The influence of amphotericin B on the permeability of mammalian erythrocytes to nonelectrolytes, anions and cations

The influence of the polyene antibiotic, amphotericin B, on the permeability of porcine and bovine erythrocytes was studied by measuring net and tracer movements of nonelectrolytes, anions and cations in these cells.

1. 1. Amphotericin B (0.5–20 μM) enhances the rates of transfer of hydrophilic nonelectrolytes (glycerol, erythritol), anions (phosphate, lactate, glycollate, Cl⁻, SCN⁻) and cation (Na⁺, K⁺). Different concentrations of the antibiotic are required for equal effects on the different transfer processes. Bovine erythrocytes respond much less to amphotericin than porcine cells.

2. 2. Nystatin enhances the transfer of all the permeants to a much lesser extent; gramicidin D, although producing a large increase of cation permeability, leaves unaltered anion and nonelectrolyte transfer.

3. 3. The amphotericin-induced enhancement of erythrocyte permeabability (ΔP) increases with time. It has a concentration dependence of the type ΔP = α · Cⁿ_A^* (n = 1.5–2.5) and becomes more pronounced at low temperatures.

4. 4. Partial depletion of membrane cholesterol, which in itself does not alter nonelectrolyte and anion permeability, reduces the effectivity of amphotericin B, indicating that in the erythrocyte membrane, too, a sterol acts as receptor for polyene antibiotics.

5. 5. The selectivity of the amphotericin-induced pathway of transfer in the erythrocyte membrane is lower than that of the normal pathways of nonelectrolyte and anion transfer in this membrane.

The results support the view that amphotericin produces the same type of molecular reorganisation of lipid constituents in biological and artificial membranes. On the other hand, the polyene-induced pathway in the erythrocyte membrane seems to differ functionally from the normal transfer pathways in this membrane.     

The temperature dependence of passive potassium permeability in mammalian erythrocytes

The effect of temperature on the "passive" permeability of mammalian plasma membranes to K+, measured as the residual flux in the presence of ouabain and bumetanide, was investigated in erythrocytes of several species. Without Ca2+ in the medium, only human red cells demonstrated the "paradoxical" rise in passive flux at low temperature (i.e., below 12 degrees C) seen by other workers. In the other species no such effect was apparent; K+ influx decreased progressively with cooling down to 0 degree C. Below 18.5 degrees C the apparent energy of activation (Ea) was very low--close to that for free diffusion in water--for red cells of all species except human. Above 18.5 degrees C the Ea was much greater and was also more variable amongst the red cells of the species chosen. Neither the inhibitors used nor cell volume changes during incubation accounted for the absence of the paradoxical effect in the species studied here. A rise in permeation of K+ with cooling can, however, be produced by the addition of Ca2+ to the medium, probably by activation of the Ca2+-sensitive K+ channel. This effect would account for previous reports of a paradoxical effect in dog and rat erythrocytes.     

The permeability of liposomes to nonelectrolytes. II. The effect of nystatin and gramicidin A.

The process of selective permeation of nonelectrolytes across liposomes of different lipid composition and amount of cholesterol has been studied. The extent of the selectivity for diffusion within the membranes has been found to be related to the physical state of the hydrocarbon chains. It has been also found that incorporation of cholesterol into egg-lecithin membranes decreases the overall permeability by affecting the dehydration step more than the subsequent diffusion of the solute. The incorpporation into liposomes of the antibiotics nystatin and gramicidin A produces changes in the selective permeation of nonelectrolytes that are consistent with the formation by these molecules of aqueous pores of fixed dimensions. Finally, comparisons are made between the process of permeation in biological membranes and in liposomes with and without antibiotics.     

Sterol specific inactivation of gramicidin A induced membrane cation permeability.

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.     

Cation permeability induced by valinomycin,gramicidin D and amphotericin B in large lipidic unilamellar vesicles studied by 31P-NMR

Permeability induced by mobile carriers and channel-forming compounds in large unilamellar lipidic vesicles (LUV) has been studied by the proteon-cation exchange method. Proton movement has been monitored by pH-stat and ³¹P-NMR techniques. pH-stat measurements indicate that, in the presence of valinomycin, the proton efflux develops with a rate dependent upon valinomycin concentration, until equilibrium is reached. ³¹P-NMR spectra, monitoring pH-dependent intravesicular phosphate ionization, show that after addition of valinomycin the initial pH peak (pH 5.5; =0.25) shifts progressively to the position corresponding to the pH at equilibrium (pH 7.4; =2.20).In the presence of the channel-forming compounds, gramicidin D or amphotericin B, permeability developed in a few minutes whatever the concentration used. The percentage of total titratable proton released depends upon the antibiotic concentration. ³¹P-NMR spectra shows two signals from internal phosphate: one signal corresponding to the initial pH and a second signal corresponding to the pH at equilibrium indicating an all-or-none mode of action; just after addition of the antibiotic, two populations of vesicles coexist in proportions that depend on ionophore concentration; after longer incubation times all vesicles are permeabilized.The results obtained primarily reflect the differences in the mode of interaction with the membrane, of valinomycin as compared to the channel-forming reagents, gramicidin D or amphotericin B.     

Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes

Summary

In order to get a better understanding in the mechanism by which tryptophan-N-formylated gramicidin (NFG) and gramicidin kill the malaria parasite Plasmodium falciparum in vitro, we studied the capacity of these peptides to change the potassium, as well as the sodium, composition of normal human erythrocytes, and their ability to cause cell lysis. It is shown that both peptides are able to induce potassium leakage from, and sodium flux into, erythrocytes in such a manner that it is most likely that they are able to form cation channels in the membrane of these cells. For both peptides, potassium efflux proceeds at a faster rate than sodium influx, but this difference is greater for NFG than for gramicidin. This explains the observation that gramicidin is more lytic than NFG is, even when comparing concentrations that show the same antimalarial activity. The finding that gramicidin is approximately 10 times more active than NFG in causing potassium efflux from normal erythrocytes, as well as in killing the malaria parasite, supports the hypothesis that peptideinduced parasite death is related to their capacity to induce potassium leakage from infected erythrocytes. Finally, the observation that erythrocytes are able to restore their normal ion contents after losing more than 50% of their potassium content by incubation with NFG or gramicidin, suggests that, in vivo, and upon treatment with drug concentrations that cause full inhibition of parasite growth, these cells would not be irreversibly damaged by action of the drugs.     

Effect of valinomycin and gramicidin d on the reflection coefficient of soybean root systems

Valinomycin and gramicidin D were used to test the hypothesis that a lipoprotein membrane is the osmotic barrier in mature root systems. Hydroponically grown soybean (var. Ranson) root systems were pressurized in steps between 0.2 and 5.0 bars at 25 C. Steady-state flow rates and exudate osmotic potentials were measured at each pressure. Valinomycin or gramicidin D to a final concentration of 2.6 or 5.0 micromolar, respectively, was injected into the nutrient solution, and steady-state values were again measured between 0.2 and 5.0 bars. Both ionophores reduced the x intercept of the straight line portion of the flux rate versus pressure curve, and increased the exudate osmotic potential at infinite flow rate. Valinomycin lowered the reflection coefficient from 0.91 to 0.76, whereas gramicidin D lowered the reflection coefficient from 0.86 to 0.81. The results support the hypothesis that one or more lipoprotein membranes in the root system function in regulation of ion movement from the ambient solution to the stele during high transpiration rates.     

The influence of phospholipid polar groups on gramicidin channels.

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of N-methylation and comprised phosphocholine, -N,N-dimethylethanolamine, -N-methylethanolamine and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude lambda of the unit conductance step and a decrease in the average channel life-time or duration tau. The series of tau-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension gamma is a relevant parameter for the prediction of tau-values.     

Changes of membrane permeability due to extensive cholesterol depletion in mammalian erythrocytes

55% of the total membrane cholesterol could be removed from porcine, bovine and human erythrocytes by incubating the cells in suspensions of lecithin liposomes. Up to 30% depletion, membrane permeability remained unaltered; more extensive depletion induced a marked increase of the transfer rates of nonelectrolytes and of organic acids penetrating by nonionic diffusion. This biphasic response of permeability to cholesterol depletion, which has not been observed in artificial lipid membranes, may be related to the heterogeneity of the erythrocyte membrane lipids or to a pool of cholesterol not interacting with the phospholipids.     

The pore dimensions of gramicidin A.

The ion channel forming peptide gramicidin A adopts a number of distinct conformations in different environments. We have developed a new method to analyze and display the pore dimensions of ion channels. The procedure is applied to two x-ray crystal structures of gramicidin that adopt distinct antiparallel double helical dimer conformations and a nuclear magnetic resonance (NMR) structure for the beta6.3 NH2-terminal to NH2-terminal dimer. The results are discussed with reference to ion conductance properties and dependence of pore dimensions on the environment.     

Effects of valinomycin on lymphocytes independent of potassium permeability

10^?7 M valinomycin affects human lymphocytes in the following manner: (1) it is non-toxic; (2) it inhibits mitogenesis; (3) it causes a reduction in cell ATP; and (4) it causes a marked increase in steady-state Na⁺ exchange. However, it has a minimal effect on cell ion (K⁺, Na⁺, Ca²⁺, Mg²⁺) contents and no effect whatever on K⁺ exchange. Neither the fast nor the slow fraction of steady-state K⁺ exchange is affected by 10^?7 M valinomycin. The various reported effects of valinomycin on lymphocyte functions cannot be assumed to be due to changes in plasma membrane K⁺ permeability. The mechanism of the increase in steady-state Na⁺ exchange, and whether or not it is related to inhibition of mitogenesis, are unsettled issues.     

Gramicidin, valinomycin, and cation permeability of Streptococcus faecalis

Gramicidin and valinomycin in concentrations of 10(-7) and 10(-6)m, respectively, inhibited the growth of Streptococcus faecalis. Inhibition of growth was associated with loss of Rb(+) and K(+) from the cells, and could be reversed by addition of excess K(+). Cells treated with these antibiotics exhibited greatly increased permeability to certain cations; no effect was observed on the penetration of other small molecules. Unlike normal cells, cells treated with gramicidin rapidly lost internal Rb(+) by passive exchange with external cations, including H(+), all monovalent alkali metals, NH(4) (+), Mg(++), and tris(hydroxymethyl)aminomethane. Exchange was rapid even at 0 C and was independent of energy metabolism. The effect of valinomycin was more selective. Cellular Rb(+) was rapidly displaced by external H(+), K(+), Rb(+), and Cs(+); other cations were less effective. The exchange was independent of metabolism but strongly affected by temperature. Under certain conditions, polyvalent cations inhibited exchange between (86)Rb and Rb(+) induced by valinomycin. The antibiotic apparently neither stimulates nor inhibits the energy-dependent K(+) pump of S. faecalis, but exerts its effect on the passive permeability of the membrane to cations. The increased permeability to specific cations induced by gramicidin and valinomycin is a sufficient explanation for the inhibition of growth, glycolysis, and other processes.