Energy metabolism in human retinal Müller cells.

PURPOSE: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells. METHODS: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase. RESULTS: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. CONCLUSIONS: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.

Oxidative damage and age-related macular degeneration.

This article provides current information on the potential role of oxidation in relation to age-related macular degeneration (AMD). The emphasis is placed on the generation of oxidants and free radicals and the protective effects of antioxidants in the outer retina, with specific emphasis on the photoreceptor cells, the retinal pigment epithelium and the choriocapillaris. The starting points include a discussion and a definition of what radicals are, their endogenous sources, how they react, and what damage they may cause. The photoreceptor/pigment epithelium complex is exposed to sunlight, is bathed in a near-arterial level of oxygen, and membranes in this complex contain high concentrations of polyunsaturated fatty acids, all considered to be potential factors leading to oxidative damage. Actions of antioxidants such as glutathione, vitamin C, superoxide dismutase, catalase, vitamin E and the carotenoids are discussed in terms of their mechanisms of preventing oxidative damage. The phototoxicity of lipofuscin, a group of complex autofluorescent lipid/protein aggregates that accumulate in the retinal pigment epithelium, is described and evidence is presented suggesting that intracellular lipofuscin is toxic to these cells, thus supporting a role for lipofuscin in aging and AMD. The theory that AMD is primarily due to a photosensitizing injury to the choriocapillaris is evaluated. Results are presented showing that when protoporphyric mice are exposed to blue light there is an induction in the synthesis of Type IV collagen synthesis by the choriocapillary endothelium, which leads to a thickened Bruch's membrane and to the appearance of sub-retinal pigment epithelial fibrillogranular deposits, which are similar to basal laminar deposits. The hypothesis that AMD may result from oxidative injury to the retinal pigment epithelium is further evaluated in experiments designed to test the protective effects of glutathione in preventing damage to cultured human pigment epithelial cells exposed to an oxidant. Experiments designed to increase the concentration of glutathione in pigment epithelial cells using dimethylfumarate, a monofunctional inducer, are described in relation to the ability of these cells to survive an oxidative challenge. While all these models provide undisputed evidence of oxidative damage to the retinal pigment epithelium and the choriocapillaris that is both light- and oxygen-dependent, it nevertheless is still unclear at this time what the precise linkage is between oxidation-induced events and the onset and progression of AMD.

Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina.

The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Müller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1-10 mM) was added to superfusion media to inhibit glutamine synthetase, a Müller cell specific enzyme, by more than 99% within 5-10 min, thereby disrupting the conversion of glutamate to glutamine in the Müller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1-2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2-5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine-treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Müller cells and its subsequent conversion to glutamine.

Regulation of corneal endothelial barrier function by adenosine, cyclic AMP, and protein kinases.

PURPOSE: To determine which processes or factors that regulate corneal hydration are responsible for the hydration-modulating effects of adenosine. Influx of fluid to the stroma and efflux to the aqueous humor are governed, respectively, by the imbibition pressure of the stromal matrix and the transendothelial ionic gradients determined by the permeability and active transport characteristics of this monolayer. The focus of this study was to assess the effects of adenosine on these endothelial parameters. METHODS: Isolated corneas freshly dissected from rabbit eyes were used throughout. Active ion transport was assessed by measurement of 86Rb+ uptake by the endothelial cells of intact corneas incubated for 30 minutes in 25 mM HCO3(-)-Ringer with agents promoting corneal deturgescence or corneal swelling. Intracellular and extracellular fluid in the scraped endothelial cell mass was estimated from simultaneous counts of 3H-mannitol and 14C-urea, allowing calculation of tissue-to-medium (T-M) ratios of 86Rb+ in cell water. Permeability of the endothelium was determined by measuring the efflux into the superfusate of 5-carboxyfluorescein (CF) applied to the stroma of deepithelialized corneas superfused at the endothelial surface with the same media described for 86Rb+ uptake. Thickness of these corneas and of others fixed for scanning electron microscopy was monitored with a specular microscope. RESULTS: In the control medium, 25 mM HCO3(-)-Ringer, 86Rb+ was accumulated to yield a T-M ratio of 6.21. Neither adenosine nor other agents that increase cyclic adenosine monophosphate (cAMP)--that is, forskolin and dibutyryl cAMP--changed this value to a significant extent. Bumetanide had no effect, but ouabain caused a decrease in T-M to 1.30, a 79% inhibition. Elimination of Na+ or HCO3- also caused marked decreases in uptake. Permeability to CF in control medium was 3.40 x 10(-4) cm/min. A decrease of more than 20% (P < 0.05) was seen in the presence of adenosine and cAMP promoters and also with the protein kinase inhibitor H-8, whereas phorbol myristate acetate caused an increase to 4.50 x 10(-4) cm/min (P < 0.01). Ouabain caused no change, but blocked the effects of adenosine. Reducing the Ca2+ concentration of the superfusing medium caused time-dependent increases in permeability to 4.57 at 15 to 45 minutes and 12.5 at 80 to 110 minutes. At the earlier time, this increase in permeability could be prevented by the addition of adenosine or H-8. Elimination of Na+ or HCO3- ions from the medium caused a small decrease in permeability and, like ouabain, blocked the effect of adenosine. Changes in thickness of corneas were consistent, in most cases, with the observed alterations in 86Rb+ uptake or permeability to CF. Scanning electron microscopy showed contraction and rounding of endothelial cells in low Ca2+ medium, with stretching of intercellular borders, features that were largely eliminated when adenosine was also present. CONCLUSIONS: Adenosine, through increasing cAMP, decreases permeability of the corneal endothelium. This effect, rather than a change in the active transport (fluid pump) mechanism, is responsible for the promotion of deturgescence and maintenance of lower steady state thickness of corneas exposed to adenosine. The mechanism may involve the phosphorylation state of cytoskeletal proteins and seems to be dependent on an undisturbed environment of monovalent ions.

Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na(+)-K(+)-2Cl- cotransport.

The role of Na(+)-K(+)-2Cl- cotransport in ion and fluid transport of the corneal endothelium was examined by measuring changes in corneal hydration and uptake of 86Rb by the endothelial cell layer. Isolated, intact rabbit corneas maintain normal hydration when they are superfused at the endothelial surface with bicarbonate (HCO3-)-Ringer solutions as a result of equilibrium between active ion and fluid transport out of the stromal tissue and leak of fluid into stromal tissue from the aqueous humor. Furosemide and bumetanide did not alter this equilibrium when they were added to the superfusion medium. Uptake of 86Rb by the endothelium of the incubated cornea was increased 25% by bumetanide, but uptake in the presence of ouabain (70% less than that of controls) was not changed by bumetanide. In Na(+)-free medium, uptake of 86Rb was reduced by 58%, but it was unchanged in Cl(-)-free medium. Calyculin A, a protein phosphatase inhibitor and activator of Na(+)-K(+)-Cl- cotransport, was without effect on 86Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes was significantly different when bumetanide was present in the media. It is concluded that Na(+)-K(+)-2Cl- cotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport of this cell layer. Its presence in cultured endothelial cells may reflect the reported importance of this protein in growth, proliferation, and differentiation.

An assessment of rat photoreceptor sensitivity to mitochondrial blockade.

PURPOSE: To report results of functional, biochemical and structural studies of photoreceptor mitochondria in isolated rat retinas under conditions of mitochondrial inhibition. METHODS: Dark-adapted rat retinas were incubated in a modified Ringer's bicarbonate medium under aerobic and anaerobic conditions. Several different procedures were used to inhibit mitochondrial function; N2, 0.01 mM antimycin A, and 1 and 10 mM potassium cyanide (KCN). Measurements were made of lactic acid production, retinal adenosine triphosphate (ATP) content, and receptor potentials. Morphology of the inner segment mitochondria was examined by electron microscopy. RESULTS: In the presence of N2, 0.01 mM antimycin, or 1 mM KCN, lactic acid production was linear throughout the 60- minute period; and the rate was similar for each condition. Retinal ATP content and the amplitude of the receptor potential were also maintained at high levels after short-term incubations with either N2, antimycin A, or 1 mM KCN. In contrast, use of 10 mM KCN produced an entirely different set of results. These effects were studied both at the alkaline pH (8.9) found when this concentration of KCN was simply added to bicarbonate-buffered media and at the normal pH (after readjustment) of 7.4. With 10 mM KCN (pH 8.9), retinal lactate production was severely depressed, retinal ATP content was nearly depleted within 5 to 10 minutes, and the amplitude of the receptor potential rapidly declined to a low level. The deleterious effects of 10 mM KCN on these parameters were lessened to varying degrees when pH was readjusted to 7.4. Electron microscopic observations of rat rod inner segments indicated generally excellent survival of these organelles after incubation with either N2, antimycin A, or 1 mM KCN in comparison with their appearance under oxygenated conditions. However, the inner segments were significantly disrupted after incubation of retinas with 10 mM KCN. CONCLUSIONS: Findings suggest that the loss of the receptor potential and depletion of ATP observed with minutes after exposing isolated rat retinas to media containing 10 mM KCN results from the inhibition of both respiration and glycolysis by this high concentration of KCN. In contrast, when conditions are chosen so that only respiration is impaired (as with N2, antimycin A, or 1 mM KCN) photoreceptor cells are resistant to short-term episodes of mitochondrial inhibition, principally because the upregulation of glycolysis generates sufficient ATP to compensate reasonably well for the loss in mitochondrially produced ATP.

