NO/cGMP pathway activation and membrane potential depolarization in pig ciliary epithelium

PURPOSE: To investigate whether in isolated porcine ciliary processes, stimulation of the nitric oxide (NO)-guanylate cyclase (GC)-3',5'-cyclic guanosine monophosphate (cGMP) pathway modulates ciliary epithelial transmembrane potential. METHODS: Changes in transmembrane potential induced by the two NO donors, sodium nitroprusside (SNP; 100 microM) and S-nitroso-N-acetyl-penicillamine (SNAP; 100 microM), or by the cGMP-analogue 8-para-chlorophenylthioguanosine-3', 5'-cyclic guanosine monophosphate (8-pCPT-cGMP; 100 microM) were measured with microelectrodes in the presence or in the absence of the GC-inhibitor 1-H-(1,2,4)oxadiazole(4,3-alpha)quinoxalin-1-1 (ODQ; 10 microM). The effect of 8-pCPT-cGMP was also assessed in the presence of the anion channel inhibitors niflumic acid (100 microM), diisothiocyanatostilbene-2,2' disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), or the K+ channel blocker tetraethylammonium chloride (TEA; 10 mM). cGMP production was measured by immunoassay. RESULTS: Significant membrane depolarizations (P < 0.05-0.001; n = 5-8) were induced by SNP (6 +/- 1 mV; mean +/- SEM), SNAP (8 +/- 1 mV), or 8-pCPT-cGMP (13 +/- 1 mV). In presence of ODQ, the effect of SNP and SNAP were significantly inhibited (-2 +/- 0 mV and 0 +/- 0 mV, respectively; P < 0.05; n = 5-6), but not depolarizations elicited by 8-pCPT-cGMP. These were prevented (P < 0.05-0.01; n = 5) by niflumic acid (1 +/- 1 mV), DIDS (1 +/- 1 mV), or 9-AC (5 +/- 1 mV), but not by TEA (12 +/- 2 mV). The increase in cGMP production induced by SNP (9.5-fold) was inhibited by ODQ (P < 0.001; n = 6). CONCLUSIONS: Activation of the NO-GC-cGMP pathway modulates epithelial transmembrane potential in isolated porcine ciliary processes.     

Isoproterenol, forskolin, and cAMP-induced nitric oxide production in pig ciliary processes.

PURPOSE: To investigate whether isoproterenol and forskolin, two adenylylcyclase activators, or 8-bromo-cAMP, an adenosine 3',5'-cyclic monophosphate (cAMP) analog, increase nitric oxide (NO) production in isolated porcine ciliary processes. METHODS: Nitrite (an NO metabolite) was measured (Griess reaction) before and 2 hours after exposure to 0.1 to 100 microM isoproterenol (a beta-adrenoreceptor agonist), 0.01 to 100 microM forskolin, or 0.1 to 1000 microM 8-bromo-cAMP. Some experiments were conducted in the presence of 0.5 mM N(G)-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase [NOS] inhibitor), 10 microM propranolol (a beta-adrenoreceptor antagonist), or 1 microM KT 5720 (a cAMP-dependent protein kinase inhibitor). cAMP production was also measured (by immunoassay). RESULTs: Nitrite production was increased by isoproterenol (maximum, 10 microM: 164%; P < 0.001), forskolin (maximum, 10 microM: 254%; P < 0.001), and 8-bromo-cAMP (maximum, 100 microM: 184%; P < 0.001), an effect prevented by L-NAME (P < 0.05-0.001). Propranolol inhibited only isoproterenol-induced (10 microM) nitrite production (P < 0.05), whereas KT 5720 (P < 0.05) inhibited isoproterenol- (10 microM) and 8-bromo-cAMP-induced (10 microM) nitrite production. Furthermore, cAMP production evoked by isoproterenol (10 microM, P < 0.05) but not by forskolin (10 microM, P < 0.001) was inhibited by propranolol (P < 0.05). CONCLUSIONS: In isolated porcine ciliary processes, drugs activating adenylylcyclase or mimicking cAMP increase the production of NO by a mechanism that appears to involve both a cAMP-dependent protein kinase and NOS.     

