An hypothesis to account for the renewal of outer segments in rod and cone photoreceptor cells: renewal as a surrogate antioxidant.

It is undisputed that glutathione (GSH) is an important cellular antioxidant. Although it is commonly believed that GSH is present in all retinal cells, several publications show that GSH is not immunologically detectable in outer segments of rod and cone photoreceptor cells, but is present in inner retinal cells and the pigment epithelium. Using these intriguing and surprising findings as a starting point, an hypothesis is proposed that the renewal of outer segments serves as a surrogate antioxidant for GSH and that the exceptional vulnerability of photoreceptor cells to certain toxic chemicals is linked to the deficiency in GSH in outer segments as a reductant, a detoxicant, and as an enzymatic cofactor. It is suggested that this deficiency of GSH is not damaging to outer segments under normal conditions, because renewal serves to replace any damaged molecules before they increase to detrimental levels. However, when photoreceptors are stressed, the renewal of outer segments alone is not capable of overcoming the higher rates of oxidizing and detrimental chemical reactions, and the health of the entire photoreceptor cell is at risk. The hypothesis is supported by a consideration of the essential role of the NADPH-dependent retinol reductase, by the different localization within photoreceptor cells of two key metabolic enzymes that are sensitive to oxidation, glyceraldehyde-3-phosphate dehydrogenase and the sodium-potassium ATPase, and by a consideration of the effects of toxic chemicals that selectively damage photoreceptor cells.

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.

Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina.

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.

Reexamining the hyperglycemic pseudohypoxia hypothesis of diabetic oculopathy.

PURPOSE: To test the hypothesis that diabetes alters retinal NAD+-to-NADH ratios early in the course of the disease (e.g., the hyperglycemic pseudohypoxia hypothesis). METHODS: In freshly excised age-matched control and diabetic rat retinas, measurements were made of the NAD+ and NADH content as well as a surrogate marker of NAD+-to-NADH ratios obtained from lactate and pyruvate levels. In addition, the effect of various hyperglycemic levels was assessed from measurements of retinal lactate and pyruvate concentrations and the rate of lactic acid production in vitro (isolated rat retinas, monolayer cultures of human retinal pigment epithelial cells, and rabbit lens epithelial cells). RESULTS: No significant differences (P>0.05) were found between control and diabetic tissues in their amount of total NAD+ and NADH/retina, and the ratio of NAD+ to NADH, or in their content of lactate, pyruvate, and adenosine triphosphate (ATP) or in the ratio of lactate to pyruvate. The content of lactate and pyruvate in retinas incubated for 2 hours in media containing 10 or 30 mM glucose was the same as found in fresh tissues, but the levels of these metabolites in retinas incubated in media containing 5 mM glucose declined in comparison to the fresh values. There were no significant differences in lactate content in cultured retinal and lens cells that were exposed to 5 or 30 mM glucose-containing media. DISCUSSION: The present results do not support the hyperglycemic pseudohypoxia hypothesis of diabetic retinopathy.

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 Dulbecco's 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.

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells.

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.

Metabolic mapping in mammalian retina: a biochemical and 3H-2-deoxyglucose autoradiographic study.

It has long been known that mammalian retinas metabolize glucose aerobically to lactic acid and carbon dioxide. The classical view holds that glucose is the primary substrate for energy metabolism in all retinal cells, and that photoreceptor cells have the highest rates of glycolysis and respiration. A different and more recent view is that the Müller cells are the principal, if not sole aerobic producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and other retinal neurons. In this paper, we have examined these two competing hypotheses in rat and guinea pig retinas by identifying the cellular sites of glucose uptake and phosphorylation via hexokinase by means of autoradiographic studies with 3H-2-deoxyglucose (3H-2DG). The rat retina serves as a vascular model and the guinea pig retina serves as an avascular model. Rat and guinea pig eyecups were incubated in oxygenated, bicarbonate-buffered media containing glucose in the presence of labeled and unlabeled 2DG. Biochemical measurements of lactate production and ATP content were made on rat retinas incubated with different concentrations of glucose and 2DG in order to establish the optimal condition for conducting the autoradiographic studies with 3H-2DG. The optimal substrate concentrations were 1mM glucose and 0.25 mM 2DG. Results showed that following incubation of dark-adapted rat eyecups for 1 hr in media containing 1mM glucose/0.25 mM 2DG and supplemented with 3H-2DG, the label was distributed throughout all the layers of the retina, from the ganglion cell layer to the retinal pigment epithelium, with denser label associated with the outer retina (photoreceptors) relative to the density of label in the inner retina, as evaluated by counts of silver grains in individual retinal layers. Exposure of rat eyecups to light did not alter the relative distribution of label, but did increase total grain counts by 70%. However, uptake of labeled 2DG, as measured by scintillation counting of radioactivity in trichloroacetic acid extracts, was not significantly different between light- and dark-adapted rat retinas. In guinea pig eyecups, labeled 2DG was distributed throughout all the retinal layers. Addition of 10mM lactate or pyruvate to the glucose/2DG media produced no measurable change in the density or distribution of label in the eyecups. Measurements of the activity of hexokinase in rat retinas revealed that this enzyme was present in both the mitochondrial and cytosolic fractions. The present results suggest that as long as the availability of ambient glucose is adequate, retinal neurons use glucose, rather than glial-derived lactate, as the major substrate for the production of high energy phosphates.