In vitro metabolic competence of the frog retina: effects of glucose and oxygen deprivation.

The metabolic competence and histological integrity of the frog retina in vitro were evaluated as a function of the presence/absence of exogenous glucose and of oxygen tension. Dark- and light-adapted frog neural retinas were incubated for 1-8 hr at 23 degrees C in a modified Ringer's-bicarbonate medium under aerobic and anaerobic conditions, in the presence and absence of 10 mM glucose. Control retinas (+glucose, aerobic conditions) maintained ATP levels comparable to those of freshly excised tissue (ave. 17 nmol mg protein-1), produced minimal lactate (ave. 0.12 mumol mg protein-1 hr-1), and exhibited normal histology. In the absence of any exogenous carbon source, retinas incubated aerobically maintained ATP levels, produced lactate, incorporated [3H]acetate into nonsaponifiable lipids, and exhibited histology comparable to controls. In the presence of 1 mM iodoacetate, aerobic ATP levels declined markedly, with or without exogenous glucose. Under anaerobic conditions with glucose present, lactate production increased ca. 8.5-fold, while ATP levels were maintained at control levels, demonstrating a marked Pasteur effect; under these conditions, retinas exhibited only moderate histopathological changes. However, in the absence of both glucose and oxygen, ATP levels declined precipitously, with concomitant massive cytological deterioration. No major differences in the biochemical measurements or histological appearance were observed as a function of light adaptation. These results demonstrate the remarkable resilience of the frog retina to anoxia and hypoglycemic stress. Aerobically, with or without exogenous glucose, ATP production and de novo lipid synthesis are maintained, apparently by recruitment of an endogenous carbohydrate substrate (e.g., glycogen).

Glucose dependence of glycolysis, hexose monophosphate shunt activity, energy status, and the polyol pathway in retinas isolated from normal (nondiabetic) rats.

PURPOSE: To measure glucose-dependent metabolic activities and selected parameters of the polyol pathway in retinas isolated from normal rats to test the hypothesis recently proposed by Van den Enden et al that incubation of whole retinas for 2 hours with elevated concentrations of glucose results in activation of the polyol pathway, which is the cause of a redox imbalance, as measured by an increase in the retinal cytosolic lactate-pyruvate ratio and a diabetic-like state. METHODS: Retinas obtained from nondiabetic rats and separated from other ocular tissues were incubated for several hours in incubation medium containing glucose at concentrations ranging from 5 to 30 mM. Measurements were made under aerobic and anaerobic conditions of lactic acid production, retinal adenosine triphosphate (ATP), lactic acid content, the hexose monophosphate shunt pathway, aldose reductase activity, and levels of sorbitol and galactitol. Morphology was examined by light microscopy at the end of the incubations. RESULTS: Incubation of isolated rat retinas with 20 mM glucose increased lactic acid production by approximately 25% in comparison to the rate observed in 5mM glucose under aerobic and anaerobic conditions. The content of ATP and lactate in the retinas after a 2-hour incubation in the presence of oxygen and 20 mM glucose was equal to the amounts found in fresh tissues, whereas these metabolites declined, respectively, by 25% and 45% when 5 mM glucose was used. The activity of the hexose monophosphate shunt pathway in isolated rat retinas was not increased increased significantly when the concentration of glucose was raised from 5 to 30 mM. Aldose reductase activity and polyols were below our limits of detection, 0.5 nmol/minute.mg protein and 3.5 nmol/retina, respectively, under all conditions tested. The morphologic appearance of the retina was similar in the presence of normal and high concentrations of glucose. CONCLUSIONS: These results show that incubation of isolated rat retinas, obtained from nondiabetic rats, with elevated concentrations of glucose for 2 hours leads to increases in glycolysis and a higher tissue content of lactic acid and ATP in comparison to values obtained with 5 mM glucose. However, the magnitude of the glucose-dependent increase in the retinal level of lactate in the current study and in that of Van den Enden et al is six to seven times greater than the calculated flux of glucose through the polyol pathway. These results, therefore, do not support the hypothesis of Van den Enden et al. Rather, it is suggested that supranormal concentrations of glucose yield more lactate and ATP in a whole retina because they optimize the supply of this essential nutrient to cells throughout the tissue by overcoming diffusional limitations that result when the retina is separated from its normal choroidal and intraretinal blood supplies.

