceptor outer segment disc structures fail to develop. In retinas of both rds and control (Balb/c) mice, cyclic AMP levels peak at 10-15 days (20-25 pmol mg-1 protein). The level drops to about 10 pmol mg-1 at about one month in normal retinas but remains high in affected retinas. Cyclic GMP levels increase five-fold in Balb/c retinas as ROS develop whereas, in affected retinas, the levels remain constant and low (about 5 pmol mg-1). In RPE-choroid, cyclic nucleotide levels are similar in control and affected mice. Cyclic AMP phosphodiesterase (PDE) activity is somewhat higher in affected than in control retinas; conversely, cyclic GMP-PDE is lower. Both cyclic AMP-PDE and cyclic GMP-PDE activities are different in normal and affected RPE-choroid. Thus, although the rds (020/A) mouse belongs to the early-onset photoreceptor dysplasia group of hereditary retinal degenerations on a morphological basis, it does not exhibit high retinal cyclic GMP levels and thus appears to be distinct from other animals exhibiting early postnatal photoreceptor dysfunction.     

Localization of an endogenous substrate for cyclic AMP-stimulated protein phosphorylation in retina

Incubation of mouse or rabbit whole retina homogenates in the presence of [gamma 32P]-ATP and Mg2+ leads to the phosphorylation of various proteins, as demonstrated using SDS-polyacrylamide gel electrophoresis and autoradiography. The phosphorylation of one protein (ca. approximately equal to 31000 mol. wt) was increased by cyclic AMP in both species, with half-maximal stimulation at 5 x 10(-7)M. Cyclic GMP was also active, but much less potent. Protein phosphorylation patterns were compared in retina homogenates from normal mice (C57BL/6J), from adult C57BL/6J mice homozygous for the retinal degeneration gene (rd/rd) in which rod photoreceptor cells are absent, and from 21-day-old 020/Cpb mice homozygous for the retinal degeneration slow gene (rds/rds) in which only the outer segments of the rod photoreceptors are missing. The 31 K protein was only present in normal and in 21-day-old rds/rds mice. When rabbit retina was microdissected into outer segment, inner segment plus outer nuclear, and inner retina layers, cyclic AMP-stimulated phosphorylation of the 31 K protein was evident only in the inner segment plus outer nuclear layer. These data indicated the presence of a specific, endogenous substrate for a cAMP-dependent protein kinase which is found in the inner portions of rod photoreceptor cells.     

Potentiation of ATP-induced Ca2+ mobilisation in human retinal pigment epithelial cells

Interaction of signalling pathways directs the functional output of many cells. This study investigated the consequences of activating adenosine and adrenergic receptors on ATP-induced Ca2+ responses in human retinal pigment epithelial (RPE) cells. Intracellular Ca2+ concentration ([Ca2+]i) of human RPE cells in primary culture was monitored using Fura-2. Cyclic adenosine monophosphate (cAMP) concentration was measured using an enzyme-linked immunosorbent assay. Both ATP and UTP (10 microM) increased [Ca2+]i in human RPE cells. Adenosine (10 nM-10 microM) had no effect on resting [Ca2+]i, but potentiated a sub-threshold response to ATP (100 nM) when ATP was added in the presence of adenosine. The potentiation occurred with other G-protein receptor agonists such as acetylcholine. Potentiation persisted in Ca-free medium, but was blocked by prior application of thapsigargin. The A1 and A2 adenosine receptor antagonists, DPCPX and MRS1706 (100 nM) respectively, inhibited potentiation in 76+/-7 and 23+/-12% of cells, respectively, but the A3 antagonist MRS1191 had no effect. Conversely, agents that activate the cAMP pathway, including isoproterenol (10 microM), forskolin (10 microM), and the protein kinase A (PKA) activator Sp-cBIMPS (1 microM), potentiated the ATP-induced response in the RPE cells. Agents that are known to inhibit the production of cAMP in other systems also caused potentiation, including clonidine (10 microM) and the Gi-activator mastoparan (10 microM). Under resting conditions, cAMP concentration in RPE cells was 7.1+/-0.5 pmol mg(-1) protein. Isoproterenol (10 microM) and forskolin (10 microM) increased levels to 104.6+/-5.2 and 113.7+/-4.2 pmol mg(-1) protein, respectively, while adenosine, clonidine, and mastoparan (all 10 microM) had no significant effect on cAMP levels. These data indicate that whilst activation of A1 and A2 adenosine receptors and alpha2 and beta adrenergic receptors does not influence basal Ca2+ levels, stimulation of these receptors can potentiate Ca2+ signalling by cAMP dependent and independent mechanisms in human RPE cells.     