The role of nitric oxide and cGMP in somatostatin's protection against retinal ischemia

PURPOSE: To investigate whether nitric oxide (NO) and/or cGMP protects the retina from chemical ischemia and underlie somatostatin's neuroprotective effects. METHODS: Eyecups of female Sprague-Dawley rats were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanate (25 mM)] in the absence or presence of (1) arginine (0.05-2.0 mM), the substrate of nitric oxide synthase (NOS); (2) the NO donors sodium nitroprusside (SNP; 0.25-4.0 mM), 3-morpholinosydnonimine (SIN-1; 0.1, 0.3, 1.0 mM), SIN-1 (0.1 mM)/L-cysteine (5 mM, peroxynitrite scavenger), and NONOate (1, 5, 10 microM, slow NO releaser); (3) 8-Br-cGMP (0.1, 0.5, 1.0 mM); (4) BIM23014 (sst(2) receptor agonist; 1 microM), alone or in the presence of (5) the NOS inhibitor N(gamma)-monomethyl-L-arginine (NMMA; 0.5 mM); or (6) the guanylyl cyclase inhibitors 1H-[1,2,4]oxadiazolol [4,3-a]quinoxalin-1-one (ODQ;100 microM) and NS2028 (50 microM) for 60 minutes, at 5%CO(2)/air in 37 degrees C. The effect of SIN-1 (0.1, 0.3, 1.0, or 3.0 mM) on the retina was also examined. Subsequently, the eyecups were fixed and sectioned for choline acetyltransferase (ChAT) immunoreactivity and TUNEL staining. RESULTS: Arginine and SNP had no effect on the chemical ischemia-induced toxicity. SIN-1, NONOate, and 8-Br-cGMP produced a concentration-dependent protective effect, as shown by ChAT immunoreactivity. TUNEL staining also confirmed the neuroprotective effect of these agents. L-cysteine partially reduced the SIN-1-induced protective effect. SIN-1 alone was toxic only at the highest concentration used (3 mM). NMMA, ODQ, and NS2028 reversed the protective effect of BIM23014. CONCLUSIONS: The results suggest that a NO/peroxynitrite/cGMP mechanism may be important in the protection of the retina from ischemic insult. Furthermore, the NO/sGC/cGMP pathway is involved in the neuroprotective effects of sst(2) ligands against retinal ischemia.     

Membrane potential depolarization in the porcine ciliary epithelium evoked by nitric oxide

BACKGROUND: The present work studies if in porcine ciliary body epithelium the intracellular signal transduction pathway nitric oxide (NO)--guanylate cyclase (GC)--3',5'-cyclic guanosinemonophosphate (cGMP) can change the membrane potential of the epithelium of the ciliary body. MATERIAL AND METHODS: Recordings of membrane potentials were done by means of intracellular microelectrodes in the presence of the NO donor Sodium Nitroprusside (SNP; 100 microM; n = 5) or the membrane permeable cGMP analogue 8-parachlorophenyl-thioguanosine-3',5' cyclic monophosphate (8-pCPT-cGMP; 100 microM; n = 5). To test whether the GC is involved in this process, recordings were repeated in both groups in presence or in absence of the GC inhibitor 1-H-(1,2,4)oxadiazole-(4,3-a)quinoxalin-1-one (ODQ; 10 microM; n = 4). RESULTS: SNP and 8-pCPT-cGMP both induced significant membrane potential depolarizations (7.7 +/- 1.8 mV and 13.1 +/- 1.3 mV, mean +/- SEM). Membrane depolarizations induced by SNP were significantly inhibited by ODQ (p < 0.01), whereas depolarizations induced by 8-pCPT-cGMP were not altered by the presence of ODQ (p > 0.2). CONCLUSIONS: Nitric oxide induces depolarizations of the membrane potential in the isolated porcine ciliary body. This process is transduced by activation of the GC. We conclude that nitric oxide might be involved in the regulation of permeability of the cellular membrane for ions in the ciliary body.     

PGI2 opens potassium channels in retinal pericytes by cyclic AMP-stimulated, cross-activation of PKG

