Studies with Composite Membranes: II. Measurement of Asymmetric Properties

Electrical potentials arising across composite membranes when they separate the same concentration of a (1:1) electrolyte or electrolytes have been measured. These potentials have been shown to arise from differences in the transport number of counterions contacting the two faces of the membrane which contained in its body a high concentration of electrolyte and polyelectrolyte. When the concentration of this trapped electrolyte or polyelectrolyte is low, the asymmetry potentials are small. Although measurements of current-voltage relations provided evidence for the existence of asymmetry between the two faces of the membrane, osmotic flow of water in either direction across the membrane and the salt flow in the two directions were symmetrical. These solvent and solute flux measurements lasted more than 30 hr. Short-term (about 4 hr) flux measurements, however, using tritiated water (THO), gave flows which were different in the two directions. Similarly, the salt flows measured using ²²Na isotope were different in the two directions. The usefulness of the present system as a model to use for studies concerned with carrier transport problems in biology has been pointed out.     

The effect of temperature on membrane hydraulic conductivity

The objective of this study was to use the temperature dependence of water permeability to suggest the physical mechanisms of water transport across membranes of osmotically slowly responding cells and to demonstrate that insight into water transport mechanisms in these cells may be gained from easily performed experiments using an electronic particle counter. Osmotic responses of V-79W Chinese hamster fibroblast cells were measured in hypertonic solutions at various temperatures and the membrane hydraulic conductivity was determined. The results were fit with the general Arrhenius equation with two free parameters, and also fit with two specific membrane models each having only one free parameter. Data from the literature including that for human bone marrow stem cells, hamster pancreatic islets, and bovine articular cartilage chondrocytes were also examined. The results indicated that the membrane models could be used in conjunction with measured permeability data at different temperatures to investigate the method of water movement across various cell membranes. This approach for slower responding cells challenges the current concept that the presence of aqueous pores is always accompanied by an osmotic water permeability value, P(f)>0.01 cm/s. The possibility of water transport through aqueous pores in lower-permeability cells is proposed.     

On the large error introduced in the estimate of the density of membrane pores from permeability measurements when diffusion in "unstirred layer" within the cells is disregarded

After the development of the "black lipid membrane" techniques, studies of the permeability of labeled water and nonelectrolytes across these artificial membranes have yielded permeability constants comparable in magnitude to those obtained from tracer studies of living cell membranes. This general agreement has affirmed the belief that the living cell membranes are indeed closely similar to these bilayer phospholipid membranes. In this report, we draw attention to a hidden assumption behind such comparisons made: the assumption that labeled material passing through the cell membrane barriers instantly reaches diffusion equilibrium inside the cell. The permeability constants to labeled water (and nonelectrolytes) across lipid layers were obtained using setups in which the lipid membrane was sandwiched between aqueous compartments both of which were vigorously stirred. In studies of permeability of living cell membranes only the outside solution was stirred, the intracellular water remained stationary. Yet the calculations of permeability constants of the cell membrane were made with the tacit assumption, that once the labeled materials pass through the cell membrane, they were instantly mixed with the entire cell contents as if a stirrer operating at infinite speed had been present inside the cells. Ignoring this unstirred condition of the intracellular water, in fact, lumped all the real-life delay due to diffusion in the cytoplasm and added it to the resistance to diffusion of the membrane barrier. The result is an estimated membrane permeability to labeled water (and nonelectrolytes) many times slower than it actually is. The present report begins with a detailed analysis of a specific case: tritiated water diffusion from giant barnacle muscle fibers and two non-living models, one real, one imagined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies with Composite Membranes: I. Preparation and Measurement of Impedances

Simple and composite membranes have been prepared from 2% collodion solutions containing different amounts of polystyrenesulfonic acid (PSSA). Various membrane parameters such as water content, electrolyte uptake, exchange capacity, and permselectivity of these membranes have been determined. The resistance and capacitance of simple membranes have been measured as functions of both external electrolyte concentration and internal fixed charge density. The impedance characteristics of composite membranes also have been determined and discussed in terms of the resistance and capacitance characteristics of simple membranes from which the composite structures have been formed.     

