Catherine A. Starnes

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.

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.

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 NH4+ 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 NH4+ 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 NH4+ 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.