Osmotic flow caused by polyelectrolytes

Osmotic flow of water caused by high concentrations of anionic polyelectrolytes across semipermeable membranes, permeable only to solvent and simple electrolyte, has been measured in a newly designed flow cell. The flow cell features small solution and solvent compartments and an efficient stirring mechanism. We have demonstrated that, while the osmotic pressure of the anionic polyelectrolytes is determined primarily by micro-counterions, the osmotic flow is determined by solution-dependent properties as embodied in the hydrodynamic frictional coefficient which is determined by the polymer backbone segment of the polyelectrolyte. The variation of the osmotic permeability coefficient, L(p)(o), with concentration and osmotic pressure closely correlated with the concentration dependence of this frictional coefficient. These studies confirm previous work that the kinetics of osmotic flow across a membrane impermeable to the osmotically active solute is primarily determined by the diffusive mobility of the solute.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles. Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restriction of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restrictions of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.     

Desformylgramicidin: a model channel with an extremely high water permeability

The water conductivity of desformylgramicidin exceeds the permeability of gramicidin A by two orders of magnitude. With respect to its single channel hydraulic permeability coefficient of 1.1.10(-12) cm(3) s(-1), desformylgramicidin may serve as a model for extremely permeable aquaporin water channel proteins (AQP4 and AQPZ). This osmotic permeability exceeds the conductivity that is predicted by the theory of single-file transport. It was derived 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. From solvent drag experiments, approximately five water molecules were found to be transported by a single-file process along with one ion through the channel. The single channel proton, potassium, and sodium conductivities were determined to be equal to 17 pS (pH 2.5), 7 and 3 pS, respectively. Under any conditions, the desformyl-channel remains at least 10 times longer in its open state than gramicidin A.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

Patterns of Nonelectrolyte Permeability in Human Red Blood Cell Membrane

The permeability of human red cell membrane to 90 different molecules has been measured. These solutes cover a wide spectrum of nonelectrolytes with varying chemical structure, chain length, lipid solubility, chemical reactive group, ability to form hydrogen bonds, and other properties. In general, the present study suggests that the permeability of red cell membrane to a large solute is determined by lipid solubility, its molecular size, and its hydrogen-bonding ability. The permeability coefficient increases with increasing lipid solubility and decreasing ability to form hydrogen bonds, whereas it decreases with increasing molecular size. In the case of small solutes, the predominant diffusion factor is steric hindrance augmented by lipid solubility. It is also found that replacement of a hydroxyl group by a carbonyl group or an ether linkage tends to increase permeability. On the other hand, replacement of a hydroxyl group by an amide group tends to decrease the permeability coefficient.     

Probability Density Function of the Red Cell Membrane Permeability Coefficient

The distribution of a random variable is determined by the probability density functions (PDF) of all other random variables with which the variable in question is jointly distributed. If the PDF of the random variable of interest is normal, or skewed normal, then the distributions with which it is jointly distributed determine its mean and standard deviation. In the case described here (where hemolysis time of the red blood cell is a function of the permeability coefficient and geometric variables of the cell) the mean and standard deviation of the permeability coefficient and the known distributions of the geometric variables on which the hemolysis time depends determine a predicted distribution of hemolysis time. An observed distribution of the hemolysis time is obtained spectrophotometrically. By choosing the mean and standard deviation of the permeability coefficient so that the predicted PDF of the hemolysis time matches the observed PDF best by least-squares criterion, the complete distribution of the permeability coefficient is determined.     

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.     

Water transport and ion-water interaction in the gramicidin channel

The diffuse permeability and the diffusion coefficient of water (Dw) in the gramicidin channel is determined from the osmotic water permeability of the channel and "single file" pore theory. Dw is about 7% of the self-diffusion coefficient of bulk water. The diffusion coefficient of a single water molecule alone in the channel is also determined and is about equal to the value in bulk water. This provides an estimate of the mobility of water on the channel walls in the absence of water-water interaction. Since the gramicidin channel walls should be representative of uncharged polar protein surfaces, this result provides direct evidence that the presence of a cation in the channel reduces the hydraulic water permeability by a factor ranging from 60 for Tl+ to 5 for Na+. The diffusion coefficient of a cation (Dc) in the channel is estimated and compared with Dw. For Na+ it is found that Dc approximately equal to Dw, which implies that the movement of the row of water molecules through the channel determines the local mobility of Na+. Thus, it seems that short range ion-wall interactions are not important in determining the channel conductance for Na+. In contrast, for Li+, local ion-wall interactions probably do limit the conductance.     

