Relations between intracellular ions and energy metabolism: a study with monensin in synaptosomes, neurons, and C6 glioma cells

Abstract

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.

Linkage of aerobic glycolysis to sodium-potassium transport in rat skeletal muscle. Implications for increased muscle lactate production in sepsis.

“…Monensin caused a change in intracellular pH of 0.25Ϯ0.01 unit (meanϮSE, n ϭ 3). This degree of alkalinization is consistent with effects reported in other tissues (27).…”

“…Moreover, the rate of lactate production by diaphragm was found to be stimulated in proportion to Na ϩ concentration in the medium (20). In several cell types, increased activity of the Na ϩ , K ϩ -ATPase leads to increased glycolysis and lactate production under fully oxygenated conditions (22)(23)(24)(25)(26)(27)(28). Aerobic glycolysis in vascular smooth muscle has been studied extensively.…”

“…Treatment of C6 glioma cells with the Na ϩ -selective ionophore monensin at a concentration sufficient to result in a doubling of [Na ϩ ] i also resulted in a doubling of lactate production (27). Monensin may stimulate glycolysis either ( a ) by intracellular alkalinization and, consequently, disinhibition of 6-phosphofructo-1-kinase or ( b ) by increasing ATP consumption secondary to stimulation of the Na ϩ -K ϩ pump.…”

“…Monensin may stimulate glycolysis either ( a ) by intracellular alkalinization and, consequently, disinhibition of 6-phosphofructo-1-kinase or ( b ) by increasing ATP consumption secondary to stimulation of the Na ϩ -K ϩ pump. Monensinstimulated glycolysis in C6 cells was largely inhibited by ouabain, suggesting that increased pump activity, rather than alkalinization, was the primary stimulus to glycolysis (27). In brain, astrocytes take up glutamic acid released by glutamatergic neurons, thus inactivating this neurotransmitter by removing it from the synaptic cleft.…”

Characterization of Ca2+‐activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa‐channel toxins

“…Cells were then dissolved in 0.1 M NaOH for the determination of total cell protein (Lowry et al, 1951) (Poenie, 1990). Ion-sensitive electrode measurements indicate that the free Ca2+ concentration in resting C6 glioma cells is 40 nM (Erecinska et al, 1991 remaining were re-extracted and the combined extracts diluted to 500 ml with 20 mM NH4OAc (pH 7.0) (buffer A). Extracts were filtered (0.2 jm nylon filter) and loaded at 10 ml min-' on to a 4 ml cation exchange h.p.l.c.…”