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Age-related macular degeneration and other eye diseases, such as diabetic retinopathy, are probably linked to the effects of oxygen radicals derived from light or metabolic reactions. We have investigated the effects of hypoxia on bovine retinal pigmented epithelial cells (RPE) and the response of these cells to two antioxidants that have previously shown a beneficial action against free radical-linked senescent involution. The main results of the study were as follows: (i) Hypoxia induced apoptotic damage on RPE cells, with LDH leakage and ATP reduction; (ii) both vitamin C (VC) and N-acetyl-cysteine (NAC) treatment protected against hypoxia-induced apoptosis, with less DNA fragmentation. In our opinion, these findings justify further experimental and clinical work to investigate the role of hypoxia in the mechanisms of age-related RPE injury and death as well as the potential of antioxidant administration to prevent or delay retinal degenerative processes caused by oxygen-dependent pathophysiological conditions.

Retinal pigmented epithelium (RPE) is an ocular tissue that performs several important functions such as light perception and phagocytosis. This study investigated the effects of hypoxia and serum and glucose deprivation of the RPE tissue. The possible protective effects of antioxidants were also studied by determining cell viability, ATP content, and DNA fragmentation. The results demonstrate that antioxidants can protect RPE tissue from death through the inhibition of some mechanisms that lead to cellular apoptosis.

Retinal pigmented epithelium (RPE) is an ocular tissue that performs a variety of functions including metabolism of photoreceptors. It constitutes an electrical and chemical barrier regulating the movement of solutes and ions between neural retina and the choriocapillary network. Reactive oxygen intermediate (ROI) generation has been implicated in the etiology of certain neurodegenerative diseases of the eye, which may be positively altered by treatment with antioxidant drugs. Cell viability under oxidative stress, hypoxia, and ischemic conditions in RPE were studied in vascular epithelium (VE) cells in culture to explore the possible role of vitamin C and N-acetyl-cysteine to prevent cell injury. The results of this study show that RPE cells may be protected against free radicals and products of peroxidation by endogenous scavengers by treatment with antioxidant drugs.

The aim of this work was to study the effect of various anti-inflammatory drugs on PGE2 synthesis in cultured bovine corneal endothelial cells (BCECs) stimulated with calcium ionophore A23187 or lipopolysaccharide (LPS) of Salmonella typhimurium. NSAIDs were more potent in inhibiting LPS-stimulated PGE2 synthesis. Diclofenac was more potent than indomethacin, although both drugs showed a 98% maximal inhibitory effect. Dexamethasone inhibited 80% of the A23187-stimulated PGE2 synthesis and only 53% of the LPS-stimulated PGE2 synthesis. Prednisolone did not show an inhibitory effect. The results demonstrate the inhibitory effect of NSAIDs and show differences between the activity of glucocorticoids on PGE2 synthesis in BCECs. Prednisolone could not inhibit PGE2 synthesis in these cells in our experimental conditions.

Nitric oxide (NO) synthase inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME), have been shown to attenuate endotoxin-induced uveitis (EIU) but they could increase leukocyte adhesion to the vascular endothelium. We hypothesize that a concomitant treatment with the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) in 50% dimethylsulfoxide (DMSO, a hydroxyl radical scavenger) could improve the anti-inflammatory activity of L-NAME. EIU was induced in albino rabbits by intravitreal injection of 100 ng lipopolysaccharide. Animals were treated with multiple intraperitoneal injections of 50% DMSO in phosphate-buffered saline (PBS), NDGA (10 mg/kg) in 50% DMSO, L-NAME (50 mg/ kg) in PBS, or the combination NDGA+L-NAME. Uveitis was assessed by slit lamp examination, protein levels in aqueous humor, and myeloperoxidase (MPO) activity in the iris/ciliary body 6 h after induction. Nitrite, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), platelet-activating factor (PAF) and interleukin-1 beta (IL-1 beta) levels in aqueous humor were also determined. NDGA or L-NAME alone did not show a significant reduction of uveitis intensity, although a significant decrease in MPO or in proteins was found, respectively. The combination NDGA+L-NAME significantly reduced the uveitis intensity, MPO in the iris/ciliary body, and the levels of nitrites, LTB4, PGE2, and PAF in aqueous humor. IL-1 beta levels were lower than the detection limit of the radioimmunoassay in all treatment groups. We conclude that concomitant treatment with NDGA in DMSO improves the anti-inflammatory activity of L-NAME during the early phase of EIU, suggesting that the inhibition of NO synthesis could enhance leukocyte infiltration and the release of oxygen free radicals.

The role of protein phosphorylation in catecholamine secretion from bovine adrenomedullary chromaffin cells was studied using different protein kinase inhibitors. Naphthalenesulfonamide derivatives as ML9 and ML7, more specific for the myosin light chain kinase, and the calmodulin antagonist W7 inhibited catecholamine secretion 20 and 40% respectively in digitonin-permeabilized chromaffin cells. ML9 also decreased calcium evoked protein phosphorylation of different proteins including tyrosine hydroxylase in permeabilized cells. These naphthalenesulfonamide derivatives showed also an effect in intact cells, ML9 and W7 produced 50% inhibition in catecholamine secretion and 45Ca2+ uptake, however H8 had no effect. The partial [3H]nitrendipine binding displacement of these drugs to adrenomedullary membranes suggests that these sulfonamide derivatives could interact directly with L-type calcium channels in intact cells. The results obtained in permeabilized cells suggest a possible role of protein phosphorylation in the regulation of catecholamine secretion in chromaffin cells.