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(d-Val-l-Pro-l-Val-d-Pro)3

The cyclic dodecapeptide PV, cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃, a structural analogue of the ion-carier valinomycin, increase the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na⁺ < NH₄⁺ < K⁺ < Cs⁺ < Rb⁺. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanims). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.     

The entrance of water into beef and dog red cells

The rate constants for diffusion of THO across the red cell membrane of beef and dog, and the rate of entrance of water into the erythrocytes of these species under an osmotic pressure gradient have been measured. For water entrance into the erythrocyte by diffusion the rate constants are 0.10 ± 0.02 msec.^-1 (beef) and 0.14 ± 0.03 msec.^-1 (dog); the permeability coefficients for water entrance under a pressure gradient of 1 osmol./cm³ are 0.28 See PDF for Equation These values permit the calculation of an equivalent pore radius for the erythrocyte membrane of 4.1 A for beef and 7.4 A for dog. In the beef red cell the change in THO diffusion due to osmotically produced cell volume shifts has been studied. The resistance to THO diffusion increases as the cell volume increases. At the maximum volume, (1.06 times normal), THO diffusion is decreased to 0.84 times the normal rate. This change in diffusion is attributed to swelling of the cellular membrane.     

Water permeability of isolated cuticular membranes: The effect of cuticular waxes on diffusion of water

Summary The water permeability of astomatous cuticular membranes isolated from Citrus aurantium L. leaves, pear (Pyrus communis L.) leaves and onion (Allium cepa L.) bulb scales was determined before and after extraction of cuticular waxes with lipid solvents. In pear, the permeability coefficients for diffusion of tritiated water across cuticular membranes (CM) prior to extraction [P _d(CM)] decreased by a factor of four during leaf expansion. In all three species investigated P _d(CM) values of cuticular membranes from fully expanded leaves varied between 1 to 2×10^-7 cm^-3 s^-1·P _d(CM) values were not affected by pH. Extraction of cuticular waxes from the membranes increased their water permeability by a factor of 300 to 500. Permeability coefficients for diffusion of THO across the cutin matrix (MX) after extraction [P _d(MX)] increased with increasing pH. P _dvalues were not inversely proportional to the thickness of cuticular membranes. By treating the cutin matrix and cuticular waxes as two resistances acting in series it was shown that the water permeability of cuticles is completely determined by the waxes. The lack of the P _d(CM) values to respond to pH appeared to be due to structural effects of waxes in the cutin matrix. Cuticular membranes from the submerse leaves of the aquatic plant Potamogeton lucens L. were three orders of magnitude more permeable to water than the cuticular membranes of the terrestrial species investigated.Abbreviations CM cuticular membrane - MX cutin matrix - WAX waxes This study was supported by a grant from the Deutsche Forschungsgemeinschaft.     

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.     

Effect of Geometrical and Chemical Constraints on Water Flux across Artificial Membranes

Studies have been made on the temperature dependence of both the hydraulic conductivity, L_p, and the THO diffusion coefficient, ω, for a series of cellulose acetate membranes (CA) of varying porosity. A similar study was also made of a much less polar cellulose triacetate membrane (CTA). The apparent activation energies, E_a, for diffusion across CA membranes vary with porosity, being 7.8 kcal/mole for the nonporous membrane and 5.5 kcal/mole for the most porous one. E_a for diffusion across the less polar CTA membrane is smaller than E_a for the CA membrane of equivalent porosity. Classical viscous flow, in which the hydraulic conductivity is inversely related to bulk water viscosity, has been demonstrated across membranes with very small equivalent pores. Water-membrane interactions, which depend upon both chemical and geometrical factors are of particular importance in diffusion. The implication of these findings for the interpretation of water permeability experiments across biological membranes is discussed.     