Adenosine promotes regulation of corneal hydration through cyclic adenosine monophosphate.

PURPOSE: To investigate the cellular mechanisms whereby adenosine increases net transendothelial fluid transport by the endothelial cells of the cornea. METHODS: Rabbit corneas were isolated and the endothelial surface was superfused while thickness was measured with the specular microscope. Cyclic adenosine monophosphate (cAMP) was measured in endothelia from fresh and incubated corneas, and adenylyl cyclase and phosphodiesterase activities were measured in homogenates or the particulate fraction of endothelia from bovine or rabbit. Adenosine, adenosine-receptor agonists, dibutyryl cAMP, forskolin, and phosphodiesterase inhibitors were used to modulate physiological and biochemical parameters. RESULTS: Adenosine, N-ethyl(carboxamido)adenosine, dibutyryl cAMP, forskolin, and phosphodiesterase inhibitors all promoted deturgescence of swollen corneas and maintained fresh corneas at lower steady state thicknesses than in controls. These effects were abolished in the presence of ouabain or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or after complete removal of HCO3- from the media. Intracellular cAMP was significantly increased by forskolin and phosphodiesterase inhibitors and, to a lesser extent, by agonists. Increases in cAMP concentration declined rapidly with time. Cyclase activity in the bovine tissue was enhanced by agonists and by G-protein activators. Dose-response curves of corneal swelling indicated a greater sensitivity to N-ethyl(carboxamido)adenosine than to the A2 alpha specific agonist CGS 21680. CONCLUSIONS: Adenosine increases net endothelial fluid transport through an increase in cAMP. The effects are mediated by stimulation of adenylyl cyclase through a G-protein coupled to an adenosine receptor, which is most probably of the A2 beta subtype. Results suggest that the regulation of corneal hydration by adenosine is more probably through stimulation of active transport than through a change in permeability, involving either transmembrane fluxes of Na+ or HCO3- or another step tightly coupled to these primary events in fluid movement.

A protective role for glutathione-dependent reduction of dehydroascorbic acid in lens epithelium.

PURPOSE: In view of the antioxidant role of ascorbic acid and the glutathione redox cycle in the lens, the authors have studied the relationship of the cycle to reduction of the oxidized product of ascorbic acid, dehydroascorbic acid (DHA), in lens epithelium. METHODS: Cultured dog lens epithelial cells and intact rabbit lenses were exposed to various concentrations of DHA in experiments performed at 20 degrees C to minimize hydrolysis of the compound (t1/2 of 5 minutes at 37 degrees C). Levels of glutathione (GSH) and oxidized glutathione (GSSG) were measured in lens cells and whole lens epithelial by electrochemical detection. RESULTS: Treatment of lens cells with 1 mM DHA for 0.5 to 3 hours in the absence of glucose (glucose is required for the reduction of GSSG through the glutathione redox cycle) produced from 60% to complete oxidation of GSH (controls contained negligible GSSG) and distinct morphologic changes (cell contraction and blebbing), as shown by scanning electron microscopy. Glucose prevented these effects and allowed nearly immediate recovery of GSH after DHA exposure in the absence of glucose. A dose-dependent response was observed for the formation of GSSG in cultured cells from 0.05 to 0.5 mM DHA in the absence of glucose. The results of experiments performed with DHA plus an inhibitor of glutathione reductase mimicked those obtained using DHA minus glucose. DHA produced a 3- to 10-fold stimulation of hexose monophosphate shunt activity in cultured lens cells and whole lenses, which was prevented by the inhibition of glutathione reductase. Treatment of whole lenses with DHA minus glucose also produced oxidation of epithelial GSH and was accompanied by the loss of lens transparency. No evidence was found for dehydroascorbate reductase activity in the lens epithelium. CONCLUSIONS: The exposure of lenses and lens epithelial cells to DHA under conditions in which the glutathione redox cycle was compromised resulted in the disappearance of GSH in the tissues and the appearance of GSSG. The reduction of DHA was shown to be linked to the glutathione redox cycle by a nonenzymatic interaction between GSH and DHA. Reduction of DHA in the lens is important because of the potential toxicity of this oxidant and/or its degradation products.