人工高眼压家兔血液,虹膜及房水cAMP,cGMP含量的关系

目的研究家兔血液、虹膜及房水中环腺苷酸（ｃｙｃｌｉｃａｄｅｎｏｓｉｎｅｍｏｎｏｐｈｏｓｐｈａｔｅ，ｃＡＭＰ）和环鸟苷酸（ｃｙｃｌｉｃｇｕａｎｏｓｉｎｅｍｏｎｏｐｈｏｓｐｈａｔｅ，ｃＧＭＰ）含量与高眼压之间的关系。方法用放射免疫方法测定家兔血浆、虹膜和房水中ｃＡＭＰ、ｃＧＭＰ含量。结果ｃＡＭＰ含量（ｎｍｏｌ／Ｌ）：血浆为７２．８１±７．６７，虹膜为１４１．０８±１６．７０，房水中为２８．４０±０．４９；ｃＧＭＰ含量（ｎｍｏｌ／Ｌ）：血浆为１０．３７±６．１５，虹膜为３５．６４±７．０２，房水中为１２．９０±０．６１。ｃＡＭＰ含量高于对照组（Ｐ＜０．０１），ｃＧＭＰ低于对照组（Ｐ＜０．０１）。结论高眼压与ｃＡＭＰ、ｃＧＭＰ含量关系显著。     

Analysis of protein kinase activities in rabbit ciliary processes: identification and characterization using exogenous substrates

Protein-kinase activities in rabbit ciliary process tissue were characterized and quantitated using histone, casein, and myosin light chain as substrates. At least four different protein-kinase activities were separated and identified in the supernatant (soluble) and in the particulate fraction using DEAE-cellulose ion-exchange chromatography. Typical activities of the protein kinases in ciliary processes dissected from one eye were as follows: in the supernatant fraction; protein kinase C, 185.0 pmol min-1; cyclic AMP-dependent protein kinase type II, 34.0 pmol min-1; casein kinase type II, 85.1 pmol min-1; protein kinase M, 9.8 pmol min-1: in the particulate fraction; protein kinase C, 55.1 pmol min-1; cyclic AMP-dependent protein kinase type II, 12.5 pmol min-1; casein kinase type II, 13.4 pmol min-1, and protein kinase M, 5.5 pmol min-1. No cyclic GMP-dependent and no calmodulin-dependent protein-kinase activities were detectable using histone, casein or myosin light chain as substrates. The apparent molecular weight of protein kinase C as estimated by exclusion chromatography on a column of Sephadex G-200 was about 90,000. Inhibitory and stimulatory effects of recently synthesized isoquinolinesulfonamide derivatives (H-7 and H-8), heparin, and polylysine were studied in ciliary process protein kinases. H-7 and H-8 were potent inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and protein kinase M, (IC50 less than 10 microM) but had no inhibitory effects on casein kinase. Heparin at 4 micrograms ml-1 inhibited casein kinase activity almost completely without affecting cyclic AMP-dependent or protein kinase C activities. Poly D- or L-lysine were both found to activate (approximately double) casein kinase activity at 40 micrograms ml-1, but did not significantly activate cyclic AMP-dependent protein kinase or protein kinase C. These results provide basic information on the protein kinase enzymes in the ciliary process and show that protein kinase C is the major kinase in this tissue. This suggests a possible role of the Ca2+ and protein kinase C system in transport functions of ciliary processes and in the regulatory mechanism of aqueous-humor formation additional to the already established importance of the cyclic AMP-dependent protein-kinase enzyme.     

