valinomycin and Glioma

The effects of several K(+)-selective neutral ionophores on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells were examined. Specifically, alterations in membrane resting potential (V(m)), input resistance (R(in)) and electrically-induced action potential generation were determined upon bath application of enniatin (0.1-10 microg/ml), nonactin (0. 1-10 microM) and valinomycin (0.1-10 microM). Although some cells exhibited a slight hyperpolarization and/or reduced R(in), i.e. membrane electrical correlates of enhanced K(+) loss, neither V(m) nor R(in) were significantly altered by any of the ionophores. However, valinomycin and especially nonactin affected action potentials induced by electrical stimulation. This was apparent in the ablation of action potentials in some cells and in the occurrence of degenerative changes in action potential shape in others. The simultaneous administration of the neutral ionophores and the protonophore CCCP or the superfusion of enniatin, nonactin or valinomycin in high (50 mM) glucose-containing physiological solution did not yield more extensive alterations in V(m) or R(in). These data suggest that the neutral ionophores are unable to materially enhance K(+) flux above the relatively high resting level in NG108-15 cells. Thus, alterations in action potentials appear to be unrelated to K(+) transport activity.

Topics: Action Potentials; Analysis of Variance; Animals; Anti-Bacterial Agents; Cell Membrane; Depsipeptides; Dose-Response Relationship, Drug; Electric Stimulation; Glioma; Gramicidin; Hybrid Cells; Ion Transport; Ionophores; Macrolides; Membrane Potentials; Mice; Neuroblastoma; Neurons; Nigericin; Peptides; Potassium; Rats; Tumor Cells, Cultured; Valinomycin

Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca2+, and the demonstration of an increase in phospholipase A2 (PLA2) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA2 in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCl inhibited both AA release and the increase in [Ca2+]i triggered by Et-1, whereas valinomycin, which causes K+ efflux, not only caused a rapid rise in [Ca2+]i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA2 in this cell type requires calcium influx dependent on K+ efflux.

Topics: Animals; Arachidonic Acid; Calcimycin; Calcium; Endothelins; Enzyme Activation; Glioma; Ion Channel Gating; Ionophores; Phospholipases A; Phospholipases A2; Potassium; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Valinomycin

The effects of the flavonoid quercetin on cell proliferation and voltage-dependent K+ current were studied on mouse neuroblastoma x glioma hybrid cells. In the presence of 1% fetal calf serum, quercetin inhibited both cell proliferation and K+ current with effective doses inducing half-maximum effects of 10 microM and 70 microM respectively. Valinomycin (1 nM) antagonized 80% of the growth-inhibitory effects of 10 microM quercetin. The results suggest that at least a part of the anti-proliferative effect of quercetin is mediated by a K+ channel blockade. They further confirm a role for K+ channels in mitogenesis and cell proliferation.

Topics: Adenosine Triphosphate; Animals; Cell Division; DNA; Electric Conductivity; Fetal Blood; Glioma; Hybrid Cells; Mice; Neuroblastoma; Potassium Channels; Quercetin; Tumor Cells, Cultured; Valinomycin

Transport pathways for spermidine (Spd) were characterized in mammalian cells in culture of different origin, i.e. L 1210, P 388, C 6, U 251, Balb/c 3T3 normal and transformed by virus SV40 (SV40/3T3). The kinetic constants (Km and Vmax) for 14C-Spd uptake were found to be different in these cells. Spd uptake was inhibited by spermine and putrescine in all cells. Preloading of these cells with system A and other amino acids, including ornithine, usually did not affect Spd uptake, except in L 1210 and C 6 cells, where Spd uptake was accelerated by 2-aminoisobutyric acid, demonstrating that in these two cell lines the polyamines share the system A pathway. Iso-osmotic replacement of Na+ by choline chloride in the assay medium resulted in a decrease in Spd uptake which suggests that Spd uptake is Na+ activated. In all cells, Spd uptake was inhibited by gramicidin and the Ca2+ ionophore A 23187. The degree of inhibition varied among the cells. Valinomycin (K+ ionophore) inhibited Spd uptake by C 6, P 388, Balb/c 3T3 and SV40/3T3 but not by L 1210 and U 251 cells. Treatment with N-ethylmaleimide or p-L 1210, C 6, Balb/c 3T3 and SV40/3T3 cells did not affect appreciably the uptake process. Some newly synthesized polyamine analogues inhibited the Spd uptake of all cells.

Topics: Amino Acids; Animals; Biological Transport; Calcimycin; Fibroblasts; Glioma; Gramicidin; Humans; Leukemia L1210; Leukemia P388; Mice; Mice, Inbred BALB C; Polyamines; Sodium; Spermidine; Sulfhydryl Reagents; Tumor Cells, Cultured; Valinomycin

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.