Solvent drag across gramicidin channels demonstrated by microelectrodes

The competition of ion and water fluxes across gramicidin channels was assessed from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double-barreled microelectrodes in the immediate vicinity of a planar bilayer. Because water movement across the membrane led to accumulation of solutes on one side of the membrane and depletion on the other, the permeable cation was not only pushed by water across the channel (true solvent drag); it also flowed along its concentration gradient (pseudo-solvent drag). For the demonstration of true solvent drag, a difference between the bulk concentrations on the hypertonic and the hypotonic sides of the membrane was established. It was adjusted to get equal cation concentrations at both membrane/water interfaces. From the sodium and potassium fluxes measured along with membrane conductivity under these conditions, approximately five water molecules were found to be transported simultaneously with one ion through the channel. In diphytanoyl phosphatidylcholine membranes, a single-channel hydraulic permeability coefficient of 1.6 x 10(-14) cm(3) s(-1) was obtained.     

OZONE-INDUCED MEMBRANE PERMEABILITY CHANGES

The effect of 0.5 ppm ozone for 0.5-1 hr on plant cell membrane permeability was ascertained. Permeabilities to both water and solutes were estimated by measuring leaf disc weight changes and following tritiated water and ⁸⁶Rb fluxes. Measurements were made immediately after ozone exposure and 24 hr after exposure. The reflection coefficient, σ, an index of solute permeability, decreased in ozone-treated primary leaves of pinto bean (Phaseolus vulgaris). The latter indicates an increase in membrane solute permeability or internal solute leakage. Water and THO flux estimates both indicated a decrease in membrane permeability to water; both the hydraulic conductivity (L_p) and the water diffusional coefficient (L_D) apparently decreased, an anomaly which is discussed. These data indicate that ozone has a direct effect on membrane function by altering permeability characteristics. We assume from these data that cell membranes are primary target sites for ozone injury.     

Effect of membrane charge on flow and protein transport during ultrafiltration

Although several recent studies have demonstrated the importance of electrostatic interactions in ultrafiltration, there have been few quantitative studies of the effects of membrane charge density on protein transport and membrane hydraulic permeability. Data were obtained using a series of charge-modified cellulose membranes, with the surface charge density controlled by varying the extent of addition of a quaternary amine functionality. The membrane charge was evaluated from streaming potential measurements. Protein transmission decreased by a factor of 100 as the membrane zeta potential increased from 0.3 to 6.6 mV. The protein sieving data were in good agreement with a partitioning model accounting for electrostatic effects, while the hydraulic permeability data were consistent with a flow model accounting for the effects of counter-electroosmosis. The results provide the first quantitative analysis of the effects of membrane charge density on the performance of ultrafiltration membranes.     

Barriers to water flow in vasopressin-treated toad urinary bladder

Summary Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.     

Self-excitation in a porous membrane doped with sorbitan monooleate (Span-80) induced by an Na+/K+ concentration gradient

The electrical potential across a fine-pore membrane doped with sorbitan monooleate (Span-80) imposed between aqueous solutions of NaCl and KCl was studied. It was found that this system showed rhythmic and sustained oscillations of electrical potential between the two aqueous solutions. These oscillations were attributed to the change of permeability of Na+ and K+ across the membrane, which originated from the phase transition of Span-80 molecules within the fine pores. Impedance measurement across the membrane also suggested a change in permeability. It was found that this membrane exhibited the property of differential negative resistance. In relation to this, it was shown that Na+ and K+ have different effects on the aggregation of Span-80 molecules. The mechanism of oscillation is discussed in relation to the ability of Span-80 molecules to behave as a dynamic channel through the membrane. This oscillatory phenomenon is interesting because in biological nervous membranes a difference between the concentrations of Na+ and K+ across the membranes is essential for excitability.     

Effects of solute hydrophobicity and phospholipid composition on permeability of composite membranes containing phospholipids

Permeabilities of several solutes through the composite membranes containing phospholipids have been measured. They were inversely proportional to the content of the phospholipids in the membrane. Both the permeability of solutes and the degree of permeability change around the phase transition temperature of the phospholipids for the hydrophobic solutes such as n-butanol and salicylamide were larger than those for the hydrophilic solutes such as amino acids and pyridoxine. These results suggest thatthe permeation path of hydrophobic solutes is different from that of hydrophilic ones. The addition of phosphatidyl ethanolamine, phosphatidyl serine, or phosphatidic acid to the composite membrane influenced the solute permeability due to the introduced negative charge and/or the change in the molecular packing of phospholipid.     

