The presence of these inhibitors had no effect on DETA-NO-triggered neuronal apoptosis (Fig

The presence of these inhibitors had no effect on DETA-NO-triggered neuronal apoptosis (Fig. that glycolytically generated ATP is required to maintain the m (9). This obtaining may explain why treatment of astrocytes with lipopolysaccharide and IFN-, which induces, among other things, the generation of NO, inhibited cytochrome oxidase activity (10), and stimulated the rate of glycolysis, whereas no indicators of cell death were detected (11). Thus, the different susceptibility of cells to NO-mediated apoptosis PP58 may be a function of the ability of the cells to increase their glycolytic activity after inhibition of mitochondrial respiration by NO. We have now carried out further studies in neurons in which inhibition of mitochondrial respiration is known to be accompanied by mitochondrial depolarization (12C14) and have compared their responses to NO with those of astrocytes. We have found that NO-mediated inhibition of cellular respiration is followed by mitochondrial depolarization and cell death in neurons but is usually followed by hyperpolarization in astrocytes. Furthermore, we show that an increase in m at the expense of glycolytically generated ATP prevents apoptotic death in astrocytes. Materials and Methods Reagents. DMEM, poly(d-lysine), horse serum, cytosine arabinoside, carbonyl cyanide 0.05 was considered significant. Results Inhibition of Cellular Respiration by NO Stimulates Glycolysis in Astrocytes but Not in Neurons. Untreated control astrocytes and neurons were found to consume O2 at a similar rate (Fig. ?(Fig.1).1). This obtaining is in agreement with previous results obtained in intact cells or isolated mitochondria (16, 21). Incubation of both of these cell types with the NO donor DETA-NO inhibited, in a dose- and time-dependent manner, the rate of O2 consumption at O2 concentrations ranging between 175 and 200 M. Rabbit polyclonal to IPO13 In both cell types, the concentration of DETA-NO that inhibited respiration by 85% was 0.5 mM, which corresponded to a continuous release of NO to maintain a concentration of 1 1.4 M NO. Open in a separate window Physique 1 Inhibition of cellular respiration by NO stimulates glycolysis in astrocytes but not in neurons. Cell suspensions (2 PP58 106 cells per ml) were incubated at 37C in buffered Hanks’ answer either in the absence (control) or presence of DETA-NO for the indicated occasions. Oxygen consumption experiments were performed at an initial O2 concentration of 200 M. For ATP and lactate concentrations, aliquots of the cell suspensions were lysed in HClO4, neutralized with KHCO3, and utilized for metabolite determinations in the supernatants as explained in 0.05 versus appropriate control values. NO-Dependent Glycolytic Activation Determines Mitochondrial Membrane Potential. Astrocytes, but not neurons, contain a considerable amount of glycogen (19), the catabolism of which might provide sufficient glucose-1-phosphate for further glycolytic metabolism in these cells. Cells were therefore preincubated for 45 min in glucose-free buffered Hanks’ PP58 answer, after which glycogen was measured and its content in astrocytes was found to be depleted (in nmols of glucosyl residues per 2 106 cells, 45.0 1.0 at = 0, and 2.0 0.1 at = 45 min). Glucose deprivation was found to enhance further the NO-mediated decrease in astrocytic ATP concentrations, reaching values much like those found in DETA-NO-treated neurons (Fig. ?(Fig.3).3). Moreover, glucose deprivation prevented the NO-mediated increase in lactate concentrations in astrocytes; indeed, such treatment caused a reduction in lactate concentrations in astrocytes to values much like those found in the neurons (Fig. ?(Fig.3).3). Glucose deprivation prevented NO-mediated hyperpolarization in astrocytes and instead caused depolarization in these cells (Fig. ?(Fig.3).3). In contrast, incubation in the absence of glucose experienced no effect on NO-dependent fall in ATP concentration, lactate production, or mitochondrial depolarization in the neurons (Fig. ?(Fig.3). 3). Finally, glucose-depleted cells were incubated in the presence of.

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