Determination of the membrane permeability coefficient and the reflection coefficient by the two-dimensional laminar flow model for intestinal perfusion experiments

We performed single perfusion experiments in the small intestine of rats in order to prove that the two-dimensional laminar flow model is suitable to determine the membrane permeability coefficient and the reflection coefficient. We used progesterone as an aqueous-diffusion-limited drug, urea as a membrane transport-limited drug and the tritiated water as an intermediate substance. The membrane permeability coefficient for progesterone was calculated to be 3.6 X 10(-4) cm/s. This value did not change when the thickness of the aqueous diffusion layer was altered by increasing the perfusion rate 10-fold. It was directly demonstrated that the two-dimensional laminar flow model was suitable to analyze the data of intestinal perfusion experiments. Membrane permeability coefficients for urea and tritiated water were determined to be 3.4 X 10(-5) cm/s and 8.9 X 10(-5) cm/s, respectively. In the presence of water absorption with the hypotonic perfusion solution, the reflection coefficient for urea was 0.84. This value is thought to be theoretically reasonable, suggesting the usefullness of the two-dimensional laminar flow model to obtain the reflection coefficient in the intestinal membrane.     

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.     

Effects of cold on vascular permeability and edema formation in the isolated cat limb.

We investigated the effects of cold temperatures on microvascular protein permeability in the isolated constant-flow perfused cat hindlimb. The perfusates were 20% cat plasma-80% albumin-electrolyte solution (low-viscosity perfusate, approximately 1 cP) or whole blood (high-viscosity perfusate, approximately 4 cP). The time at low temperature (less than 10 degrees C) was less than 3 h (short term) or greater than 5 h (long term). Decreases in the solvent drag reflection coefficient (sigma f) indicated increases in permeability. The sigma f's were determined with the integral-mass balance method from measurement of changes in protein concentration and hematocrit induced by fluid filtration into the tissues. Short-term cold exposure did not increase permeability with either a low- or a high-viscosity perfusate, whereas long-term exposure with limb temperatures of approximately 5 degrees C significantly increased permeability when the perfusate was whole blood. In addition, we verified our previous prediction that flow had to be reduced to 6-8 ml.min-1.100 g-1 to avoid the hydrostatic edema caused by short-term perfusion with whole blood at approximately 5 degrees C. Also, we found that at approximately 3 degrees C histamine's permeability-increasing effect was totally abolished, whereas at approximately 20 degrees C this effect was partially inhibited. Hence, constant-flow perfusion at low temperature with whole blood can cause edema by a pressure-dependent mechanism, whereas long-term perfusion with this perfusate at low temperatures can cause a permeability increase that further compounds edema formation. Histamine is not responsible for this permeability increase.     

Transport of small anionic and neutral solutes through chitosan membranes: dependence on cross-linking and chelation of divalent cations

Chitosan membranes were prepared by solvent casting and cross-linked with glutaraldehyde at several ratios under homogeneous conditions. The cross-linking degree, varying from 0 to 20%, is defined as the ratio between the total aldehyde groups and the amine groups of chitosan. Permeability experiments were conducted using a side-by-side diffusion cell to determine the flux of small molecules of similar size but with different chemical moieties, either ionized (benzoic acid, salicylic acid, and phthalic acid) or neutral (2-phenylethanol) at physiological pH. The permeability of the different model molecules revealed to be dependent on the affinity of those structurally similar molecules to chitosan. The permeability of the salicylate anion was significantly enhanced by the presence of metal cations commonly present in biological fluids, such as calcium and magnesium, but remained unchanged for the neutral 2-phenylethanol. This effect could be explained by the chelation of metal cations on the amine groups of chitosan, which increased the partition coefficient. The cross-linking degree was also correlated with the permeability and partition coefficient. The change in the permeation properties of chitosan to anionic solutes in the presence of these metallic cations is an important result and should be taken into consideration when trying to make in vitro predictions of the drug release from chitosan-based controlled release systems.     

