gramicidin-a and Carcinoma--Ehrlich-Tumor

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Amino Acids; Amino Acids, Diamino; Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Cell Membrane; Gramicidin; Hydrogen-Ion Concentration; Kinetics; Mice; Models, Biological; Oligomycins; Potassium; Sodium; Time Factors; Valinomycin

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.

Topics: Animals; Annexin A2; Annexin A3; Annexin A5; Annexins; Calcium; Carcinoma, Ehrlich Tumor; Cattle; Gramicidin; Hydrogen-Ion Concentration; Lipid Bilayers; Lung; Mice; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Potassium Chloride; Swine

The K+ and Cl- currents activated by Ca2+-ionophore treatment or by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the patch-clamp technique. A charybdotoxin-inhibitable K+ current was activated by increasing intracellular Ca2+ concentration. In contrast, the K+ current activated by cell swelling was insensitive to charybdotoxin as well as to apamin, suggesting that channels different from those sensitive to Ca2+ are responsible for regulatory volume adjustments in these cells. The magnitude of the K+ and Cl- currents activated by hypotonic challenge was markedly temperature-dependent, possibly reflecting the temperature-dependence of enzymes involved in the intracellular signalling of cell volume regulation.

Topics: Animals; Apamin; Calcium; Carcinoma, Ehrlich Tumor; Cell Size; Charybdotoxin; Chloride Channels; Gramicidin; Ionomycin; Ionophores; Membrane Potentials; Osmotic Pressure; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Temperature; Tumor Cells, Cultured

The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl- but inhibited by addition of MK196 (anion channel blocker) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl-, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a "Mini Cl- channel." The Cl- efflux via this Cl- channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS and inhibited by arachidonic acid and oleic acid. It is suggested that the swelling-activated "Mini Cl- channel" and the swelling-activated taurine channel in the Ehrlich cell represent two distinct types of channels.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Arachidonic Acid; Carcinoma, Ehrlich Tumor; Cell Size; Chloride Channels; Chlorides; Gramicidin; Indans; Ion Channels; Kinetics; Membrane Potentials; Mice; Osmolar Concentration; Taurine; Tumor Cells, Cultured

The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and

Topics: Animals; Calcimycin; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Electric Conductivity; Fluorescent Dyes; Gramicidin; Ion Channels; Membrane Potentials; Mice; Quinine; Thiocyanates; Valinomycin

The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl- transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl- transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl- conductance is increased 14- and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl- conductance is rate limiting. An A23187-induced activation of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: inhibited by quinine which blocks the Ca2+-activated K+ channel, unaffected by substitution of NO-3 or SCN- for Cl-, and inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl- conductance. The Cl- conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl- transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl- transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl- transport pathway. The activated anion transport pathway is unselective, carrying both Cl-, Br-, NO-3, and SCN-. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl- transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN-) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from 42K flux measurements at high external K+, and from net K+ flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Topics: Animals; Biological Transport, Active; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Egtazic Acid; Female; Gramicidin; Ion Channels; Kinetics; Mice; Mice, Inbred Strains; Pimozide; Potassium; Quinine; Thermodynamics; Valinomycin

The Effect of gramicidin S (GS), a polypeptide antibiotic, on the growth of murine tumor cells such as allotransplantable sarcoma 180 (S180) and Ehrlich ascites carcinoma (EAC) and Meth A fibrosarcoma (Meth A) was studied. GS inhibited the proliferation of EAC cells in culture and its effect on cell viability was dependent on the concentration of GS. On exposure to GS at concentrations ranging from 1 to 100 micrograms/ml for 10 min, EAC cells lost their transplantability in ddY mice depending on the concentration of GS. In particular, the transplantability of EAC cells was completely missing on exposure to GS at a concentration of 100 micrograms/ml. In the in vivo experiments, a daily intraperitoneal injection of GS exhibited a high inhibitory effect not only on the growth of subcutaneously implanted S180 in ICR mice but on the growth of subcutaneously implanted syngeneic Meth A in BALB/c mice. In the studies using radioactively labeled DNA, RNA and protein precursors, GS at high concentrations inhibited the incorporation of all the precursors into EAC cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Division; Cells, Cultured; DNA, Neoplasm; Dose-Response Relationship, Drug; Female; Fibrosarcoma; Gramicidin; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Neoplasm Proteins; Neoplasm Transplantation; RNA, Neoplasm; Sarcoma 180; Time Factors

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Female; Gramicidin; Kinetics; Mice; Mice, Inbred ICR; Sodium; Thiamine

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Chloromercuribenzoates; Ethylmaleimide; Fluorouracil; Gramicidin; Iodoacetates; Iodoacetic Acid; Kinetics; p-Chloromercuribenzoic Acid; Sodium; Sodium Fluoride; Time Factors

Trans-stimulation of glycine uptake by cellular glycine in Ehrlich cells is a Na+-dependent phenomenon. In contrast trans-stimulated methionine or leucine uptake is Na+-independent. Trans-stimulated uptake of glycine does not show any characteristics of an ex change process but rather appears to be due to changes in membrane potential which occur as a result of a net Na+-dependent loss of cellular amino acids. Trans-stimulated influx of glycine occurs during the time of net loss of cellular glycine and is absent when the cellular amino acid level is at steady or when the cell is depolarized. Exchange of leucine or methionine occurs when the amino acid level is at steady state and it is not directly affected by depolarizing agents such as gramicidin.

Topics: Amino Acids; Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Choline; Diffusion; Glycine; Gramicidin; Kinetics; Leucine; Membrane Potentials; Methionine; Mice; Sodium

Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+.

Topics: Amino Acids; Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Cell Membrane; Glycine; Gramicidin; Inositol; Kinetics; Mice; Sodium; Temperature

Topics: Adenosine Triphosphate; Amino Acids; Aminoisobutyric Acids; Animals; Biological Transport, Active; Carbon Radioisotopes; Carboxylic Acids; Carcinoma, Ehrlich Tumor; Cell Membrane; Choline; Dinitrophenols; Gramicidin; Kinetics; Lysine; Membrane Potentials; Methods; Mice; Models, Biological; Piperidines; Sodium; Time Factors

Topics: Aminoisobutyric Acids; Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Cell Membrane; Cesium; Ethanolamines; Gramicidin; Hydrogen-Ion Concentration; Kinetics; Lithium; Mathematics; Membrane Potentials; Methylglucosides; Mice; Osmolar Concentration; Ouabain; Potassium; Radioisotopes; Rubidium; Sodium; Sodium Isotopes; Time Factors; Valinomycin

Topics: Adenosine Triphosphatases; Amino Acids; Animals; Antimetabolites; Arginine; Carcinoma, Ehrlich Tumor; Cell Membrane; Glycine; Gramicidin; Kinetics; Leucine; Oligomycins; Ornithine; Ouabain; Piperidines; Potassium; Sodium; Valinomycin