The roles of bicarbonate and CO2 in transendothelial fluid movement and control of corneal thickness.

PURPOSE: To determine whether maintenance of corneal hydration is dependent on bicarbonate ions and whether these ions can be derived from metabolic or exogenous CO2, and to investigate the relationship of transendothelial fluid movement to control of hydration. METHODS: The thickness of intact or deepithelialized rabbit corneas was measured while superfused on the endothelial surface with either 33 mM HGO3-/5% CO2 buffered media or 10 mM HPO4- buffered media in the presence and absence of inhibitors of ion transport and respiration. The corneal surface was covered with either silicone oil ("normal" corneas) or with the same media used for superfusion ("swollen" corneas). ATP and Na+,K(+)-ATPase activity were measured in endothelia scraped from the tissues after superfusion. RESULTS: Intact and deepithelialized corneas covered with oil swelled at a negligible rate (4 to 8 microns/hour) in 33 mM HCO3- medium but at 45 to 60 microns/hour in HPO4- medium. Antimycin A altered neither of these swelling rates, but ethoxzolamide (0.1 mM) caused swelling in HCO3-/CO2 (approximately 12 microns/hour above controls) with no change of rate in HPO4-. Ouabain (0.1 mM) increased swelling to 45 to 50 microns/hour in HCO3-/CO2 but had no effect in HPO4-. Saturating the oil on deepithelialized corneas with 5% CO2, or putting HCO3-/CO2 medium on the epithelial surface of intact corneas, did not alter the swelling rates seen with HPO4- superfusion. The equilibrium thickness of deepithelialized corneas swollen with HCO3-/CO2 on both surfaces was 35 microns less than that of corneas swollen in HPO4-. The difference was abolished by ouabain, which caused corneas in HCO3-/CO2 to swell an additional 30 microns but did not alter the equilibrium thickness of corneas swollen in HPO4-. Ethoxzolamide and DIDS (0.2 mM) increased the thickness in HCO3-/CO2 but not in HPO4-. Na+,K(+)-ATPase activities of endothelia were similar after HCO3-/CO2 and HPO4- superfusions, but the concentration of ATP in the HPO4(-)-superfused tissues was increased 35%. CONCLUSIONS: Normal corneal thickness can be maintained in vitro only in media that contain HCO3- at concentrations of more than 20 mM. Neither metabolic CO2 nor CO2 present in air-equilibrated, nominally HCO3(-)-free media can supply this requirement for HCO3-, even though these sources support the presumably related processes of transendothelial fluid movement and intracellular pH regulation.

The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective.

This article provides a comprehensive analysis of the redox reaction between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid. It includes an historical perspective of the progression of the experiments, first begun more than 60 years ago and continuing today with heightened importance. Indeed, the antioxidant capacity of glutathione and ascorbic acid, whether singly or in combination, linked via the redox couple, is a subject of intense interest for studies by bench scientists and clinicians, particularly because a growing body of evidence suggests that free radicals may be involved in a variety of diseases. The authors begin with a detailed summary of "test tube" experiments (the chemical perspective) that have revealed the conditions that regulate the rate of the redox coupling between glutathione and dehydroascorbic acid and that promote or inhibit the decomposition of dehydroascorbic acid in ordinary, buffered aqueous media; results obtained in the authors' laboratory are used for illustration purposes and uniformity of presentation. The authors then proceed to a critical examination of the extent to which the redox couple between glutathione and ascorbic acid operates in a cell, using the often published antioxidant cascade (See Fig. 1) as the model for the analysis (the physiological perspective). The evidence for and the evidence against the presence of the enzyme dehydroascorbate reductase in animal cells is outlined in a balanced way in an attempt to make sense of this continuing controversy. Next, the authors carefully document the many studies showing that exogenous dehydroascorbic acid is transported into cells where it is reduced to ascorbic acid by glutathione. Finally, they probe the functional significance and efficiency of the redox couple in monolayer cultures of human retinal pigment epithelial (RPE) cells, as a prototypical cellular model. The authors include the results of new experiments showing that incubation of RPE cells with a nitroxide, TEMPOL, leads to the selective oxidation of intracellular ascorbic acid. This approach is desirable because it dissects the cascade at a specific site and permits measurements of the levels of ascorbic acid and glutathione in the cells before, during, and after oxidation. The results show that only partial regeneration of ascorbic acid is obtained when control conditions are restored. However, if either ascorbic acid or dehydroascorbic acid is added to the media during the recovery period following treatment of cells with TEMPOL, then full recovery of ascorbic acid is observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium.

