The Water and Nonelectrolyte Permeability Induced in Thin Lipid Membranes by the Polyene Antibiotics Nystatin and Amphotericin B

Nystatin and amphotericin B increase the permeability of thin (<100 A) lipid membranes to ions, water, and nonelectrolytes. Water and nonelectrolyte permeability increase linearly with membrane conductance (i.e., ion permeability). In the unmodified membrane, the osmotic permeability coefficient, P_f, is equal to the tagged water permeability coefficient, (P_d)_w; in the nystatin- or amphotericin B-treated membrane, P_f/(P_d)_w ≈ 3. The unmodified membrane is virtually impermeable to small hydrophilic solutes, such as urea, ethylene glycol, and glycerol; the nystatin- or amphotericin B-treated membrane displays a graded permeability to these solutes on the basis of size. This graded permeability is manifest both in the tracer permeabilities, P_d, and in the reflection coefficients, σ (Table I). The "cutoff" in permeability occurs with molecules about the size of glucose (Stokes-Einstein radius 4 A). We conclude that nystatin and amphotericin B create aqueous pores in thin lipid membranes; the effective radius of these pores is approximately 4 A. There is a marked similarity between the permeability of a nystatin- or amphotericin B-treated membrane to water and small hydrophilic solutes and the permeability of the human red cell membrane to these same molecules.     

Temperature Dependence of Nonelectrolyte Permeation across Red Cell Membranes

The temperature dependence of permeation across human red cell membranes has been determined for a series of hydrophilic and lipophilic solutes, including urea and two methyl substituted derivatives, all the straight-chain amides from formamide through valeramide and the two isomers, isobutyramide and isovaleramide. The temperature coefficient for permeation by all the hydrophilic solutes is 12 kcal mol^-1 or less, whereas that for all the lipophilic solutes is 19 kcal mol^-1 or greater. This difference is consonant with the view that hydrophilic molecules cross the membrane by a path different from that taken by the lipophilic ones. The thermodynamic parameters associated with lipophile permeation have been studied in detail. ΔG is negative for adsorption of lipophilic amides onto an oil-water interface, whereas it is positive for transfer of the polar head from the aqueous medium to bulk lipid solvent. Application of absolute reaction rate theory makes it possible to make a clear distinction between diffusion across the water-red cell membrane interface and diffusion within the membrane. Diffusion coefficients and apparent activation enthalpies and entropies have been computed for each process. Transfer of the polar head from the solvent into the interface is characterized by ΔG^‡ = 0 kcal mol^-1 and ΔS^‡ negative, whereas both of these parameters have large positive values for diffusion within the membrane. Diffusion within the membrane is similar to what is expected for diffusion through a highly associated viscous fluid.     

Nonelectrolyte diffusion across lipid bilayer systems

The permeability coefficients of a homologous series of amides from formamide through valeramide have been measured in spherical bilayers prepared by the method described by Jung. They do not depend directly on the water:ether partition coefficient which increases regularly with chain length. Instead there is a minimum at acetamide. This has been ascribed to the effect of steric hindrance on diffusion within the bilayer which increases with solute molar volume. This factor is of the same magnitude, though opposite in sign to the effect of lipid solubility, thus accounting for the minimum. The resistance to passage across the interface has been compared to the resistance to diffusion within the membrane. As the solute chain length increases the interface becomes more important, until for valeramide it comprises about 90% of the total resistance. Interface resistance is also important in urea permeation, causing urea to permeate much more slowly than an amide of comparable size, after allowance is made for the difference in the water:ether partition coefficient. Amide permeation coefficients have been compared with relative liposome permeation data measured by the rate of liposome swelling. The ratios of the two measures of permeation vary between 3 and 16 for the homologous amides. The apparent enthalpy of liposome permeation has been measured and found to be in the neighborhood of 12 kcal mol-1 essentially independent of chain length. Comparison of the bilayer permeability coefficients with those of red cells shows that red cell permeation by the lipophilic solutes resembles that of the bilayers, whereas permeation by the hydrophilic solutes differs significantly.     