Chloride Transport in Porous Lipid Bilayer Membranes

This paper describes dissipative Cl_- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10^-7 M amphotericin B. P_DCl (cm·s^-1), the diffusional permeability coefficient for Cl^-, estimated from unidirectional ³⁶Cl^- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω^-1·cm^-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, P_DCl was 1.36 x 10^-4 cm·s^-1 when Gm was 0.02 Ω^-1·cm^-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of P_DCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ~90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl^- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the P_DNa/P_DCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness.     

Organotin and osmoregulation: quantifying the effects of environmental concentrations of sediment-associated TBT and TPhT on the freshwater-adapted European flounder, Platichthys flesus (L.)

Chronic (5 weeks) exposure of freshwater-adapted European flounder, Platichthys flesus (L.), to environmental concentrations of sediment-associated tri-n-butyltin chloride (TBTCl) and triphenyltin chloride (TPhTCl) caused significant changes to hydromineral fluxes and membrane permeability, mechanisms that maintain osmotic homeostasis. The half-time of exchange of tritiated water (THO) in TBTCl- and TPhTCl-exposed fish was significantly increased during the first 2 weeks of the experiment and then decreased steadily, eventually reaching the level that the control group had constantly maintained throughout the experiment. This change in apparent water permeability was accompanied by a significant decrease in diffusional water flux across the membranes. Passive Na(+)-efflux across the gills was increased significantly but effluxes in the control group were near constant over the same time span. Drinking rates in the organotin groups increased significantly while the rate of urine production did not change. This lead to an increased net water balance in the organotin groups and consequently to a significant reduction of the blood osmolality of both organotin groups when compared to a control. There would appear to be a metabolic cost attached to the changes produced by exposure to environmental levels of organotin compounds which are manifested as a minimal increase in body length compared to the controls.     

Water permeabilities and salt reflection coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule

The mechanisms of water transport across the rabbit renal proximal convoluted tubule were approached by measuring osmotic permeabilities and solute reflection coefficients of the brush-border and the basolateral membranes. Plasma and intracellular membrane vesicles were isolated from rabbit renal cortex by centrifugation on a Percoll gradient. Three major turbidity bands were obtained: a fraction of purified basolateral membranes (BLMV), the two others being brush-border (BBMV) and endoplasmic reticulum (ERMV) membrane vesicles. The osmotic permeability (Pf) of the three types of vesicle was measured using stop-flow techniques and their geometry was determined by quasi-elastic light scattering. Pf was equal to 123 +/- 8 microns/s (n = 10) for BBMV, 166 +/- 10 microns/s (n = 10) for BLMV and 156 +/- 9 microns/s (n = 4) for ERMV (T = 26 degrees C). A transcellular water permeability, per unit of apical surface area, of 71 microns/s was calculated considering that the luminal and the basolateral membranes act as two conductances in series. This value is in close agreement, after appropriate normalizations, with previously reported transepithelial water permeabilities obtained using in vitro microperfusion techniques thus supporting the hypothesis of a predominantly transcellular route for water flow across rabbit proximal convoluted tubule. The addition of 0.4 mM HgCl2, a sulfhydryl reagent, decreased Pf about 60% in three types of membrane providing evidence for the existence of proteic pathways. NaCl and KCl reflection coefficients were measured and found to be close to one for plasma and intracellular membranes suggesting that the water channels are not shared by salts.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

X-ray scattering from labeled membranes.

We present a new method for the determination of structural parameters in biological membranes. Recording the continuous scattering of heavy-atom labeled membranes and applying elementary Fourier methods we obtain the scattering of the heavy-atom distribution alone. The details of this distribution are explored by developing a simple model and testing for cases relevant to biological membranes. We find that the intensity distribution is highly sensitive to many key parameters. The increased signal from heavy-atom labeling and the use of an improved x-ray system make it possible to record patterns from dilute membrane suspensions. Thus determination of these parameters is possible in the same environment where many membrane biochemical studies are performed. Application of the method is made to a model lipid bilayer membrane, dipalmitoyl phosphatidylcholine by labeling with UO2++ ions. We determine the precise distance between UO2++ layers on either side of the membrane as well as the width of the label on each side. This determination permits estimation of phosphate separation across single labeled bilayers in an aqueous suspension.