To further know the signaling pathways involved in hypoxia-induced apoptosis in retinal pigmented epithelial (RPE) cells and to improve the understanding of the antioxidant N-acetyl-cysteine (NAC) treatment effect.We analyzed the expression levels of several apoptosis-related genes by semiquantitative reverse transcriptase-polymerase chain reaction in RPE after 72 h of maintained hypoxia, with or without 10 mM NAC treatment.Under hypoxic conditions, we detected a higher expression level of p53 and CASP8. Cell treatment with NAC 10 mM prevented this increase. Other apoptosis-related genes such as bax, CASP3, CASP4, CASP7, and fas did not show an increase in expression levels in hypoxia.NAC prevents the increased expression levels of p53 and CASP8 induced by long-term maintained hypoxia. The supply of antioxidants could be a useful preventive approach in protecting RPE from the effects of chronic oxygen stress, which is of great interest in oxygen stress-related diseases such as age-related macular degeneration and other senescence-associated pathologies.

The aim of this work was to study the regulation of LPS-stimulated PGE 2 synthesis by traditional NSAIDs (piroxicam and diclofenac) and a selective COX-2 inhibitor (NS-398), in cultured bovine corneal endothelial cells and retinal pigmentary epithelial cells. The IC50 values of piroxicam and diclofenac were compared with IC50 values of NS-398, diclofenac, in both types of cells, showed higher potency than piroxicam. Diclofenac seemed to be a COX-2 inhibitor because its IC50 values were similar to the IC50 values of NS-398. We suggest that this in vitro cell assay system could be useful for identifying compounds that selectively inhibit COX-2 in ocular tissues.

The ocular pharmacokinetics of topical diclofenac sodium was studied in two experimental models of ocular inflammation and compared to physiological conditions. Keratitis or uveitis were induced by intrastromal injection of clove oil or by intravitreal lipopolysaccharide in rabbits. The control eyes were not inflamed. Simultaneously to the induction of inflammation, 30 µl of 0.1% diclofenac were applied topically in the right eye. Diclofenac levels were measured by HPLC in the cornea, aqueous humor (AH), iris/ciliary body (ICB) and plasma 30 min, 1, 3, 6 and 12 h after application. In physiological conditions, diclofenac reached a peak level in the cornea and ICB at 30 min slowly decreasing afterwards. Low levels of diclofenac were found in AH. In keratitic eyes, two peak levels which were significantly higher than in the controls were found in the cornea 30 min and 3 h after application. Diclofenac concentrations in keratitic AH and ICB were lower than in controls. In uveitic eyes, corneal and ICB levels peaked at 30 min, being significantly higher than in controls, and decreased quickly to very low levels at 1 h after application. In uveitic AH, diclofenac levels were lower than in controls. Plasma levels were very low (less than 0.1 µg/ml) in all experimental groups. It is concluded that the ocular pharmacokinetics of topical diclofenac is affected by inflammatory processes in the eye, reaching higher levels in the target tissues.

The anti-inflammatory effect of the inhibition of nitric oxide synthase (NOS) or 5lipoxygenase (5-LO), and a concomitant inhibition of both pathways were investigated on the endotoxin-induced uveitis (EIU) model in the rabbit. EIU was induced in albino rabbits by intravitreal injection of 100 ng lipopolisaccharide (LPS) in both eyes. Four groups of rabbits were treated intraperitoneally with phosphate buffered saline (PBS) and 50% DMSO (control group), 10 mg/kg nordihydroguayaretic acid (NDGA) in 50% DMSO, 50 mg/kg NG-nitro-L-arginine methyl ester (L-NAME) in PBS, or with the combination NDGA+L-NAME. The severity of uveitis was graded under slit lamp examination. Six hours after the induction of EIU, animals were sacrificed, and protein (Bio-Rad assay), PGE2and LTB4(RIA), and nitrites were determined in aqueous humor. Leukocyte infiltration in iris/ciliary body was estimated by myeloperoxidase (MPO) activity. NDGA or L-NAME alone did not reduce the severity of EIU when compared to the control group. NDGA reduced MPO in iris/ciliary body correlating with a reduction in LTB4levels, but did not change protein, nitrite and PGE2levels. L-NAME produced a slight decrease in MPO activity and LTB4levels, and reduced protein, nitrite, and PGE2levels. The combination NDGA+L-NAME significantly reduced the severity of uveitis as well as all inflammatory parameters and mediators. We conclude that an additive effect between NO and LO-metabolites plays a role in the development of EIU, and we speculate that interactions between NO and oxygen free radicals could modulate the inflammatory response.

