Effect of phloretin on the permeability of thin lipid membranes

Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules.     

The effect of amphotericin B on the water and nonelectrolyte permeability of thin lipid membranes

This paper reports the effects of amphotericin B, a polyene antibiotic, on the water and nonelectrolyte permeability of optically black, thin lipid membranes formed from sheep red blood cell lipids dissolved in decane. The permeability coefficients for the diffusion of water and nonelectrolytes (P_DDi) were estimated from unidirectional tracer fluxes when net water flow (J_w) was zero. Alternatively, an osmotic water permeability coefficient (P_f) was computed from J_w when the two aqueous phases contained unequal solute concentrations. In the absence of amphotericin B, when the membrane solutions contained equimolar amounts of cholesterol and phospholipid, P_f was 22.9 ± 4.6 µsec^-1 and P _DDHDH2O was 10.8 ± 2.4 µsec^-1. Furthermore, P_DDi was < 0.05 µsec^-1 for urea, glycerol, ribose, arabinose, glucose, and sucrose, and σ_i, the reflection coefficient of each of these solutes was one. When amphotericin B (10^-6 M) was present in the aqueous phases and the membrane solutions contained equimolar amounts of cholesterol and phospholipid, P _DDHDH2O was 18.1 ± 2.4 µsec^-1; P_f was 549 ± 143 µsec^-1 when glucose, sucrose, and raffinose were the aqueous solutes. Concomitantly, P_DDi varied inversely, and σ_i directly, with the effective hydrodynamic radii of the solutes tested. These polyene-dependent phenomena required the presence of cholesterol in the membrane solutions. These data were analyzed in terms of restricted diffusion and filtration through uniform right circular cylinders, and were compatible with the hypothesis that the interactions of amphotericin B with membrane-bound cholesterol result in the formation of pores whose equivalent radii are in the range 7 to 10.5 A.     

Water and nonelectrolyte permeability of lipid bilayer membranes

Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n- butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility- diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).     

Vasopressin and water permeability of artificial lipid membranes

Vasopressin markedly stimulated the water permeability of bilayer lipid membranes: a two-fold increase was measured at 25° in presence of 1.7·10⁻⁹ M (50 μunits/ml) vasopressin. Oxytocin and a mixture of the amino acids comprising the vasopressin molecule could not substitute for vasopressin at comparable concentration. The experimental activation energy of water transport was reduced in the presence of vasopressin from 14 to 4 kcal/mole, in agreement with the effect of the hormone on water permeability of toad bladder.     

The effect of endotoxin on thin lipid bilayer membranes

Summary The effect of endotoxin fromSalmonella typhimurium orEscherichia coli was studied on bilayer lipid membranes (1% lecithin–1% cholesterol in n-decane) formed in buffered 0.1m NaCl solution (pH 6.8). Endotoxin was added to the buffered solution either prior to membrane formation or after stable membranes were formed. In both cases, concentrations of 110 to 720 g/ml endotoxin initiated a decrease in the electrical resistance of the membranes followed by their rupture. A 50 g/ml concentration of the agent was unable to elicit any response. Also, the addition of an equal volume of buffer solution, serving as a control, caused no decrease in membrane resistance or survival time. Treatment of the endotoxin with alkaline hydroxylamine to remove esterand amide-bound fatty acids likewise abolished the membrane effect. This is the first report of an endotoxin effect on lipid bilayer membranes. The potential correlation of this interaction of bilayer and endotoxin with the diverse biologic effects of endotoxin is discussed.     

Water permeability of gramicidin A-treated lipid bilayer membranes

In membranes containing aqueous pores (channels), the osmotic water permeability coefficient, P f, is greater than the diffusive water permeability coefficient, P d. In fact, the magnitude of P f/P d is commonly used to determine pore radius. Although, for membranes studied to date, P f/P d monotonically declines with decreasing pore radius, there is controversy over the value it theoretically assumes when that radius is so small that water molecules cannot overtake one another within the channel (single-file transport). In one view it should equal 1, and in another view it should equal N, the number of water molecules in the pore. Gramicidin A forms, in lipid bilayer membranes, narrow aqueous channels through which single-file transport may occur. For these channels we find that P f/P d approximately 5. In contrast, for the wider nystatin and amphotericin B pores, P f/P d approximately 3. These findings offer experimental support for the view that P f/P d = N for single-file transport, and we therefore conclude that there are approximately five water molecules in a gramicidin A channel. A similar conclusion was reached independently from streaming potential data. Using single-channel conductance data, we calculate the water permeability of an individual gramicidin A channel. In the Appendix we report that there is a wide range of channel sizes and lifetimes in cholesterol-containing membranes.     