On simultaneous transport of water and solute through plant cell membranes: Evidence for the absence of solvent drag effect and insensitivity of the reflection coefficient

The simultaneous efflux of tritiated water and ¹⁴C labelled ethanol from inner epidermal cells of the bulb scale of Allium cepa was measured with a specially designed efflux chamber. It was found that water and ethanol moved essentially independently. Rates of efflux of tritiated water and ¹⁴C ethanol were essentially the same in the presence or absence of a simultaneous influx of water. Using the same technique the efflux of tritiated water from the epidermal cells was measured during a simultaneous flow of nonlabelled ethanol. When tritiated water and ethanol moved in opposite directions, the water permeability values became slightly reduced depending upon the concentration of ethanol. When ethanol and tritiated water moved in the same direction, however, no effect on water permeability values could be detected. These results are best explained by the molecular theory of diffusion across lipid bilayer membranes, and are consistent with the above findings of lack of interaction between water and ethanol as they are transported across the cell membrane. In another study, the solute permeability coefficients (K_s) for non-electrolytes such as urea and methyl urea were measured by plasmolyzing the epidermal cells and transferring them to equimolal solutions of urea and methyl urea. This method was also used to measure the reflection coefficient (σ) for these nonelectrolytes. The K_s values for methyl urea were 16 times greater than the ones for urea. The values of σ for both of these solutes, however, were very close to 1. Using the K_s data available in the literature for the subepidermal cells of the Pisum sativum stem basis, the σ values were calculated for malonamide, glycerol, methyl urea, ethyl urea, dimethyl urea, and formamide. Again the K_s values for these nonelectrolytes varied by several orders of magnitude, whereas all σ values were found to be close to 1. These findings point out that σ is an insensitive parameter and that K_s, the solute permeability constant, has to be used for characterizing solute transport through the membrane. The present study shows that fast (e.g. ethanol, formamide) as well as slowly permeating molecules do not interact with water as they are transported across the cell membrane. Aqueous pores for the simultaneous transport of water and solutes, therefore, are absent in the plant cell membranes investigated here.     

Dimensions of polar pathways through rabbit gallbladder epithelium

Summary The permeability of rabbit gallbladder to hydrophilic nonelectrolytes, with molecular weights from 20 to 60,000, has been studied. Restriction in the diffusion of the small electrolytes is very significant up to glycerol, which suggests permeation through aqueous pores with equivalent radii of 4 Å. An extracellular pathway is responsible for the permeation of the larger solutes. This extracellular pathway shows no restriction in diffusion of molecules up to the size of inulin. Dextran (15,000 to 17,000 mol wt) is significantly restricted. Albumin permeability is <10^–8 cm sec^–1. These observations can be equated with equivalent, pore radii of 40 Å for the shunt pathway.Increasing osmolarities of the incubation medium cause decreased cell-membrane permeability and increased shunt permeability. 0.5mm phloretin induces a 60% reduction in urea permeability and a 168% increase in antipyrine permeability. No effect on the osmotic water permeability or on the shunt permeability is observed in the presence of phloretin. The apparent activation energy of urea permeation changes from values consistent with diffusion in bulk water, to values consistent with diffusion through hydrocarbon regions. This suggests that the polar route for urea permeation is blocked by phloretin.The contribution of the shunt pathway to osmotic flow induced by sucrose or NaCl gradients is smaller than 16% according to Poiseuille's flow calculations. Tetraethylammoniumchloride and albumin have been shown to be osmotically more effective than sucrose, suggesting a greater shunt contribution to the total water flow.     

