Permeability of Alkali Metal Cations in Lobster Muscle : A comparison of electrophysiological and osmometric analyses

Single muscle fibers from lobster walking legs are effectively impermeable to Na, but are permeable to K. They shrink in hyperosmotic NaCl; they swell in low NaCl media which are hyposmotic or which are made isosmotic with the addition of KCl. In conformity, the membrane potential is relatively insensitive to changes in external Na, while it responds according to the Nernst relation for changes in external K. When the medium is made isosmotic or hyperosmotic with RbCl the volume and membrane potential changes are of essentially the same magnitudes as those in media enriched with KCl. The time courses for attaining equilibrium are slower, indicating that Rb is less permeant than K. Substitution of CsCl for NaCl (isosmotic condition) produces no change in volume of the muscle fiber. Addition of CsCl (hyperosmotic condition) causes a shrinkage which attains a steady state, as is the case in hyperosmotic NaCl. Osmotically, therefore, Cs appears to be no more permeant than is Na. However, the membrane depolarizes slowly in Cs-enriched media and eventually comes to behave as an ideal Cs electrode. Thus, the electrode properties of the lobster muscle fiber membrane may not depend upon the diffusional relations of the membrane and ions, and the osmotic permeability of the membrane for a given cation may not correspond with the electrophysiologically deduced permeability. Comparative data on the effects of NH₄ and Li are also included and indicate several other degrees of complexity in the cell membrane.     

Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure

Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.     

Isosmotic volume reabsorption in rat proximal tubule

A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model.     

Osmotic Flow of Water across Permeable Cellulose Membranes

Direct measurements have been made of the net volume flow through cellulose membranes, due to a difference in concentration of solute across the membrane. The aqueous solutions used included solutes ranging in size from deuterated water to bovine serum albumin. For the semipermeable membrane (impermeable to the solute) the volume flow produced by the osmotic gradient is equal to the flow produced by the hydrostatic pressure RT ΔC, as given by the van't Hoff relationship. In the case in which the membrane is permeable to the solute, the net volume flow is reduced, as predicted by the theory of Staverman, based on the thermodynamics of the steady state. A means of establishing the amount of this reduction is given, depending on the size of the solute molecule and the effective pore radius of the membrane. With the help of these results, a hypothetical biological membrane moving water by osmotic and hydrostatic pressure gradients is discussed.     

Influence of membrane heterogeneity on kinetics of nonelectrolyte tracer flows

Summary In a composite membrane with heterogeneous channels, prevention of net volume flow with hydrostatic pressure differences and/or impermeant osmotic solutes may induce positive isotope interaction (coupling of isotope flows) consequent to circulation of volume flow. The permeability coefficient for net flow will then exceed the tracer permeability coefficient. A permeant osmotic solute will induce either positive or negative isotope interaction, according to whether membrane heterogeneity is more marked for the test solute or the osmotic solute, respectively. Thus membrane heterogeneity may account for phenomena commonly attributed to single file diffusion or exchange diffusion. For sufficiently small flows the general flux ratio relationship for homogeneous membranes will continue to apply.     

The Response of Duck Erythrocytes to Nonhemolytic Hypotonic Media : Evidence for a volume-controlling mechanism

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells'' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

Analysis of proximal tubule salt and water transport in standing droplets.

A mathematical model has been developed describing solute and water movement in the renal proximal tubule standing droplet experiment. The model explicitly incorporates the constraint of isosmotic reabsorption. Solute asymmetry due to the unequal distribution of protein, bicarbonate and other solutes between plasma and the standing droplet is shown to be one of the major reabsorptive forces; however, the introduction of an additional reabsorptive mechanism into the system equations is required in order to obtain a quantitative fit with experimental observations. The model demonstrates that limiting concentration gradients can be obtained in the absence of active transport and that their magnitudes will vary inversely with the permeability of the poorly permeant solute. Conversely, situations can occur where active transport will not elicit a limiting gradient. Consequently, previous interpretations of the meaning of limiting gradients and their magnitudes need to be reconsidered. The model further predicts that the technique for measuring non-electrolyte permeability using standing droplet experiments is likely to underestimate the true permeability. Finally, it is shown that a previous theoretical model of standing droplets, which does not explicitly include the constraint of isomotic reabsorption, cannot fit experimental data from proximal tubules.     

