No abstract

No abstract

The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK‐21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [Pi] ([ATP]i, [ADP]i, and [Pi]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [Pi], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose‐1,6‐bisphosphate] and [glyceraldehyde‐3‐phosphate] and lower [glucose‐6‐phosphate] and [fructose‐6‐phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [Pi] rose under all pHe conditions. Simultaneously [glucose‐6‐phosphate] and [fructose‐6‐phosphate] decreased whereas [fructose‐1,6‐bisphosphate] and [glyceraldehyde‐3‐phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4. Alterations in concentrations of cations other than H+ were small and unlikely to contribute to the regulation of glycolysis. It is concluded that (a) under intracellular conditions, in the presence of a high [ATP] and a low [ADP] and [AMP], a fall in [H+] powerfully inhibits phosphofructokinase; lactate production correlates with the levels of glycolytic intermediates. (b) A small decline in cellular energy state is sufficient to release phosphofructokinase inhibition by protons. (c) Once activated by the decrease in energy level, the steady‐state glycolytic rate is dependent on other factors, such as glycolytic intermediates and enzymes.

Treatment of rat brain synaptosomes with 10 microM monensin stimulated activity of the Na/K pump, which enhanced oxygen consumption and lactate production. Glycolytic flux was also increased independently of the pump activation by a fall in [H+]i. Under such conditions, glycolysis provided 26% of ATP for the ouabain-sensitive ATPase, a value substantially greater than the 4% obtained in veratridine-treated preparations (Erecińska and Dagani, 1990). In C6 glioma cells, a glia-derived line endowed with high rates of aerobic lactate synthesis, the cytosolic and mitochondrial ATP generation contributed 50% each for the support of the pump in the presence of 10 microM monensin. The fraction of energy utilized by the pump was greater in synaptosomes than in C6 cells. Enhancement of ion movements was accompanied by changes in the levels of high-energy phosphate compounds. Measurements with ion-sensitive microelectrodes in C6 cells and cultured neurons showed that monensin caused an increase in pHi by 0.4-0.5 unit and a parallel rise in [Na+]i. The increases in [Na+]i were about twofold in both types of cells, but the absolute values attained were much higher in neurons (40-50 mM) than in C6 cells (10-12 mM). Membrane potentials transiently declined by less than 10 mV and returned to their original values after 20 min of treatment. Rises in [Ca2+]i were small in neurons as well as in C6 cells. These changes could be explained by the known mechanism and/or consequences of monensin action. In contrast, in synaptosomes monensin caused an internal alkalinization of 0.1-0.15 pH unit, a large depolarization of the plasma membrane, and massive leakage of potassium into the external medium. The decrease in plasma membrane potential was accompanied by an increase in [Ca/+]i and release of the neurotransmitter amino acids GABA, aspartate, and glutamate. The depolarization and loss of K+ were unaffected by calcium withdrawal, replacement of chloride with gluconate, and addition of 1 mM 4-acetamido-4'-isothiocyanostilebene-2,2'-disulfonic acid (SITS), but was markedly attenuated by elimination of Na+. It is proposed that in synaptosomes monensin and/or the consequences of its action open a nonspecific cation channel that allows Na+ entry and K+ exit, with a consequent decrease in membrane potential.

Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i, [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 microM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+]i and [Na+]i. The increase in [Na+]i in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+]i depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.