Hyperosmolarity and electroretinogram (ERG) potentials in isolated rat retinas: possible implications in diabetic models.

In this report, the effects of increases in the osmolarity of media superfusing isolated rat retinas on the a-wave, b-wave and oscillatory potentials of the electroretinogram (ERG) were examined. Osmolarity of the media was raised from 310 milliosmoles (control) to 340 and 370 milliosmoles by addition of NaCl or sucrose. Increases in osmolarity led to rapid decreases in the amplitudes of the b-wave and oscillatory potentials with little change in the amplitude of the a-wave. Excellent recovery of the ERG potentials was observed when control conditions were restored. The implications of these effects of an hyperosomotic load on ERG potentials in vitro are discussed with regard to a possible role of this load in models of experimental diabetes.

Modulation of the Pasteur effect in retinal cells: implications for understanding compensatory metabolic mechanisms.

The purpose of the present experiments was to enhance understanding of the factors that are critical for the survival of retinal cells exposed to mitochondrial inhibition. Confluent cultures of Müller cells (rMC-1) and human retinal pigment epithelial cells (hRPE) were incubated in Dulbecco's minimal essential medium in the presence and absence of 1x10(-5)M Antimycin A, an inhibitor of mitochondrial electron transport. To modulate the rates of aerobic and anaerobic glycolysis, cells were incubated in media containing varying concentrations of glucose and 1-100 micro M of iodoacetic acid (IAA), an inhibitor of glyceraldehdye-3-phosphate dehydrogenase (G3PDH). Measurements were made of G3PDH, lactic acid production, and cellular ATP levels, along with an examination of cellular morphology, the latter providing an index of cellular viability. Control rMC-1 and hRPE produced lactate aerobically, respectively, at 0.48 and 1.50 micro molhr(-1)/10(6) cells. Anaerobically, lactate production increased 2-fold in rMC-1 and 3-fold in hRPE. Anaerobic ATP levels in both types of cells were maintained at control levels over 8hr. Experimental conditions were sought that would modulate only the capacity of rMC-1 and hRPE to increase glycolysis following mitochondrial inhibition, i.e. alter their Pasteur effect. We used low concentrations of IAA to partially inhibit G3PDH. Incubation of rMC-1 with IAA for 6hr caused a graded inhibition of G3PDH: 70% inhibition with 1 micro M, 90% with 5 micro M, 97% with 10 micro M, and 100% with 100 micro M. While the aerobic and anaerobic rates of lactic acid production were not altered by 1 micro M IAA, both were suppressed completely by 100 micro M IAA. However, incubation of rMC-1 with 5 micro M IAA caused a decrease of 30% in the rate of anaerobic lactic acid production but no change in the rate of aerobic glycolysis. Moreover, with 5 micro M IAA, rMC-1 incubated aerobically maintained ATP levels, but anaerobic ATP content decreased to a low level and cell morphology and viability were compromised. Essentially similar results were observed with hRPE. Both rMC-1 and hRPE are remarkably resistant to mitochondrial inhibition. This resistance is linked directly to the magnitude of the increase in the Pasteur effect. When the capacity of rMC-1 and hRPE to generate a Pasteur effect is selectively curtailed, these cells no longer are resistant to mitochondrial inhibition. It is suggested that in an intact tissue the ability of a cell to withstand a metabolic challenge will depend very much on the adequacy of the supply of glucose. Even a small limitation in the availability of this utilizable substrate and in the rate of the compensatory increase in the rate of anaerobic glycolysis could put the cell at greater risk during the challenge.