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.

Pharmacologic induction of posterior vitreous detachment in the rabbit.

OBJECTIVE: To assay the proteolytic activity of plasmin on the vitreoretinal junction and to assess a potential facilitating effect on posterior vitreous detachment. METHODS: We injected 1 U of plasmin into the vitreous of rabbits. Some eyes underwent vitrectomy after plasmin injection. Electroretinography and electron microscopy were performed. RESULTS: In plasmin-treated eyes, electroretinography displayed a transient (3 days) decreased b-wave amplitude. Histologic examination demonstrated posterior vitreous detachment in eyes that received intravitreal plasmin followed by vitrectomy. CONCLUSION: Plasmin may prove to be a useful biochemical adjunct to mechanical vitrectomy.

Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid.

Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.

The pH of antibiotic vitreous infusion combinations: a potential cause of retinal toxicity.

Experiments were undertaken to measure the pH of solutions of commonly used intravitreal antibiotics in various irrigating solutions to determine whether a change in pH might be a factor in antibiotic-induced retinal toxicity. Such retinal toxicity has been particularly damaging when solutions of gentamicin have been used. The pHs of the following solutions were measured: gentamicin, amikacin, methicillin, tobramycin, and vancomycin, combined with balanced salt solution (BSS) PLUS (bicarbonate buffer), BSS (citrate/acetate buffer), and lactated Ringer's solution (lactate buffer). Each of these antibiotics induced a concentration-dependent decrease in pH of the solutions; gentamicin, amikacin and tobramycin produced the largest shifts. The results also demonstrated that BSS PLUS acts as the strongest buffer and lactated Ringer's as the weakest. We conclude that it is important to determine the pH of intraocular antibiotic irrigating solutions, not just the pH of the antibiotics.

Chloride is required for fluid transport by the rabbit corneal endothelium.

The role of chloride in fluid transport of the rabbit corneal endothelium was examined by measuring changes in corneal thickness following ion substitutions or addition of ion transport inhibitors in media superfusing the isolated tissue. Normal fluid transport is indicated by maintenance of constant thickness in a fresh cornea or thinning (deturgescence) of a preswollen deepithelialized cornea to its initial thickness at approximately 40 microns/h. These patterns are seen when tissues are superfused with HCO(3-)-Ringer containing 114 mM Cl-. When Cl- was substituted with gluconate, glucuronate, or SO4(2-) fresh and preswollen corneas immediately thinned at greater than 150 microns/h to a value less than 300 microns and then began to swell at 30 microns/h to above their original thickness. Substitution of Cl- with NO3- or Br- had a negligible immediate thinning effect, but fresh corneas subsequently swelled and preswollen corneas failed to deturgesce fully. The rapid thinning (called a "downtransient") observed with gluconate, glucuronate, and SO4(2-) also occurred in these media when ion and fluid transport were completely inhibited with ouabain or stilbenes or by absence of HCO3-, indicating that the thinning results from osmotic gradients induced by ionic reflection coefficients different from that of Cl-. When the downstransient was avoided in deepithelialized corneas by preswelling with the same Cl(-)-free media on both sides of the cornea, corneas maintained a constant but swollen thickness in gluconate and in NO3- or Br- deturgesced slowly and incompletely; ouabain or stilbenes caused further swelling in all media. We conclude that absence of Cl- partially impairs fluid transport, most probably via its role in a Cl(-)-HCO3- exchanger which has been proposed in a recent model of endothelial fluid transport.

Effects of oxidized glutathione on ATPase activities in rat retina.

