Middle: The appearance of Mller cells after a 4-hour incubation with glucose and 1 10?5 M antimycin A

Middle: The appearance of Mller cells after a 4-hour incubation with glucose and 1 10?5 M antimycin A. in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Mller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, Bmp5 pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after AZD5582 mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. Conclusions These results suggest that, in the presence of glucose and oxygen, cultured Mller AZD5582 cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Mller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Mller cells are less susceptible than neurons to ischemia or hypoglycemia. The principal glial cell in the retina is the radially oriented Mller cell, which extends from the vitreal surface to 50% to 70% of retinal depth. Interest in the physiological properties of Mller cells began many years ago when Faber1 and Miller and Dowling2 first proposed that the b-wave of the electroretinogram (ERG) was generated by the Mller cells. This suggestion was based in part on findings in the central nervous system of the leech and the optic nerve of the frog and for 10 minutes. An aliquot of the supernatant was diluted 200-fold, and the ATP content was measured using a firefly luciferase-based spectrofluorometric assay (Turner Systems, Mountain View, CA). Protein was determined with a BCA assay kit (Pierce, Rockford, IL). Mitochondrial Glucose Oxidation Cells were grown in special 75-mm2 flasks, each containing an extra side arm capped with a rubber septum. The incubation medium was the same (e.g., serum free) as during the other biochemical experiments except for the addition of 5 mM 14C-3,4 glucose or 1 mM 14C-1 glutamate (specific activity was approximately 50,000 counts per minute/mole for each substrate). Five milliliters of medium was present in each flask. The incubator was equilibrated with 20% O2-5% CO2-75% N2. At the end of the incubations, which lasted from 1 to 4 hours, the reaction was stopped and the 14CO2 released by addition of 1 1 ml of 2 N H2SO4 through the rubber septum and the 14CO2 collected in 0.5 ml hyamine contained in a vial inserted into the culture flask. Radioactivity was determined in a liquid scintillation spectrometer. Appropriate blanks and background measurements were performed in each experiment. Enzyme Activities Measurements were made of selected enzymes of glycolysis and the hexose monophosphate shunt (hexokinase, glyceraldehyde-3-phosphate dehydrogenase ([G3PDH], glucose-6-phosphate dehydrogenase [G6PDH], and lactic acid dehydrogenase [LDH]) and other metabolic enzymes (malate dehydrogenase, aspartate aminotransaminase, glutamate dehydrogenase, and GS). The standard straightforward procedures found in Bergmeyer29 were used for the measurements of all these enzymes except GS. Typically, culture dishes were rinsed three times with saline, 0.6 ml of an appropriate buffer (e.g., 0.1 M NaPO4 or 0.1 M triethanolamine) was added, and cells were AZD5582 scraped and collected in the buffer. The suspension was sonicated and centrifuged at 20,000for 20 minutes. Aliquots of the supernatant were used for measurements of cytosolic enzyme activities using standard assay constituents and changes in OD340, reflecting an increase or decrease in the concentration of reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH), were monitored to obtain linear rates of reactions. Appropriate blanks (no substrate added) were monitored, and background rates were subtracted from the rates obtained with the substrate. The pellet was resuspended in buffer containing 0.2% Triton-X and was subsequently used for measurements of mitochondrial activities. GS activity was assayed by the method described by Thorndike and ReifCLehrer30 after sonication and centrifugation of cells in 1 ml of a buffer mixture containing several protease inhibitors (phenylmethylsulfonyl fluoride, pepstatin A, and leupeptin). Inclusion of these inhibitors was necessary to prevent loss in activity of GS during the preparative stages. Results Figure 2.