The variations of the spectral irradial of sunlight and the increasing use of artificial sources of light that emits UV-radiation, have increased the incidence of nocive effects of UV-radiation. Preliminary studies demonstrated the photochemical damage to ocular tissues caused by solar radiation and other UV sources. Since ocular tissues do not develop tolerance to UV exposure, excessive exposure of the ocular surface might be nocive to the eye. Corneal epithelium is the external layer of the eye directly exposed to the UV radiation of the sun. To determine the effect of UV radiation on corneal epithelium, rabbit corneal epithelial cells in culture were irradiated during 6 days at 370 or 525 nm with different intensities and the role of an absorption filter was also investigated.

The involvement of nitric oxide (NO) and prostaglandin E2 (PGE2) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed respectively by nitrite and PGE2 levels in aqueous humor. Treatment with inhibitors of NOS (NG-nitro-L-arginine methyl ester, L-NAME, 50 mg/kp i.p.) or COX (diclofenac, 30 micrograms, topically), alone or in combination, were compared to a saline-treated group. Diclofenac or L-NAME alone reduced or delayed the intensity of uveitis, and partially decreased the protein concentration in aqueous humor; diclofenac, but not L-NAME, partially reduced the polymorphonuclear leukocyte infiltration in the iris ciliary body as indicated by the MPO activity. Treatment with both inhibitors in combination diminished the clinical uveitis, the disruption of the blood-aqueous barrier and the MPO activity in the iris-ciliary body. We conclude that NO and PGE2 have additive effects in endotoxin-induced uveitis in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis.

The effect of Ca2+ channel-acting drugs on bovine adrenal mitochondria Ca2+ movements was investigated. Mitochondrial Ca2+ uptake is performed by an energy-driven Ca2+ uniporter with a Km of 20.9 ± 3.2 μM and V max of 148.1 ± 7.2 nmol 45 Ca 2+ min −1 mg −1 · Ca 2+ release is performed through an Na+/Ca2+ antiporter with a Km for Na+ of 4.2 ± 0.5 mM, a Vmax of7.5 ± 0.4 nmol45Ca2+ min−1 mg−1, and a Hill coefficient of 1.4 ± 0.2· Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger was inhibited by several dihydropy-ridines (nitrendipine, felodipine, nimodipine, (+)isradipine) and by the benzothiazepine diltiazem with similar potencies. In contrast, neither CGP 28392, Bay-K-8644, amlodipine, nor verapamil had any effect on Ca2+ efflux. Nitrendipine at 20 μM modified neither the Km nor the Hill coefficient for Na+, whereas the Vmax was reduced to 2.9 nmol 45Ca2+ min−1 mg−1, thus demonstrating noncompetitive modulation of the Na+/Ca2+ exchanger. None of the Ca2+ channel-acting drugs assayed at 100 μM affected Ca2+ influx through the uniporter. Ca2+ channel blockers inhibited the Na+/Ca2+ antiporter and displaced the specific binding of [3H]nitrendipine to intact mitochondria with Ki values similar to the IC50s obtained for the inhibition of the Ca2+ efflux. Ca2+ channel-acting drugs that did not inhibit the Na+/Ca2+ exchanger (amlodipine, CGP 28392, Bay-K-9644, and verapamil, at concentrations of 100 μM or higher) had no effect on [3H]nitrendipine binding. These results suggest that the adrenomedullary mitochondrial dihydropyridine receptor is associated with the Na+/Ca2+ exchanger.

1. 1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/ protein ratio of 2.5, during 45 min at 4°C. 2. 2. From the rate constants of association (8.10 ± 0.25 × 10 4 M −1 min −1 ) and dissociation (0.022 ± 0.001 min −1 a K d of 275 nM was calculated, while from saturation experiments a K d of 270 ± 30 nM and a density of receptors of 106 ± 9 pmol/mg protein was obtained. 3. 4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds. 4. 5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [ 3 H]Azidopine.

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of different selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60-80%) correspond to M2 muscarinic subtypes, and the rest is constituted by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.

The myosin-light-chain (MLC) phosphorylation accompanying catecholamine release in chromaffin cells was investigated with the objective of assessing the possible role of this contractile protein in catecholamine secretion. The electrophoretic characteristics of adrenomedullary MLC were determined by immunochemical techniques using two different specific antibodies. The identified 22 kDa phosphoprotein was mainly present in the cytosol, as demonstrated by ultracentrifugation and immunocytochemical analysis. A part of this protein was located on, or close to, the plasma membrane. Cell stimulation by secretagogues resulted in a Ca2(+)-dependent 32P incorporation into MLC, the time course of this process being related to catecholamine release. These findings were supported by a two-dimensional gel-electrophoretic analysis by which means this protein was resolved into two acidic forms. A role for Ca2(+)-calmodulin and Ca2(+)-phospholipid kinases in adrenomedullary MLC phosphorylation is reported. The results obtained suggest a regulatory role for such a protein in the underlying exocytotic event., This work was supported by grants from the Comision Asesora para la Investigacion Cientifica y Tecnica (CAICYT 976/84; CICYT PB87-93) and the U.S.-Spanish Joint Committee (CCA 8411029). L. M.G. is a Fellow of the Fondo de Inversiones Sanitarias de la Seguridad Social.