Topics: Arachidonic Acid; Arachidonic Acids; Biological Transport; Cell Membrane; Cell Membrane Permeability; Electrochemistry; Fatty Acids; Fatty Acids, Unsaturated; Glioma; Glycine; Membrane Potentials; Neuroglia; Potassium; Serum Albumin, Bovine; Thermodynamics; Tumor Cells, Cultured; Valinomycin

C6 glioblastoma cells in culture were employed to isolate plasma membrane vesicles. After disruption of the glioblastoma cells by homogenization, membrane fractions were obtained by centrifugation on a discontinuous Ficoll density gradient. Fragmented membranes were found mainly in vesicular form. Transport of glycine has been demonstrated in membrane vesicles, using artificially imposed ion gradients as the sole energy source. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a Cl- gradient (out greater than in) when the other essential ion is present. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of ionophore valinomycin and anions with different permeabilities. The kinetic analysis shows that glycine is accumulated by two systems with different affinities.

Topics: Biological Transport; Cell Line; Cell Membrane; Glioma; Glycine; Kinetics; Nigericin; Onium Compounds; Organophosphorus Compounds; Sodium-Potassium-Exchanging ATPase; Valinomycin

Cyclic AMP efflux was measured following hormonal stimulation of adenylate cyclase in a variety of animal cells including C-6 rat glioma cells, WI-38 human fibroblasts, and avian erythrocytes. Using a variety inhibitors of mitochondrial function and glycolysis, a correlation was noted between cellular ATP levels and the rate of cyclic AMP efflux in all cells examined. A relationship between the efflux rate and the magnitude of the membrane potential was not observed. Pharmacological agents which inhibited cyclic AMP egress in these cells without reducing ATP levels included several prostaglandins (A greater than B greater than E greater than F) and probenecid. The characteristics of the cyclic AMP efflux system resemble those of the organic anion transport system.

Topics: Animals; Biological Transport; Cell Line; Columbidae; Cyclic AMP; Erythrocytes; Glioma; Kinetics; Membrane Potentials; Potassium; Prostaglandins; Turkeys; Valinomycin

Rat glioma cells grown in culture secrete cyclic adenosine 3':5'-monophosphate (cyclic AMP) into the culture medium following stimulation by beta-agonistic catecholamines. Agents which reduced cellular ATP levels such as valinomycin, oligomycin, and uncouplers of oxidative phosphorylation, inhibited cyclic AMP efflux. Secretion of cyclic AMP was also prevented by prostaglandin A-1 and pharmacological agents including probenecid and papaverine. Of the latter agents, only papaverine reduced ATP levels. These results suggest that the transport of cyclic AMP across animal cell membranes is energy-dependent and subject to regulation.

Topics: Animals; Biological Transport; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Cells, Cultured; Clone Cells; Cyclic AMP; Glioma; Neoplasms, Experimental; Oligomycins; Papaverine; Potassium Chloride; Probenecid; Propranolol; Prostaglandins A; Prostaglandins E; Prostaglandins F; Rats; Time Factors; Valinomycin

The transport and metabolism of glucose was examined in monolayers of C-6 glioma cells. 1) Glucose transport appeared to have both a low (Km = 7.74 mM) and a high (Km = 1.16 mM) affinity site in C-6cells; whereas 2-deoxyglucose had only one (Km = 3.7 mM). 2) A large portion of the accumulated glucose was rapidly metabolized to the two glycolytic end products, lactate and pyruvate, and then extruded into the medium. The temperature-dependent efflux of lactate and pyruvate was linear up to 2 hrs with 6 to 10 times more lactate being extruded into the medium than pyruvate. 3) The efflux of lactate and pyruvate increased with increasing extracellular (medium) pH. The presence of 5 percent CO2 not only inhibited the acid efflux but also inhibited the short-term uptake of glucose. The CO2 effect was attributed to a lowering of the medium pH since bicarbonate alone either increased or did not inhibit efflux. 4) Valinomycin increased the levels of cellular lactate but not those of pyruvate by almost three-fold. Lactate efflux was stimulated while that of pyruvate was inhibited. The addition of 5 percent CO2 increased the cellular levels of both lactate and pyruvate, but unlike valinomycin decreased the acid efflux. Idoacetate inhibited the acid efflux by 50 percent suggesting that glycolysis is necessary for efflux.

Topics: Acetone; Biological Transport, Active; Cell Line; Fluoroacetates; Glioma; Glucose; Glycolysis; Hydrogen-Ion Concentration; Iodoacetates; Kinetics; Lactates; Phenobarbital; Pyruvates; Valinomycin