The activities of Mg(2+)-dependent and Na(+)-K(+)-stimulated ATPase in homogenates of rat retina were measured in the presence of increasing concentrations of oxidized glutathione (GSSG). The Mg(2+)-ATPase was not inhibited by GSSG at any of the concentrations tested. The Na(+)-K(+)-stimulated ATPase was not inhibited by 1 mM GSSG, but its activity was decreased by 20 and 35%, respectively, in the presence of 5 and 10 mM GSSG. Other enzymatic measurements using supernatant fractions of rat retina showed that 1-10 mM GSSG did not inhibit the activities of hexokinase, glucose-6-phosphate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase. These results suggest that GSSG is not likely to exert significant deleterious changes on cellular processes, at least in cells and tissues in which normal glutathione (GSH) concentration is 2 mM or lower.

Relative contributions of epithelial cells and fibers to rabbit lens ATP content and glycolysis.

The adenosine triphosphate (ATP) content was measured independently in separated capsule-epithelium and fibers from whole rabbit lenses, both fresh and after incubation under various combinations of glucose and oxygen deprivation. Lactate production was also measured during aerobic and anaerobic incubations of whole lenses and of monolayers of cultured epithelial cells. The fresh capsule-epithelium contained 3.3 nmoles ATP, whereas the decapsulated lens contained 410 nmoles ATP, a value that was indistinguishable from that of the whole, intact lens. In the presence of glucose, the fibers and epithelium each maintained their respective ATP content under aerobic and anaerobic conditions. In the absence of glucose, the ATP content in each fraction declined with time, but only in the epithelium was the rate of decline of ATP significantly faster in nitrogen than in oxygen. In whole lens, the rates of anaerobic and aerobic lactate production were similar, whereas in the cultured epithelial monolayers, the anaerobic rate was two-fold greater than in oxygen. From this it is concluded that approximately 50% of the ATP of the epithelial cells is derived from oxidative metabolism. A Pasteur response shown here for the first time with the cultured epithelium allows these cells to compensate for the loss of ATP production when mitochondrial oxidation is curtailed. The epithelium does not contribute to the ATP content of the lens fibers under aerobic or anaerobic conditions.

Strong Pasteur effect in rabbit corneal endothelium preserves fluid transport under anaerobic conditions.

1. The hydration and transparency of the mammalian cornea are maintained by a metabolically dependent fluid transport system located in the endothelial cell layer. The purpose of the study was to determine whether this pump activity is dependent upon aerobic or anaerobic metabolism. 2. The ability of the cornea, superfused in vitro with a bicarbonate-Ringer solution containing glucose and adenosine, to maintain normal hydration (thickness) when respiration is inhibited has been examined in intact and de-epithelialized preparations and correlated with glycolytic activity and cellular concentrations of ATP. 3. In respiring intact and de-epithelialized corneas thickness was maintained for periods up to 5 h during superfusion with the control Ringer solution. 4. KCN (10(-3) M) or antimycin A (10(-6) M) caused the intact cornea to swell at 15 +/- 3 microns h-1, whereas the de-epithelialized tissue maintained normal thickness under these conditions. This swelling of the intact tissue appears to be due to the osmotic effect of increased epithelial lactate production under anaerobic conditions. 5. Pre-swollen de-epithelialized corneas deturgesced fully to original thickness at a rate of 43 +/- 7 microns h-1 under aerobic conditions, but with KCN or antimycin they deturgesced at only 65% of that rate and generally failed to return to their original thickness. 6. Ouabain (10(-4) M) caused de-epithelialized corneas to swell in the presence of KCN or antimycin, as it did under aerobic conditions, showing that maintenance of hydration or deturgescence are pump-dependent processes under both conditions. 7. Lactate production was markedly stimulated by KCN or antimycin in intact and de-epithelialized preparations, but not in the stroma alone. The magnitude of the Pasteur effect was calculated to be 5-fold in the endothelium and 2.5-fold in the epithelium. Ouabain inhibited anaerobic lactate production in the endothelium by 50%. 8. ATP content of the epithelium following 5 h superfusion was 22.0 nmol cm-2 in control (aerobic) corneas, but fell to 1.9 nmol cm-2 in the presence of 10(-3) M-KCN, whereas the endothelial value fell only from 1.1 to 0.7 nmol cm-2 under these conditions. 9. Omission of glucose from the medium containing KCN or antimycin caused immediate swelling of tissues and a rapid decline of ATP content to less than 1% of that in control conditions.(ABSTRACT TRUNCATED AT 400 WORDS)