Glucose metabolism in freshly isolated Müller glial cells from a mammalian retina.

Abstract

Glucose metabolism was studied in isolated retinal Müller glial cells from the juvenile guinea pig. Cells, once enzymatically isolated and purified, were identified by morphological criteria, positive vimentin immunoreactivity, and histochemical staining for glycogen. Purified suspensions of Müller cells were obtained in quantities sufficient for biochemical analysis (approximately 2 x 10(5)/pair of retinas) and light microscopic autoradiography. In bicarbonate-buffered Ringer's medium containing 3H-2-deoxyglucose and no glucose, greater than or equal to 80% of the glucose analogue taken up intracellularly by Müller cells was phosphorylated to 3H-2-deoxyglucose-6-phosphate. In autoradiographs, this non-metabolized product provided visual evidence of glucose phosphorylation: the distribution of cell grains mirrored the morphology of individual Müller cells in situ. Exposure to the glycolytic inhibitor iodoacetate (500 microM) caused an 85% decrease in adenosine triphosphate (ATP) content; concomitantly, 3H-2-deoxyglucose-6-phosphate decreased by 90% and paralleled a dramatic decrease of cell labelling in autoradiographs, while levels of 3H-2-deoxyglucose did not change. In the continual absence of glucose, glycogen content decreased with time and this decrease was slowed by 36% in the presence of iodoacetate. This indicated that, in control conditions, glycosyl units from glycogen sustain cellular metabolism, and hence 3H-2-deoxyglucose phosphorylation. 3H-2-deoxyglucose-6-phosphate concentration was 43-fold less than that of ATP in the control conditions so that depletion of ATP during iodoacetic acid (IAA)-blocked glycolysis was not due to hexokinase activity. These results demonstrate that this preparation is adequate for quantitative studies of glucose metabolism at the cellular and molecular level in an important metabolic compartment of the mammalian retina.