Transport Parameters in a Porous Cellulose Acetate Membrane

The transport parameters of a cellulose acetate membrane prepared from a mixture of cellulose acetate, formamide, and acetone, 25:25:50 by weight, were studied. The membrane consists of a thin, porous layer, the skin, in series with a thick, highly porous layer, the coarse support. In the skin the diffusional permeability coefficient, ω, of a number of small amides and alcohols depends critically upon the partition coefficient, K_s, the size of the molecule, and the apparent hydrogen-bonding ability, N_s, of the solute. These observations are in general agreement with our earlier conclusions on the properties of nonporous membranes. On the other hand, the corrected reflection coefficient, σ', is not a very sensitive function of either N_s or K_s taken separately. The correlation between σ' and molecular diameter is reasonably good; however, it is much improved when both N_s and K_s are taken into consideration. Isotope interaction was also studied in the present preparation and was found to provide only a small (5–8%) contribution to the diffusional permeability coefficient of ethylene glycol. The contribution of solute-water friction was found to be less than 24% of the total solute friction.     

Comparative transport efficiencies of urea analogues through urea transporter UT-B

Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes also permeate various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. In this study, we examined UT-B-facilitated passage of urea analogues and other neutral small solutes by comparing transport properties of wildtype to UT-B-deficient mouse erythrocytes. Stopped-flow light-scattering measurements indicated high UT-B permeability to urea and chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with P_s > 5.0 × 10^− 6 cm/s at 10 °C. UT-B genetic knockout and phloretin treatment of wildtype erythrocytes similarly reduced urea analogue permeabilities. Strong temperature dependencies of formamide, acetamide, acrylamide and butyramide transport across UT-B-null membranes (E_a > 10 kcal/mol) suggested efficient diffusion of these amides across lipid bilayers. Urea analogues dimethylurea, acryalmide, methylurea, thiourea and methylformamide inhibited UT-B-mediated urea transport by > 60% in the absence of transmembrane analogue gradients, supporting a pore-blocking mechanism of UT-B inhibition. Differential transport efficiencies of urea and its analogues through UT-B provide insight into chemical interactions between neutral solutes and the UT-B pore.     

Effects of sulfhydryl and other reagents on the diffusional permeability of dog erythrocytes to small solutes

The effects of p-chloromercuriphenylsulfonic acid (PCMBS), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), phloretin and thiourea on the diffusional permeability of dog erythrocytes to tritiated water and to small 14C-labeled lipophilic and hydrophilic solutes were measured at 37 degrees C by means of the linear diffusion technique. Permeability to 3HHO was significantly decreased by PCMBS but was not affected by the other reagents. The permeability to the small hydrophilic solutes acetamide and urea was decreased by phloretin and thiourea but only the permeability to acetamide was reduced to a statistically significant extent by PCMBS. The permeability to the lipophilic solutes methanol, ethanol and antipyrine was not affected by any of these agents. We interpret these results as an indication that the small lipophilic solutes probably move through lipid areas, that the small hydrophilic solutes probably move through protein associated areas in the erythrocyte membrane and that pathways for the small hydrophilic solutes are distinct from those for water. While the pathways for water may be associated with membrane protein they do not appear to be associated specifically with band 3 protein as has been suggested for human erythrocytes. Diffusional water movement through the dog erythrocyte occurs by two distinct pathways.     

Nonelectrolyte diffusion through lecithin-water lamellar phases and red-cell membranes

The longitudinal diffusion of a homologous series of monoamides through lecithin-water lamellar phases with aqueous channel widths of 16–27 Å has been studied. The diffusion coefficients relative to water of the hydrophilic amides, formamide and acetamide, depend logarithmically on solute molar volume, as previously demonstrated in human red cells. Aqueous diffusion of amides in red-cell membranes is similar to that in a lecithin-water phase of aqueous channel width less than 16 Å, the smallest channel width used. Partition coefficients of the lipophilic amides, valeramide and isovaleramide, between lecithin vesicles and water are 1.64 and 1.15 at 20 °C. These data enabled us to compute a valeramide diffusion coefficient of 6.5 · 10⁻⁷cm² · s⁻¹ at 20 °C in the lipid region of a lamellar phase containing 30% water about one order of magnitude greater than the diffusion coefficient of spin-labelled analogs of phosphatidylcholine. The discrimination between the permeability coefficients of valeramide and isovaleramide is more than twice as great in the human red cell as between lipid diffusion coefficients in a phase containing 8% water. This suggests that the lipid region of the human red cell is more highly organized than lipid in the lecithin-water lamellar phase.     