Water permeability of bilayer lipid membranes: Sterol-lipid interaction

Summary Bilayer lipid membranes were generated in an aqueous medium from synthetic, egg or plant phosphatidyl choline (PC) or from plant monogalactosyl diglyceride (MG). The water permeability of the black membranes was determined by measuring the net volume flux produced by a NaCl gradient. The osmotic permeability coefficient,P _os, was markedly affected by the number of double bonds in the fatty acid conjugates of the lipids: the greater the degree of unsaturation, the higher the value ofP _os. The temperature dependence ofP _os of the lipid membranes was studied over a range of 29 to 40°C. The experimental activation energy,E _a , estimated from the linear plots of log (P _os)versus 1/T, was significantly higher for MG membranes (17 kcal/mole) than for the various PC membranes (11 to 13 kcal/mole), probably owing to hydrogen bonding between MG and water molecules. In comparison with PC membranes, the membranes generated from PC and cholesterol (11 molar ratio) had lowerP _os but similarE _a values. Likewise, either stigmasterol or -sitosterol decreasedP _os of MG membranes, whileE _a was not affected by the sterols. MG-cholesterol membranes were specifically characterized by a unique value ofE _a (–36 kcal/mole) thus indicating temperature dependent structural changes.     

The action of gramicidin S on the ionic permeability of bilayer lipid membranes

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.     

The effect of prymnesin on the electric conductivity of thin lipid membranes

Summary A proteolipidic toxin, prymnesin, when added to the aqueous solutions around thin lipid membranes causes a marked increase in membrane conductance. The toxin-treated membrane is cation-permselective. The extent of cation permselectivity is dependent upon ionic strength of the aqueous solutions in a fashion similar to the dependence of cation permselectivity of a cation exchanger containing about 100mm of fixed negative sites. Dose-response relationship studies reveal a linear relation between log prymnesin concentration and log membrane conductance. The slope of the curve is around 3 if the toxin is applied to one side of the membrane and is around 7 if the toxin is applied to both sides of the membrane. The membrane treated with toxin on one side only is clearly asymmetric in its properties. These characteristics are expressed by an asymmetric current-voltage relationship, and by asymmetric sensitivity of membrane conductance to pH and to salt concentration. The conductance of the toxin-treated membrane is inversely proportional to temperature. It is suggested that aggregates of toxin moieties assemble in the membrane to form negatively charged aqueous pores. There is roughly a good correlation between the increase in membrane conductance and the increase in membrane permeability to urea if both were attributed to the formation of aqueous channels in the membrane.     

A new method of measurement of permeability of lipid membranes

A new method, based on potential measurements to obtain permeabilities and mobilities of ionic species passing through membranes, under the influence of concentration gradients is proposed. This method is applied to three different lipid membranes in aqueous KCl solutions. The values of permeabilities and mobilities obtained by this simple method is in good agreement with those reported in literature     

Monitoring the permeability profile of lipid membranes with spin probes

The rate of reaction of the ascorbate ion with the nitroxide group of spin probes intercalated in lipid bilayers has been studied to examine the mechanism of transport of solutes across membranes. The loss of electron spin resonance (ESR) signal follows first-order kinetics. For a given bilayer system, the half-time of the process increases with the distance of the reacting group from the aqueous interface, according to an approximately linear permeation profile. The dependence on phospholipid headgroup is that which would be predicted from the net charge; addition of negatively charged headgroups increases the half-time of reaction, and positively charged headgroups decrease it, compared with bilayers having no net charge. Addition of cholesterol, which is known to decrease the fluidity of the hydrocarbon core of the bilayer, is found to increase the half-time of reaction. The results have been analyzed in terms of a partition-diffusion mechanism. It is suggested that the rate-limiting step for partitioning the solute into the bilayer might be removal of water of hydration. Cholesterol increases the activation energy, most probably by increasing the height of the barriers to diffusion. Quantitation of the changes in reaction rates gives an estimate of the change in bilayer surface potential on changing the headgroup composition. Examination of the permeation profile supports a diffusive mechanism, from which it can be estimated that the diffusion coefficient is approximately halved on adding 35 mol% cholesterol to egg lecithin bilayers.     

Optical and electrical studies on dansyllysine-valinomycin in thin lipid membranes

Summary Dansyllysine-valinomycin, a fluorescent analogue of the ionophore valinomycin was synthesized and incorporated into black lipid membranes. Its concentration inside the membrane was measured fluorometrically and was also determined from electrical relaxation experiments, which were analyzed on the basis of a previously proposed carrier model. The results of both methods agreed within less than one order of magnitude. This appears satisfactory in view of the sources of error inherent in both procedures.A conductance increment per carrier molecule of about 3 · 10^{–17 –1} was obtained for dansyllysine-valinomycin in diphytanoyllecithin membranes at 25 C and 1M RbCl in the aqueous phases. This is about 400 times smaller compared to unmodified valinomycin in monoolein membranes. The difference is mainly caused by the change in the membrane properties and to a smaller extent by the structural modification of the carrier.