Pericytes exert an important influence on the control of retinal blood flow; however, little is known regarding the molecular basis of retinal pericyte excitability. The purpose of this study was to elucidate the signaling pathway of how prostacyclin (PGI2), an important endogenous regulator of retinal blood flow, stimulates potassium channel activity in retinal pericytes. Retinal pericytes were isolated from porcine eyeballs and plated on glass coverslips. Immunocytochemistry was performed to verify expression of the pericyte-specific ganglioside marker, 3G5 and smooth muscle alpha-actin. Activity of the large-conductance, voltage- and calcium-activated potassium (BKCa) channel was measured in retinal pericytes via single-channel patch-clamp, and channel identification was confirmed via biophysical and pharmacological characterization. PGI2 (10 microM) or beraprost (30 microM; more stable prostacyclin analog) dramatically stimulated the activity of BKCa channels isolated in cell-attached patches. These experiments strongly suggested that PGI2 stimulated BKCa channel activity via a diffusible second messenger. Similarly, chlorophenylthio (CPT)-cAMP (100 microM; membrane permeable cAMP derivative) induced a significant increase in BKCa channel activity; however, inhibition of the cAMP-dependent protein kinase (PKA) with 300 nM KT5720 could not reverse the stimulatory effect of either PGI2 or CPT-cAMP. In contrast, activation of BK(Ca) channels with either CPT-cAMP or PGI2 was abolished by 300 nM KT5823 (n=5, p<0.01), an inhibitor of the cGMP-dependent protein kinase (PKG). In addition, PGI2-stimulated channel activity was also attenuated by Rp-8-CPT-cGMPS, which inhibits PKG activity via a different mechanism. These findings demonstrate that prostacyclin, the most abundant prostanoid in the retinal circulation, is a potent stimulator of BKCa channel activity in retinal pericytes. Interestingly, this response appears to involve cAMP-stimulated cross-activation of PKG, and not PKA. Taken together, these findings could explain, at least in part, the cellular/molecular basis for PGI2-induced pericyte relaxation and augmentation of blood flow in the retina. Further, we propose PKG-dependent stimulation of BKCa channel activity as a new potential therapeutic target to combat decreased retinal blood flow seen in some disease states (e.g., diabetic retinopathy).     

Effect of cAMP on porcine ciliary transepithelial short-circuit current, sodium transport, and chloride transport

PURPOSE: To investigate the effects of 8-bromo-cAMP (cAMP) on porcine ciliary transepithelial short-circuit current (Isc) and transport of chloride (Cl-) and sodium (Na+). METHODS: With Ussing-type chambers, cAMP-induced changes in Isc, electrical resistance (ER), and transepithelial 36Cl- and 22Na+ fluxes were measured. Drugs were applied to the nonpigmented epithelium (NPE) and/or pigmented epithelium (PE) side(s). The effect of IBMX (1, 5, or 10 microM; 3-isobutyl-1-methylxanthine) on Isc-increase induced by 8-bromo-cAMP on the PE side was also tested. RESULTS: On the NPE side, a single concentration (10 microM, 100 microM, or 1 mM) of 8-bromo-cAMP induced a biphasic (transient peak followed by sustained plateau) Isc increase. On the PE side, 8-bromo-cAMP induced a similar but delayed biphasic Isc increase at 1 mM, a slight plateau-Isc increase at 100 microM, and no Isc increase at 10 microM. In the concentration-response curve, the cAMP-induced peak-Isc increase became significant at a concentration 10,000 times lower on the NPE than on the PE side. At 10 microM, the cumulative cAMP-induced Isc-increase reached its maximum on the NPE side, but was virtually nonexistent on the PE side. IBMX (a phosphodiesterase inhibitor) but not 8-CPT-6-Phe-cAMP (higher permeability than 8-bromo-cAMP) significantly increased the peak-Isc concentration-response curve induced by 8-bromo-cAMP (10 nM-1 mM) on the PE side. On the NPE but not the PE side, 10 microM 8-bromo-cAMP induced a significant but transient increase in net PE-to-NPE 36Cl- flux (1.03 +/- 0.18 microEq/min per square centimeter; P < 0.001). Neither ER nor transepithelial 22Na+ flux was changed after cAMP exposure. CONCLUSIONS: In porcine ciliary processes, apparently on the NPE side, cAMP triggers a biphasic (transient peak followed by a sustained plateau) Isc increase. Only the peak-Isc increase involves an increase in net PE-to-NPE Cl- transport.     

Effect of VEGF on retinal microvascular endothelial hydraulic conductivity: the role of NO

PURPOSE: Vascular endothelial growth factor (VEGF) increases microvascular permeability in vivo and has been hypothesized to play a role in plasma leakage in diabetic retinopathy. Few controlled studies have been conducted to determine the mechanism underlying the effect of VEGF on transport properties (e.g., hydraulic conductivity [Lp]). This study was conducted to determine the effect of VEGF on bovine retinal microvascular endothelial LP and the role of nitric oxide (NO) and the guanylate cyclase/guanosine 3', 5'-cyclic monophosphate/protein kinase G (GC/cGMP/PKG) pathway downstream of NO in mediating the VEGF response. METHODS: Bovine retinal microvascular endothelial cells (BRECs) were grown on porous polycarbonate filters, and water flux across BREC monolayers in response to a pressure differential was measured to determine endothelial LP RESULTS: VEGF (100 ng/ml) increased endothelial LP: within 30 minutes of addition and by 13.8-fold at the end of 3 hours of exposure. VEGF stimulated endothelial monolayers to release NO and incubation of the BRECs with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 100 microM) significantly attenuated the VEGF-induced LP increase. It was observed that incubation of the monolayers with the GC inhibitor LY-83583 (10 microM) did not alter the VEGF-mediated LP: response. Addition of the cGMP analogue 8-br-cGMP (1 mM) did not change the baseline LP over 4 hours. Also, the PKG inhibitor KT5823 (1 microM) did not inhibit the response of BREC LP to VEGF. CONCLUSIONS: These experiments indicate that VEGF elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.     