Role of Hydrogen-Bonding in Nonelectrolyte Diffusion through Dense Artificial Membranes

The diffusion of two series of alcohols and amides through complex cellulose acetate membranes was studied. The thin dense part of these membranes behaves as a nonporous layer of low water content. In this layer, called the skin, the solute diffusion coefficients, ω, depend upon size, steric configuration, and the partition coefficient, K₈, between membrane and bathing solution. From the experimental values of ω and K₈, the over-all friction, f, experienced by the solutes in the membrane was computed. It was found that f depends upon the chemical nature of the solute and is related to hydrogen-bonding ability. In the coarse, porous layer of the cellulose acetate membrane, diffusion occurs mainly through aqueous channels. In this instance also the hydrogen-bonding ability of the solute seems to exercise a smaller but significant influence.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Aquaporin Expression Contributes to Human Transurothelial Permeability In Vitro and Is Modulated by NaCl

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm²), with mean diffusive water and urea permeability coefficients of 6.33×10⁻⁵ and 2.45×10⁻⁵ cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.     

Determination of Urea Permeability in Red Cells by Minimum Method : A test of the phenomenological equations

A new method has been developed for measuring the permeability coefficient, ω, of small nonelectrolytes. The method depends upon a mathematical analysis of the time course of cell volume changes in the neighborhood of the minimum volume following addition of a permeating solute to an isosmolal buffer. Coefficients determined by the minimum volume method agree with those obtained using radioactive tracers. ω for urea in human red cells was found to decrease as the volume flow, J_v, into the cell increased. Such behavior is entirely unexpected for a single uniform rate-limiting barrier on the basis of the linear phenomenological equations derived from irreversible thermodynamics. However, the present findings are consonant with a complex membrane system consisting of a tight barrier on the outer face of the human red cell membrane and a somewhat less restrictive barrier behind it closer to the inner membrane face. A theoretical analysis of such a series model has been made which makes predictions consistent with the experimental findings.     

A Physical Interpretation of the Phenomenological Coefficients of Membrane Permeability

A "translation" of the phenomenological permeability coefficients into friction and distribution coefficients amenable to physical interpretation is presented. Expressions are obtained for the solute permeability coefficient ω and the reflection coefficient σ for both non-electrolytic and electrolytic permeants. An analysis of the coefficients is given for loose membranes as well as for dense natural membranes where transport may go through capillaries or by solution in the lipoid parts of the membrane. Water diffusion and filtration and the relation between these and capillary pore radius of the membrane are discussed. For the permeation of ions through the charged membranes equations are developed for the case of zero electrical current in the membrane. The correlation of σ with ω and L_p for electrolytes resembles that for non-electrolytes. In this case ω and σ depend markedly on ion concentration and on the charge density of the membrane. The reflection coefficient may assume negative values indicating anomalous osmosis. An analysis of the phenomena of anomalous osmosis was carried out for the model of Teorell and Meyer and Sievers and the results agree with the experimental data of Loeb and of Grim and Sollner. A set of equations and reference curves are presented for the evaluation of ω and σ in the transport of polyvalent ions through charged membranes.     

