Barry S. Winkler

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.

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.

Multiple NADPH-producing pathways control glutathione (GSH) content in retina

Glutathione (GSH), together with NADPH-producing pathways and glutathione reductase, provides a defense system against oxidants. Oxidation of GSH causes stimulation of the hexose monophosphate shunt and increased production of NADPH. We have asked if hexose monophosphate shunt activity is required for the recovery of GSH following exposure of the isolated rat retina to an oxidant. Hexose monophosphate shunt activity was decreased by depleting the retina of hexose stores, before exposing the tissue to diamide (0.04-1.0mM), an oxidant for GSH, for 30 min. After exposure, retinas were transferred to either glucose-containing or glucose-free recovery medium for an additional 30 min. Control retinas kept in glucose-free, oxygenated medium (no diamide) for 90-120 min maintained GSH at 90% of the value found in retinas incubated with glucose. After exposure of hexose-depleted retinas to 0.4 mM diamide, a nearly 90% decrease in GSH was observed. When the oxidant was removed, the level of GSH returned to more than 80% of the control value in the presence or absence of glucose. In contrast, no recovery of GSH was observed after diamide treatment if the retinas were transferred to ice-cold (1-5 degrees C) media with or without glucose or if the retinas were pre-treated with 2 mM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. Measurements of two NADPH-producing cytosolic enzymes, namely NADP+-dependent malic enzyme and NADP+-dependent isocitrate dehydrogenase, revealed high activities. Optimum production of NADPH from malic enzyme was 0.90 nmol NADPH produced min-1 per retina, while with isocitrate dehydrogenase the average rate was 6.9 nmol NADPH produced min-1 per retina. We suggest that these enzymes together with a long-lived endogenous substrate (probably glutamate) are responsible for the recovery of GSH in hexose-depleted retinas. The present results suggest that more than one NADPH-producing system is capable of controlling the GSH concentration in retina. Studies that have focused on the hexose monophosphate shunt pathway as the sole source of NADPH for glutathione reductase in retina and other tissues may require re-evaluation depending on the overall metabolic capacity and substrate utilization of the particular tissue. Thus, the present findings are significant not only with respect to the retina but also for other tissues whose metabolic characteristics are similar to those found in the retina.

Cultured retinal neuronal cells and Müller cells both show net production of lactate

Glucose has long been considered the substrate for energy metabolism in the retina. Recently, an alternative hypothesis (metabolic coupling) suggested that mitochondria in retinal neurons utilize preferentially the lactate produced specifically by Müller cells, the principal glial cell in the retina. These two views of retinal metabolism were examined using confluent cultures of photoreceptor cells, Müller cells, ganglion cells, and retinal pigment epithelial cells incubated in modified Dulbeccos minimal essential medium containing glucose or glucose and lactate. The photoreceptor and ganglion cells represented neural elements, and the Müller and pigment epithelial cells represented non-neural cells. The purpose of the present experiments was two-fold: (1) to determine whether lactate is a metabolic product or substrate in retinal cells, and (2) to examine the evidence that supports the two views of retinal energy metabolism. Measurements were made of lactic acid production, cellular ATP levels, and cellular morphology over 4 h. Results showed that all cell types incubated with 5 mM glucose produced lactate aerobically and anaerobically at linear rates, the anaerobic rate being 2-3-fold higher (Pasteur effect). Cells incubated with both 5 mM glucose and 10 mM lactate produced lactate aerobically and anaerobically at rates similar to those found when cells were incubated with glucose alone. Anaerobic ATP content in the cells was maintained at greater than 50% of the control, aerobic value, and cellular morphology was well preserved under all conditions. The results show that the cultured retinal cells produce lactate, even in the presence of a high starting ambient concentration of lactate. Thus, the net direction of the lactic dehydrogenase reaction is toward lactate formation rather than lactate utilization. It is concluded that retinal cells use glucose, and not glial derived lactate, as their major substrate.

Glutathione oxidation in retina: Effects on biochemical and electrical activities

This study investigates the possible role of glutathione (GSH) in defending the retina against oxidative damage. Freshly excised rat retina was found to contain 1.2 mumol/g wet wt GSH and an undetectable level of oxidized glutathione (GSSG). Whole retinas were either incubated or superfused with various concentrations of the GSH-oxidant diamide in order to study the effects of oxidation of GSH on the activity of the hexose monophosphate shunt (HMS) and on the receptor potential of the retina. It was found that exposure of the retina to diamide produced a stimulation of HMS activity up to 26-times that of the control. Significant changes in GSH content and receptor potential were observed at concentrations of diamide that produced more than a 5.4-fold stimulation of HMS activity. The diamide-induced electrical alterations included an increase in latency and peak time of the receptor potential, a delay in the onset of the off response and an increase in the time required for the potential to return to the baseline. It was found that nearly 80% of GHS could be regenerated and that most of the electrical effects of diamide could be reversed by superfusion with normal medium. The results indicate that the retina possesses an active system for maintaining GSH in the reduced state and that this may be essential for the normal function of this tissue.