Effect of shear stress on the hydraulic conductivity of cultured bovine retinal microvascular endothelial cell monolayers

The shear stress of flowing blood on endothelial cells increases water transport (hydraulic conductivity, Lp) in several vascular beds in vivo and has been hypothesized to play a role in elevating vascular transport in ocular diseases such as diabetic retinopathy. The purpose of this study is to determine the response of Lp to varying levels of shear stress using an in vitro model of the blood-retinal barrier: bovine retinal endothelial cells (BRECs) grown on polycarbonate filters. The study also addresses the role of nitric oxide (NO) and other downstream effectors in mediating shear-induced changes in water transport. A step change in shear stress of 10 dyn/cm(2) did not produce a significant change in Lp over 3 hours, whereas a 20 dyn/cm(2) step change elevated Lp by 14.6-fold relative to stationary controls at the end of 3h of shear exposure. 20 dyn/cm( 2) of shear stress stimulated the endothelial monolayers to release nitric oxide in a biphasic manner and incubation of the BRECs with a nitric oxide synthase (NOS) inhibitor, L-NMMA, significantly attenuated the shear-induced Lp response. These experiments demonstrate that NO is a key signaling molecule in the pathway linking shear stress and Lp in BRECs. A widely studied pathway downstream of NO involves the activation of guanylate cyclase (GC), guanosine 3', 5' -- cyclic monophosphate (cGMP) and protein kinase G (PKG). It was observed that incubation of BRECs with the GC inhibitor, LY83583 (10 microM) or the PKG inhibitor, KT5823 (1 microM) did not significantly alter the shear-induced Lp response. Also the cGMP analogue, 8-br-cGMP (1mM), did not affect the baseline Lp over 4h. These results demonstrate that shear stress elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.     

Reversal of the epinephrine stimulation of Cl- transport in bullfrog cornea by phorbol esters

The effects of protein kinase C (PKC) stimulation with phorbol dibutyrate, PDBu, were determined on transepithelial net Cl- transport (Isc) across the isolated bullfrog cornea. Between 1 nM and 1 microM, PDBu had no effect on either the Isc or the conductance, gt, as well as the membrane voltage (Vsc) but it decreased the fractional apical membrane resistance (fR0). PDBu caused a dose-dependent decline and reversal of the stimulatory effect of the mixed alpha and beta adrenergic agonist, epinephrine, on the Isc. With 1 microM PDBu, 100 microM epinephrine decreased the Isc by 20% below its control value. As without PDBu, epinephrine decreased fR0 and depolarized the Vsc. Evidence that this reversal by PDBu stems from a selective stimulation of PKC is: (1) neither its vehicle nor the inactive phorbol analog, 4 alpha-phorbol didecanoate, PDD (1 microM), decreased either the fR0 or altered the stimulatory effect of epinephrine on the Isc. (2) After preincubation with 30 microM H-7, which inhibited PKC stimulation in subcellular membrane and cytosolic fractions of frog corneal epithelium, the stimulatory effect of epinephrine on the Isc was unchanged by 1 microM PDBu. Preincubation with PDBu completely inhibited the stimulatory effect of the beta adrenergic agonist, isoproterenol (10 microM), on the Isc but did not affect the stimulatory effect of 1 microM forskolin on the Isc, indicating that PKC stimulation results in uncoupling of adrenergic regulation of adenylate cyclase activity. Epinephrine did not reverse the Isc in corneas that were preincubated with either the alpha 2- -adrenergic antagonist, yohimbine (100 microM), or the general alpha adrenergic antagonist, phenoxybenzamine (100 microM). With yohimbine, epinephrine had no effect on the Isc whereas with phenoxybenzamine the stimulation by epinephrine was fully restored. These effects suggest that stimulation of PKC is dependent upon the stimulation of alpha 1-adrenergic receptors by epinephrine as well as the presence of PDBu. Reversal and stimulation of the Isc by epinephrine were both associated with a depolarization of the Vsc and similar decreases in fR0. These similar effects on fR0 suggest that PKC stimulation as well as alpha 2 adrenergic activation by epinephrine are associated with increases in basolateral membrane resistance.     