Gradients of turgor, osmotic pressure, and water potential in the cortex of the hypocotyl of growing ricinus seedlings : effects of the supply of water from the xylem and of solutes from the Phloem

To evaluate the possible role of solute transport during extension growth, water and solute relations of cortex cells of the growing hypocotyl of 5-day-old castor bean seedlings (Ricinus communis L.) were determined using the cell pressure probe. Because the osmotic pressure of individual cells (πⁱ) was also determined, the water potential (ψ) could be evaluated as well at the cell level. In the rapidly growing part of the hypocotyl of well-watered plants, turgor increased from 0.37 megapascal in the outer to 1.04 megapascal in the inner cortex. Thus, there were steep gradients of turgor of up to 0.7 megapascal (7 bar) over a distance of only 470 micrometer. In the more basal and rather mature region, gradients were less pronounced. Because cell turgor ≈ πⁱ and ψ ≈ 0 across the cortex, there were also no gradients of ψ across the tissue. Gradients of cell turgor and πⁱ increased when the endosperm was removed from the cotyledons, allowing for a better water supply. They were reduced by increasing the osmotic pressure of the root medium or by cutting off the cotyledons or the entire hook. If the root was excised to interrupt the main source for water, effects became more pronounced. Gradients completely disappeared and turgor fell to 0.3 megapascal in all layers within 1.5 hours. When excised hypocotyls were infiltrated with 0.5 millimolar CaCl₂ solution under pressure via the cut surface, gradients in turgor could be restored or even increased. When turgor was measured in individual cortical cells while pressurizing the xylem, rapid responses were recorded and changes of turgor exceeded that of applied pressure. Gradients could also be reestablished in excised hypocotyls by abrading the cuticle, allowing for a water supply from the wet environment. The steep gradients of turgor and osmotic pressure suggest a considerable supply of osmotic solutes from the phloem to the growing tissue. On the basis of a new theoretical approach, the data are discussed in terms of a coupling between water and solute flows and of a compartmentation of water and solutes, both of which affect water status and extension growth.     

Effects of Viscosity and Constraints on the Dispersion and Dissipation of Waves in Large Blood Vessels: II. Comparison of Analysis with Experiments

The theoretical predictions described in part I of this study are compared with in vivo data from anesthetized dogs. It is shown that the observed attenuation of the pressure and axial waves cannot be accounted for by fluid viscosity alone. For large values of the frequency parameter α = √ρωR²₀/μ, the analysis of part I is extended to include the effects of viscoelasticity of the vessel wall. The results indicate that the speeds of both types of waves are essentially not affected by a realistic viscoelasticity model while the attenuation per wavelength is significantly increased and becomes frequency independent. The application of this analysis to in vivo data from the carotid arteries of anesthetized dogs demonstrates partial agreement between theory and experiment and suggests that the carotid arteries are anisotropically viscoelastic.     

On the theoretical aspects of tracer exchange in red blood cells

An analytical solution is presented for isotopic exchange between red blood cells and a finite surrounding medium where the intracellular diffusion is taken into account for two models of the red blood cell, the rectangular and the spherical. The solution is applied on experimental results from the literature with exchange of water, methanol, formamide, urea and ethylene glycol. It is seen that if the diffusion coefficient for those substances is less than 10^?7 cm²/sec, then the previous assumption about exchange between two well-stirred compartments becomes doubtful.     

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solute’s oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.     

Aluminum and Temperature Alteration of Cell Membrane Permeability of Quercus rubra

This report extends research on Al-induced changes in membrane behavior of intact root cortex cells of Northern red oak (Quercus rubra). Membrane permeability was determined by the plasmometric method for individual intact cells at temperatures from 2 or 4 to 35°C. Al (0.37 millimolar) significantly increased membrane permeability to urea and monoethyl urea and decreased permeability to water. Al significantly altered the activation energy required to transport water (+32%), urea (+9%), and monoethyl urea (−7%) across cell membranes. Above 9°C, Al increased the lipid partiality of the cell membranes; below 7°C, Al decreased it. Al narrowed by 6°C the temperature range over which plasmolysis occurred without membrane damage. These changes in membrane behavior are explainable if Al reduces membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains.