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.
Winkler
Barry S
BS
Eye Research Institute, Oakland University, Rochester, Michigan 48309, USA. winkler@oakland.edu
Sauer
Michael W
MW
Starnes
Catherine A
CA
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002478:Cells, Cultured; D026603:D-Aspartic Acid / Q000493:pharmacokinetics / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000493:pharmacokinetics / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D019344:Lactic Acid / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology*; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D011922:Rats, Mutant Strains; D012160:Retina / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012964:Sodium / Q000378:metabolism
D004791:Enzyme Inhibitors; D007476:Ionophores; D019344:Lactic Acid; D018698:Glutamic Acid; D026603:D-Aspartic Acid; D010042:Ouabain; D000968:Antimycin A; D008985:Monensin; D012964:Sodium