Horizontal cell glutamate receptor modulation by NO: mechanisms and functional implications for the first visual synapse.

Neurons of the horizontal cell retinal neural network are subject to modulation by the neurotransmitter nitric oxide (NO). We have examined the effects of NO on glutamate receptor function in isolated horizontal cells from the perch (Perca fluviatilis) using the concentration ramp technique to simultaneously record receptor current and agonist concentration. Dose-response curves for glutamate (0-1 mM) and kainate (0-200 microM) were measured in the presence and absence of 1-2 mM sodium nitroprusside (SNP), 1 mM 8-Br-cGMP, 100 microM cyclothiazide or 200 microM dopamine as modulators. SNP increased the EC50 (i.e. decreased affinity) for glutamate and increased Imax (i.e. increased efficacy), whereas 8-Br-cGMP increased EC50, but not Imax. In the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor desensitization blocker cyclothiazide, the SNP-induced increase in EC50 persisted, but the increase in Imax was blocked. The increase in EC50, but not the increase in Imax was also observed when the non-desensitizing agonist kainate (100-200 microM) was applied in the presence of SNP. When 2 mM SNP and 200 microM dopamine were applied together, they increased Imax (740 vs. 2455 pA) and EC50 (422 vs. 682 microM). Our findings indicate that NO modulates horizontal cell glutamate responses by reducing the affinity of receptors for glutamate while simultaneously increasing the maximal current. The shift in affinity is cGMP-mediated and independent of desensitization. The action of NO on horizontal cell glutamate receptors is distinct from, but synergistic with. that of dopamine. Glutamate receptor modulation by NO qualitatively predicts the action of NO on horizontal cell light responses in situ and may alter transmission at visual synapses according to adaptational conditions.     

Comparison of nitric oxide donors in lowering intraocular pressure in rabbits: role of cyclic GMP.

L-arginine-nitric oxide (NO) pathway participates in the physiology and in many pathological processes in the eye, such as glaucoma. The aim of the present study was to compare the ocular hypotensive effect of different NO-donors, and to get more information on the role of cyclic guanosine 3',5'-monophosphate (cGMP) in this process. The test compounds were administered topically or intravitreally in the eye of a normotensive rabbit. Intraocular pressure (IOP) was measured with a pneumatonometer after topical anesthesia. The metabolites of NO (nitrite, nitrate, NOx) and cGMP were assayed from the aqueous humor and plasma. NO-synthase (NOS) protein expression was assayed in the ciliary body by Western blotting. The maximal lowering of IOP was achieved as follows: atriopeptin III (concentration 78 (microM, decrease in IOP 50%), atriopeptin II (84 (microM 37%). 8-Br-cGMP (90 mM, 37%), zaprinast + 8-Br-cGMP (1 mM + 90 mM, 34%), L-arginine (1 mM, 29%), SNP (40 mM, 28%), nitrosocaptopril (100 mM, 28%), S-nitrosothiol (SNOG) (10 mM, 27%), YC-1 (10 (microM, 25%), zaprinast + SNP (1 mM + 40 mM, 22%), spermine NONOate (100 mM, 20%) [corrected]. The decrease in IOP lasted for 2-5 hr, except with atriopeptin II and III, when IOP values were first normalized in 6 hr and 2 days, respectively. In conclusion, the results of the present study indicate that by increasing the activity of L-arginine/NO/cGMP-pathway it is possible to lower IOP in rabbits equally to the currently used antiglaucomatous drugs.     

Dopamine (D1) receptor activation and nitrinergic agents influence somatostatin levels in rat retina