Cyclic AMP has no effect on the generation, recovery, or background adaptation of light responses in functionally intact rod outer segments: with implications about the function of phosducin

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free beta gamma dimer of transducin (Tbeta gamma) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the beta gamma and alpha subunits. Phosducin's interaction with Tbeta gamma is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is approximately 5 microM, internal dialysis with solution containing a much higher concentration (100 microM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.     

Some properties of the cyclic AMP dependent protein kinase of epithelial cells and cortical fibers of bovine eye lens.

Cyclic AMP dependent protein kinase activity was demonstrated in extracts of epithelial cells and cortical fibers of bovine eye lens. The histone fractions can be phosphorylated by these enzymes. Highest phosphorylation was obtained with f2b and f1 histone fractions. The K_m values of the epithelial and cortical enzymes were very similar with all histone fractions. Protein kinase in the two extracts had a similar affinity to ATP and showed a pH optimum at 6·0–6·2. The enzymes were activated by cyclic AMP and the concentration of cyclic AMP necessary to half maximal activation was similar for both enzymes: 5×10^?8m for the epithelial enzyme and 9×10^?8m for the cortical enzyme. The magnitude of activation, however, was very different. The activity of the epithelial enzyme was increased 5–8-fold, while that of the cortical enzyme up to 25-fold. The high cyclic AMP sensitivity of the cortical enzyme was accompanied by a relatively high cyclic AMP binding capacity.     

Characterization of unique ADTN-catecholamine binding sites in the iris root-ciliary body of rabbits

A binding site for tritiated 2-amino-6, 7-dihydroxy-1, 2,3,4-tetrahydronaphthalene (ADTN) has been partially characterized in the rabbit iris root-ciliary body. Binding of ADTN is proportional to protein content and requires at least 60 minutes to reach equilibrium. Binding is saturable, with a Kd of 27 +/- 1 nM and a Bmax of 2.1 +/- .3 pmol/mg protein (mean +/- SEM). Dopamine competes for this site with a Ki of 100 nM and apomorphine with a Ki of 180 nM. This site is not blocked by L-timolol, phenoxybenzamine, or by several DA1 and DA2 antagonists. It appears to be a new type of catecholamine binding site, of a type not observed outside the anterior eye. It is possible that some of the effects of dopamine on intraocular pressure are mediated through this binding site.     

Stimulation of tear secretion by topical agents that increase cyclic nucleotide levels

The authors examined the effect of topical application of agents known to increase cyclic nucleotide levels on tear secretion by accessory lacrimal gland tissue in their rabbit model for keratoconjunctivitis sicca (KCS). Tear secretion was studied by changes in tear film osmolarity and tear volume caused by application of the agents relative to application of isotonic buffer solution alone. A decrease in tear film osmolarity or increase in tear volume was interpreted as an increase in tear secretion. Irritative stimulation was distinguished from pharmacologic stimulation by the prior use of topical proparacaine. The following agents significantly decreased tear film osmolarity and increased tear volume: vasoactive intestinal peptide (2 X 10(-8) to 2 X 10(-6) M); three pro-opiomelanocortin fragments alpha-, beta-, and gamma-melanocyte stimulating hormone at 10(-4), 10(-3), and 10(-3) M, respectively; the permeable cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) analogs 8-Br cAMP (0.3-3.0 X 10(-3) M) and 8-Br cGMP (1.0-10.0 X 10(-3) M); and the cyclic nucleotide phosphodiesterase inhibitor 1-isobutyl-3-methyl xanthine (0.3-3.0 X 10(-3) M). Forskolin (2 X 10(-4) M), which activates the catalytic subunits of adenyl cyclase, increased tear volume significantly. Secretin, adrenocorticotropic hormone, and pilocarpine were ineffective. The authors conclude that agents that increase either cAMP or cGMP levels pharmacologically stimulated tear secretion when applied topically to rabbit eyes with surgically induced KCS.     

Rod outer segment phosphodiesterase: Factors affecting the hydrolysis of cyclic-AMP and cyclic-GMP

Mg²⁺ and Mn²⁺ stimulate phosphodiesterase activity in isolated bovine rod outer segments. Ca²⁺ cannot substitute for these ions, rather, it inhibits hydrolysis of both cyclic-AMP (cAMP) and cyclic-GMP (cGMP). Triton X-100 at 0·01% increases hydrolysis of both cGMP and cAMP by about two-fold although increased detergent concentrations decrease activity. Sonification or freezing and thawing tends to decrease activity. Methylxanthines inhibit hydrolytic activity at millimolar concentrations; papaverine is effective (> 50% inhibition) at 10^?5m. Other inhibitors include dibutyryl cAMP and triiodothyronine. In general, hydrolysis of both cGMP and cAMP is similarly affected by the stimulators and inhibitors tested. Hydrolysis of the cyclic nucleotides is inhibited by ATP above 0·5 mm. Inhibition is greater in dark-adapted than in light-bleached preparations. With 0·1 mm-ATP, little effect is observed in dark-adapted rods but an apparent stimulatory effect is observed in light-bleached rods. ATP may thus act in this manner to control cyclic nucleotide levels in outer segments in vivo.     

Aqueous humor messengers in the transient decrease of intraocular pressure after ganglionectomy.

Intraocular pressure (IOP) decreases in rabbits 1 day after superior cervical ganglionectomy. It was hypothesized that this IOP decrease was caused by an accumulation of norepinephrine (NE) released from the iris-ciliary body into the aqueous humor during nerve degeneration. Direct measurement of aqueous humor NE concentration, however, was not successful because of the technical difficulty. In the current study, aqueous humor NE after superior cervical ganglionectomy was extracted and quantified using high-performance liquid chromatography-electrochemical detection. Twelve New Zealand albino rabbits were maintained in a daily 12-hr light-12-hr dark environment. Unilateral ganglionectomy was done on these rabbits during the light phase under halothane anesthesia. Twenty-two hours after the procedure, a significant IOP decrease occurred. The IOP was 16.1 +/- 0.6 mmHg (mean +/- the standard error of the mean) in the operated eye and 20.9 +/- 0.6 mmHg in the contralateral eye (P < 0.01). Aqueous humor NE concentration in the operated eye (475 +/- 81 pg/ml) was not different from that in the contralateral eye (469 +/- 58 pg/ml). However, the concentration of aqueous humor cyclic adenosine monophosphate (cAMP) in the operated eye (29.8 +/- 6.8 pmol/ml) was significantly higher (P < 0.05) than that in the contralateral eye (11.7 +/- 0.8 pmol/ml). These data indicate that aqueous humor NE per se does not cause the transient IOP decrease after superior cervical ganglionectomy and cAMP-mediated ocular activities may be involved in this change in IOP.     

cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells

Nitric oxide (NO) is cytoprotective to certain types of neuronal cells. The neuroprotective ability of NO in the retina was reportedly mediated by the cyclic GMP (cGMP) to protein kinase G (PKG) pathway. Cyclic AMP-responsive element binding protein (CREB) plays an essential role in the NO/cGMP/PKG-mediated survival of rat cerebellar granule cells. We tested whether CREB transduces the NO/cGMP/PKG anti-apoptotic cascade in R28 neuro-glial progenitor cells. Apoptosis was induced in R28 cells by serum deprivation for 24 h. Varying concentrations of two NO donors, sodium nitroprusside (SNP) and nipradilol, were added to medium with or without an NO scavenger, a soluble guanylyl cyclase inhibitor, or a PKG inhibitor. The cells were immunostained against activated caspase-3 and counterstained with Hoechst 33258. Apoptosis was quantified by counting activated caspase-3 positive or pyknotic cells. SNP and nipradilol rescued R28 cells from apoptosis in a dose-dependent manner, at an optimal concentration of 1.0 microM and 10 microM, respectively. Higher concentrations were cytotoxic. The NO scavenger and the inhibitors decreased the anti-apoptotic effect of the NO donors. Intracellular cGMP levels were increased after exposure to SNP and nipradilol. Western blotting showed that both NO donors increased CREB phosphorylation, which was blocked when pre-exposed to the inhibitors. Transfection with a dominant negative CREB construct defective of phosphorylation at Ser-133 interfered with the anti-apoptotic activity of SNP. These results indicate that CREB at least in part mediates the cGMP/PKG-dependent anti-apoptotic signal induced by NO in R28 cells.