gramicidin-a and Neuroblastoma

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na+ channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 microg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.

Topics: Animals; Biological Assay; Bivalvia; Chromatography, High Pressure Liquid; Fluorometry; Food Contamination; Gramicidin; Humans; Marine Toxins; Membrane Potentials; Mice; Neuroblastoma; Radioligand Assay; Reproducibility of Results; Saxitoxin; Sensitivity and Specificity; Shellfish; Spectrometry, Fluorescence; Time Factors; Tumor Cells, Cultured; Veratridine

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

Studies were conducted using a novel in vitro approach to investigate the efficacy of acetamidine hydrochloride (ACE) and guanidine hydrochloride (GUAN), previously shown to block gramicidin D (GRAM) channels in artificial membranes, in preventing the toxic effects of GRAM in NG108-15 (neuroblastoma x glioma hybrid) cells. Specifically, intracellular microelectrode techniques were employed to examine changes in membrane resting potential (Vm) and input resistance (Rin). At 1 micromol/L, ACE significantly reduced loss of Vm induced by 1 or 10 microg/ml GRAM, although higher concentrations of ACE did not afford enhanced antagonism. GUAN, in contrast, produced a concentration-dependent antagonism of GRAM-induced Vm and Rin loss, with high concentrations (10 or 100 micromol/L) completely preventing diminutions in both Vm and Rin. In control cells superfused without GRAM, ACE produced a direct, concentration-dependent reduction in Vm and Rin, whereas GUAN hyperpolarized NG108-15 cells but did not alter Rin. These data represent the initial demonstration of the reversal of GRAM toxicity in an intact cell system.

Topics: Amidines; Animals; Anti-Bacterial Agents; Drug Interactions; Electric Impedance; Electrophysiology; Glioma; Gramicidin; Guanidine; Hybrid Cells; Membrane Potentials; Neuroblastoma; Neurons; Neuroprotective Agents; Parasympathomimetics; Rats; Trypsin Inhibitors

A one-dimensional version of the model recently proposed by Läuger (1988) to explain the closed-time distribution of ionic channels in cell membranes is solved analytically. While the probability density f(t) for closed-time lengths may show a well-defined exponential behavior at short times, a power-law decay is predicted at long times. The influence of an additional random distribution of defects in the current-conducting protein is investigated and found to be dominating at long times. Explicit expressions that may be used for fitting experimental data are given for the closed-time distribution. Some of the available data are discussed and shown to be in good agreement with the predictions of the model.

Topics: Animals; Cell Line; Colon; Diffusion; Endothelium, Corneal; Glioma; Gramicidin; Hybrid Cells; Ion Channels; Mathematics; Models, Theoretical; Motor Endplate; Muscle, Smooth; Muscles; Neuroblastoma; Rabbits; Rats