Somatostatin (SRIF) influences the release of two important neuromodulators of retinal circuitry, dopamine (DA) and nitric oxide (NO). The aim of the present study was to examine whether DA and NO modulate SRIF release in rat retina, and the mechanisms involved in their actions. Retinas of adult female Sprague--Dawley rats (250--300 g) were mechanically detached from the eyecup and ex vivo experiments were performed. Retinal explants were incubated in the presence of dopaminergic [DA (10 microM, 100 microM and 200 microM), apomorphine (nonselective D1/D2 agonist, 0.50 mM, 1.0 microM and 10 microM), A68930 (D1 selective agonist, 0.50 microM, 1.0 microM and 10 microM), quinpirole (D2 selective agonist, 0.50 microM, 1.0 microM and 10 microM), SCH 23390 (D1 selective antagonist, 250 nM and 500 nM) and sulpiride (D2 selective antagonist, 100 microM and 200 microM)], and nitrinergic agents [arginine (62.5 microM--5mM), SIN-1 (50 microM, 100 microM and 500 microM) and 8-Br-cGMP (50 microM, 250 microM and 500 microM)]. SRIF levels were quantified using radioimmunoassay (RIA). Dopamine had no effect on SRIF levels. Apomorphine produced a concentration dependent decrease and increase in SRIF levels, suggestive of pre- and postsynaptic effects. A68930 (10 microM) and SCH 23390 (250 nM and 500 nM) mimicked and reversed apomorphine's postsynaptic actions, respectively. Quinpirole had no effect, but blockade of D2 autoreceptors by sulpiride (200 microM) afforded an increase in SRIF levels. Arginine and SIN-1 increased, and 8-Br-cGMP attenuated SRIF levels. These results show that dopamine D1 receptors, and NO/peroxynitrite agents modulate SRIF release in the retina suggesting that the triad SRIF--DA--NO have reciprocal interactions via which they regulate retinal circuitry and vision transduction.     

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells

PURPOSE: Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair. METHODS: The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or other agents. At prescribed time intervals, the cells were stained by Giemsa and the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured by radioimmunoassay. RESULTS: Addition of EGF (1-100 ng/ml) to RCEC stimulated cell proliferation which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 microM) or SNP (10 microM to 1 mM), in the presence of EGF, dose-dependently inhibited the growth factor-stimulated wound closure in RCEC. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 microM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibited the wound closure with a corresponding increase in cGMP contents. Treatment of the cells with ODQ (10 nM to 10 microM), a soluble guanylate cyclase inhibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, and reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Addition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited the EGF-stimulated wound closure in a dose-dependent manner. Treatment of RCEC with mitomycin C (5 microM) exerted a marked inhibitory effect on wound closure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated cells. CONCLUSIONS: The results demonstrate that ANP and SNP inhibit the EGF-stimulated wound repair in RCEC. The effect of these agents is mediated via activation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exert its inhibitory effect at the level of cell proliferation and not cell migration. The data suggest an important role for cGMP-dependent protein kinase in proliferation of RCEC stimulated by EGF.     

Down-regulation of Na-K-Cl cotransport by protein kinase C is mediated by protein phosphatase 1 in pigmented ciliary epithelial cells

The role of protein phosphatases in the regulation of Na-K-Cl cotransport was examined in human pigmented ciliary epithelial (PE) cells. Both a 37 kDa form and a 72 kDa form of protein phosphatase 1 (PP1) could be immunologically detected. The protein phosphatase inhibitor calyculin A stimulated Na-K-Cl cotransport by 89 +/- 12% at 10 n M, whereas okadaic acid had no effect at concentrations less than 100 n M. Calyculin A had no significant effect on either Na-K ATPase or ouabain-insensitive, bumetanide-insensitive 86Rb+uptake. These data suggest that PP1 plays a role in the inhibition of Na-K-Cl cotransport in PE cells. Treatment of cells with phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PKC) activator caused an 82% inhibition of Na-K-Cl cotransport. When cells were first treated for 5 min with PMA, 10 n M calyculin A stimulated Na-K-Cl cotransport by 53% compared to 101% by calyculin A alone. Treatment of cells with PMA after stimulation of Na-K-Cl cotransport by calyculin A resulted in a prompt 56% drop in cotransport activity. These data suggest that maximal inhibition of Na-K-Cl cotransport by PKC requires PP1 activity, but that a part of PKCs inhibitory effect is independent of PP1. The effect of PKC activation on PP1 was further examined by determining PP1 activity in cells pretreated with PMA. PP1 activity increased 38+/-8% in cells exposed to 1 microM PMA for 5 min. This stimulation was blocked by 100 n M staurosporine or 1 microM bisindolylmaleimide, two PKC inhibitors. An isomer which does not activate PKC (4 alpha phorbol didecanoate), did not stimulate PP1 activity. Thus PKC activation leads to an increase in PP1 activity in PE cells. Pretreatment of cells with the protein kinase A (PKA) inhibitor PHI 14-22 resulted in a partial reduction in calyculin A stimulation of cotransport, suggesting that PP1 and PKA function in a kinase-phosphatase regulatory loop. To determine whether other protein kinases might also be involved, several protein kinase inhibitors were tested, including KT5823 (protein kinase G, type II-specific), KN62 (calmodulin activated kinase-specific) and ML7 (myosin light chain kinase-specific). None prevented activation of Na-K-Cl cotransport by calyculin A, suggesting that these kinases are not involved in the activation of Na-K-Cl cotransport.     