The water permeability of toad urinary bladder. I. Permeability of barriers in series with the luminal membrane

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(- 4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.     

A Biomechanical Triphasic Approach to the Transport of Nondilute Solutions in Articular Cartilage

Biomechanical models for biological tissues such as articular cartilage generally contain an ideal, dilute solution assumption. In this article, a biomechanical triphasic model of cartilage is described that includes nondilute treatment of concentrated solutions such as those applied in vitrification of biological tissues. The chemical potential equations of the triphasic model are modified and the transport equations are adjusted for the volume fraction and frictional coefficients of the solutes that are not negligible in such solutions. Four transport parameters, i.e., water permeability, solute permeability, diffusion coefficient of solute in solvent within the cartilage, and the cartilage stiffness modulus, are defined as four degrees of freedom for the model. Water and solute transport in cartilage were simulated using the model and predictions of average concentration increase and cartilage weight were fit to experimental data to obtain the values of the four transport parameters. As far as we know, this is the first study to formulate the solvent and solute transport equations of nondilute solutions in the cartilage matrix. It is shown that the values obtained for the transport parameters are within the ranges reported in the available literature, which confirms the proposed model approach.     

Transport parameters in the human red cell membrane: solute-membrane interactions of amides and ureas

We have studied the permeability of a series of hydrophilic amides and ureas through the red cell membrane by determining the three phenomenological coefficients which describe solute-membrane interaction: the hydraulic permeability (Lp), the phenomenological permeability coefficient (omega i) and the reflection coefficient (sigma i). In 55 experiments on nine solutes, we have determined that the reflection coefficient (after a small correction for solute permeation by membrane dissolution) is significantly less than 1.0 (P less than 0.003, t-test), which provides very strong evidence that solute and water fluxes are coupled as they cross the red cell membrane. It is proposed that the aqueous channel is a tripartite assembly, comprising H-bond exchange regions at both faces of the membrane, joined by a narrower sieve-specific region which crosses the lipid. The solutes bind to the H-bond exchange regions to exchange their solvation shell with the H-bonds of the channel; the existence of these regions is confirmed by the finding that the permeation of all the amides and ureas requires binding to well-characterized sites with Km values of 0.1-0.5 M. The sieve-specific regions provide the steric restraints which govern the passage of the solutes according to their size; their existence is shown by the findings that: (1) the reflection coefficient (actually the function [1-corrected sigma i]) is linearly dependent upon the solute molecular diameter; and (2) the permeability coefficient is linearly dependent upon solute molar volume. These several observations, taken together, provide strong arguments which lead to the conclusion that the amides and urea cross the red cell membrane in an aqueous pore.     

Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes.     

Relation Between the Dielectric Constant of Hydrophobic Cation Exchange Membrane and Membrane Permeability to Counterions

Filters made of cellulose acetate-nitrate when saturated with organic solvents and interposed between aqueous solutions form membranes which behave like cation exchangers. The diffusion coefficients of counterions in such membranes are strongly dependent upon the dielectric constant of the saturating solvent. The results obtained suggest that a linear relationship between the log of the cation's diffusion coefficient (or membrane conductance) and the reciprocal value of the dielectric constant of the saturating solvent exists. There is also a good correlation between the relative membrane permeability to organic cations and the solubility of the cations in the pure solvent phase. These studies indicate that there are two routes for cation movement through the membrane: (a) the bulk hydrophobic phase and (b) continuous narrow aqueous channels.     

Measurement of the water permeability of single human granulocytes on a microscopic stopped-flow mixing system

The water permeability (Lp) of human granulocytes was measured for individual isolated cells with a novel, microscopic stopped-flow mixing system. Changes in volume were monitored as a cell was introduced suddenly into an osmotically active solution. Permeability values were determined as a function of solution osmolality from the volume versus time curves for mixing into both hypotonic and hypertonic solutions within the range of 145 to 833 mOsm. The calculated reference permeability at 25 degrees C was 1.15 micrometers/atm.min with an osmotic coefficient of 0.46 Osm/kg.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.