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.

Buffer dependence of retinal glycolysis and ERG potentials.

Using the isolated rat retina, the present experiments have examined the effects of varying the type and concentration of buffer system with and without changes in external pH on lactic acid production and ERG potentials. The buffers chosen for study included bicarbonate/CO2, HEPES, Tris+ and phosphate. High rates of retinal glycolysis were observed with either 25 mM bicarbonate and 5% CO2 (control condition) or with 30 mM quantities of the other buffers at an external pH of 7.4. Significantly lower rates of retinal glycolysis were obtained when the concentration of each buffer was reduced to 10, 5 or 3 mM without a change in external pH. Lactic acid production was also decreased when the external pH was lowered regardless of the buffer system employed. The a- and b-waves were well maintained in the presence of 25 mM bicarbonate and 5% CO2. Lowering the bicarbonate concentration at constant external pH led to a decrease in their amplitudes. With either Hepes, Tris+ or phosphate employed at concentrations between 3 and 30 mM the amplitude of the a-wave was reduced and the b-wave was lost. These results show that the nature and concentration of the buffer system used in in vitro retinal studies influences metabolic and electrical activities in this tissue and emphasize the importance of the buffer as a constituent of the incubation medium.

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 of86Rb by the endothelial cell layer. Isolated, intact rabbit corneas maintain normal hydration when they are superfused at the endothelial surface with bicarbonate ([Formula: see text])-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 of86Rb 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 on86Rb 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.

Nuclear magnetic resonance and biochemical measurements of glucose utilization in the cone-dominant ground squirrel retina.

PURPOSE To provide quantitative information on glucose utilization in cone-dominant ground squirrel retinas. METHODS Ground squirrel eyecups were incubated in medium containing (14)C-glucose, and the production of (14)CO(2) was measured. Measurements were also made of lactic acid production (glycolysis). Nuclear magnetic resonance (NMR) was used to track metabolites generated from (13)C-1 glucose. RESULTS Ground squirrel eyecups produced lactate at a high rate and exhibited normal histology. Light-adaptation reduced glycolysis by 20%. Ouabain decreased glycolysis by 25% and decreased (14)CO(2) production by 60%. Blockade of glutamate receptors had little effect on the glycolysis and (14)CO(2) produced. When metabolic responses were restricted to photoreceptors, light caused a 33% decrease in (14)CO(2) production. The rate of (14)CO(2) production was less than 10% of lactate production. Lactate was the major product formed from (13)C-glucose. Other (13)C-labeled compounds included glutamate, aspartate, glutamine, alanine, taurine, and GABA. Lactate was the only product detected in the medium bathing the ground squirrel retinas. The rod-dominant rat retina exhibited a similar pattern of metabolites formed from glucose. CONCLUSIONS Lactate, not CO(2), is the major product of glucose metabolism in both ground squirrel and rat retinas. Active Na(+) transport, however, depends more on ATP produced by mitochondria than by glycolysis. A relatively high fraction of ATP production from glycolysis and glucose oxidation continues in the absence of active Na(+) pumping and glutamatergic transmission. Major neurotransmitters are synthesized from the aerobic metabolism of glucose; anoxia-induced impairment in retinal synaptic transmission may be due to depletion of neurotransmitters.

Dependence of fast components of the electroretinogram of the isolated rat retina on the ionic environment.

Abstract Changes in the amplitudes of the a-wave , b-wave and wavelets of the electroretinogram of the isolated rat retina were observed as a function of alterations in thei onic composition of media bathing isolated rat retinas. All the components of the electroretinogram were reduced in low sodium solutions. An increase of the K + concentration from 5 to 20 mM increased the amplitude of the b-wave but reduced the amplitudes of the a-wave and wavelets. Above 30 mM K + the b-wave was also reduced. Low concentrations of Cl − abolished the b-wave and wavelets but did not alter the a-wave . The amplitudes of the a-wave and b-wave were increased while the wavelets were suppressed in Ca 2+ -free media. Implications of these ionic effects are discussed in relation to the cellular origins of these potentials.