The role of NaKCl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium

PURPOSE: To evaluate the role of NaKCl cotransport in short-circuit current (Isc) and chloride fluxes across rabbit ciliary epithelium mounted in a Ussing-type chamber. METHODS: Bilayered intact ciliary epithelium free of stroma was obtained after perfusion and dissection of rabbit eyes and mounted in an Ussing-type chamber. The effects of bumetanide and other drugs on Isc and transepithelial 36Cl fluxes in bicarbonate-containing Ringer's were determined. Immunoblot analysis was performed by standard techniques. RESULTS: Bumetanide (100 microM) applied to the blood (pigmented epithelium [PE]) side of the ciliary bilayer caused a dose-dependent decrease in Isc from 18.2 +/- 2.2 to 10.4 +/- 1.4 microA/cm2 (43%). Bumetanide applied to the aqueous (nonpigmented epithelium [NPE]) side of the tissue inhibited Isc by only 12%. Immunoblots of dissected NPE and PE tissue probed with an antibody to mammalian NaKCl cotransporter detected approximately 10 times more NaKCl cotransporter protein in PE than in NPE. 36Cl flux studies revealed a PE-to-NPE chloride flux of 180.3 +/- 37.2 microEq/cm2 per hour and an NPE-to-PE flux of 72.3 +/- 22.9 microEq/cm2 per hour, indicating a net PE-to-NPE flux of 108.0 +/- 31.3 microEq/cm2 per hour across rabbit ciliary epithelium. Bumetanide inhibited the PE-to-NPE chloride flux by 52% but did not inhibit the NPE-to-PE flux. Isoproterenol (10 microM) added to the PE side of the bilayer increased Isc by a dose-dependent 53%. Prior addition of bumetanide to the PE side blocked the increase due to isoproterenol by 37%. Isoproterenol (10 microM) stimulated the PE-to-NPE chloride flux by 75% but had no stimulatory effect on the NPE-to-PE chloride flux. 4,4'Diisothiocyanatostilbene-2,2'disulfonic acid (DIDS) inhibited Isc when added to either side of the bilayer but was more potent at low concentrations (<100 microM) when added to the NPE side and more potent at higher concentrations (>100 microM) when added to the PE side. Prior addition of 1 mM DIDS to the NPE side decreased isoproterenol stimulation of Isc by 56%. CONCLUSIONS: NaKCl cotransporters located primarily on the blood side of rabbit ciliary epithelium contribute to aqueous-negative Isc and to blood-to-aqueous chloride transport across the tissue in bicarbonate-containing medium. DIDS-inhibitable mechanisms, possibly including HCO3-Cl exchange and Cl channels, also play a role. Isoproterenol stimulation of Isc involves coordinate upregulation of PE-side NaKCl cotransport and an NPE-side DIDS-inhibitable mechanism(s).     

Transient Ca2+-activated Cl-currents with endothelin in isolated arteriolar smooth muscle cells of the choroid

PURPOSE: To characterize the effects of endothelin (ET)-1 on the Ca2+-activated Cl- conductance of choroidal arteriolar smooth muscle. METHODS: Microvascular smooth muscle cells were enzymatically isolated from choroidal arterioles from the eyes of freshly killed rabbits. Cells were voltage-clamped at -60 mV using the whole-cell perforated patch-clamp technique. Internal pipette solutions were K+ based and contained amphotericin B (200 microg/ml). The cells were bathed in a 20 mM tetraethyl-ammonium solution to block outward K+ currents. RESULTS: Within 2 to 5 seconds of adding ET-1 (10 nM), inward current pulses were generated at a frequency of around 1 Hz. These evoked transient inward currents were blocked by niflumic acid (10 microM) or anthracene-9-carboxylic acid (1 mM). They were increased 2.4+/-0.1-fold when Cl- was replaced by I in the bathing medium and lost within 4 minutes when external Cl- was reduced from 151.6 to 20 mM. The reversal potential was -1+/-2 mV with 135 mM Cl- in the recording pipette and with 54 mM Cl it was -18+/-4 mV. When gramicidin D (100 microg/ml), which maintains [Cl-]i, was used instead of amphotericin B, the reversal potential was -18+/-1 mV. Ca2+ release by caffeine (10 mM) produced a single transient inward current. Endothelin-evoked transient inward currents were slowly reduced and eventually abolished in Ca2+-free solution (approximately 2 to 3 minutes) and were eliminated after approximately 30 seconds by the sarcoplasmic reticulum Ca2+-uptake inhibitor cyclopiazonic acid (5 microM). The ET(A) receptor antagonist BQ123 (1 microM) prevented an effect by endothelin but did not inhibit the current oscillations once they had been triggered. CONCLUSIONS: In choroidal arteriolar smooth muscle ET-1 evokes transient inward Ca2+-activated Cl- currents induced through the cyclical release and re-uptake of Ca2+ from intracellular stores after ET(A) receptor stimulation.     

Unoprostone isopropyl rescues retinal progenitor cells from apoptosis in vitro

PURPOSE: Unoprostone isopropyl is an ocular hypotensive that was originally produced as a prostaglandin F2alpha analogue and is eventually recognized as a synthetic docosanoid. The compound is recently suggested to have potent neuroprotective ability in the retina. The purpose of this study is to test whether and how the biologically active metabolites of unoprostone isopropyl rescue retinal neuro-glial progenitor cells from apoptosis. METHODS: R28 cells were deprived of serum for 24 hr with or without varying concentrations of unoprostone metabolite M1 or M2 or vehicle in the presence or absence of specific inhibitors against several types of signal transduction proteins. Immunocytochemistry against activated caspase-3 with Hoechst nuclear staining was performed. RESULTS: Up to 15%of R28 cells became pyknotic and activated caspase-3 immunoreactive after 24-hr serum withdrawal. M1, but not M2, significantly reduced apoptotic cells in a dose-dependent fashion with a maximal effect at 100 microM (p < .0001). LY294002, the phosphatidylinositol 3-OH kinase (PI3K) inhibitor, and KT5823, the protein kinase G (PKG) inhibitor, reversed the antiapoptotic effect of M1. CONCLUSIONS: The unoprostone metabolite M1 protects retinal neuro-glial progenitor R28 cells from apoptosis induced by serum deprivation via the PI3K and PKG pathways.     

Diquafosol elicits increases in net Cl- transport through P2Y2 receptor stimulation in rabbit conjunctiva

The purpose of the present study was to understand the mechanisms of action of diquafosol, a stable derivative of uridine 5'-triphosphate, on Cl(-) transport across the isolated rabbit conjunctiva. Rabbit conjunctivas were isolated and mounted in a modified Ussing chamber. Under short-circuit conditions, the effects were determined of mucosal (tear) side diquafosol application on the short-circuit current (Isc). Diquafosol rapidly and dose-dependently increased the Isc at concentrations ranging from 0.1 to 968 microM when added to the mucosal side of the conjunctiva. In the absence of the serosal Cl(-), the Isc induced by 10 microM diquafosol was substantially reduced. On the contrary, in the absence of mucosal side Na(+), the diquafosol-induced increases in Isc were unchanged. Following 45-min preincubation, the P2Y(2) antagonist suramin inhibited the diquafosol-induced increases in the Isc whereas the P2Y(1) antagonist pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid had no effect. These studies suggest that diquafosol stimulates net Cl(-) secretion from the serosal to the mucosal side via stimulation of P2Y(2) receptors in the rabbit conjunctiva.     

Neuroprotective effect of nipradilol on axotomized rat retinal ganglion cells

PURPOSE: To determine whether nipradilol, a new anti-glaucoma drug, can protect retinal ganglion cells (RGCs) from secondary cell death caused by transection of the optic nerve (ON). METHODS: The ON was transected 0.7 mm from its exit from the eye in Sprague Dawley rats. Nipradilol (1 x 10(-8) - 10(-3) M), timolol, prazosin, or sodium nitroprusside (SNP) (1 x 10(-6) - 10(-4) M) was injected intravitreally fifteen-minutes before the ON transection. Control eyes received the same amount of phosphate buffered (PB). The RGCs were labeled retrogradely by placing gelfoam soaked in fluoro-gold (FG) on the stump of ON. RGCs density was determined by counting the FG-labeled RGCs in flat-mounted retinas 3 to 14 days post-transection. To determine whether the neuroprotective action of nipradilol was due to its NO-donor property, carboxy-PTIO, a NO-scavenger, or KT5832, a protein kinase G inhibitor, was injected with the nipradilol. RESULTS: After ON transection, the number of surviving RGCs after intravitreal injection of 1 x 10(-4) M nipradilol was significantly higher than that following PB injection. This protective activity was dose-dependent. Neither timolol nor prazosin had a neuroprotective effect but SNP protected RGCs in a dose-dependent manner. Carboxy-PTIO and KT5832 decreased the neuroprotective effect of nipradilol. CONCLUSIONS: These results indicate that nipradilol has a possibility of neuroprotective effect on axotomized RGCs, and the effect depended mainly on its NO-donor property.