Coupling of Solute and Solvent Flows in Porous Lipid Bilayer Membranes

The present experiments were designed to evaluate coupling of water and nonelectrolyte flows in porous lipid bilayer membranes (i.e., in the presence of amphotericin B) in series with unstirred layers. Alterations in solute flux during osmosis, with respect to the flux in the absence of net water flow, could be related to two factors: first, changes in the diffusional component of solute flux referable to variations in solute concentrations at the membrane interfaces produced by osmotic flow through the unstirred layers; and second, coupling of solute and solvent flows within the membrane phase. Osmotic water flow in the same direction as solute flow increased substantially the net fluxes of glycerol and erythritol through the membranes, while osmotic flow in the opposite direction to glycerol flow reduced the net flux of that solute. The observed effects of osmotic water flow on the fluxes of these solutes were in reasonable agreement with predictions based on a model for coupling of solute and solvent flows within the membrane phase, and considerably in excess of the prediction for a diffusion process alone.     

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index

Solute reflection coefficients sigma of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of sigma. We determined sigma NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone (Mr 40,000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a sigma NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again sigma NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.     

Osmosis and intermolecular force

A hypothesis concerning the molecular-dynamical basis of osmosis is presented. According to this hypothesis, osmosis results from the attractive force between solute and water molecules, and the exclusion of the solute from the water transport channels of the membrane. Based on this hypothesis, the osmotic process is predicted to involve a large number of short "spurts" of high magnitude force, with a spurt force per unit area of channel of RT/Vs, where R is the gas constant, T is absolute temperature, and Vs is solute molar volume.     

Single Water Channels of Aquaporin-1 Do not Obey the Kedem-Katchalsky Equations

The Kedem-Katchalsky (KK) equations are often used to obtain information about the osmotic properties and conductance of channels to water. Using human red cell membranes, in which the osmotic flow is dominated by Aquaporin-1, we show here that compared to NaCl the reflexion coefficient of the channel for methylurea, when corrected for solute volume exchange and for the water permeability of the lipid membrane, is 0.54. The channels are impermeable to these two solutes which would seem to rule out flow interaction and require a reflexion coefficient close to 1.0 for both. Thus, two solutes can give very different osmotic flow rates through a semi-permeable pore, a result at variance with both classical theory and the KK formulation. The use of KK equations to analyze osmotic volume changes, which results in a single hybrid reflexion coefficient for each solute, may explain the discrepancy in the literature between such results and those where the equations have not been employed. Osmotic reflexion coefficients substantially different from 1.0 cannot be ascribed to the participation of other 'hidden' parallel aqueous channels consistently with known properties of the membrane. Furthermore, we show that this difference cannot be due to second-order effects, such as a solute-specific interaction with water in only part of the channel, because the osmosis is linear with driving force down to zero solute concentration, a finding which also rules out the involvement of unstirred-layer effects. Reflexion coefficients smaller than 1.0 do not necessitate water-solute flow interaction in permeable aqueous channels; rather, the osmotic behaviour of impermeable molecular-sized pores can be explained by differences in the fundamental nature of water flow in regions either accessible or inaccessible to solute, created by a varying cross-section of the channel.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Fluid mechanical and electrostatic study on the osmotic flow through cylindrical pores

When two solutions differing in solute concentration are separated by a porous membrane, the osmotic pressure will generate a net volume flux of the suspending fluid across the membrane; this is termed osmotic flow. We consider the osmotic flow across a membrane with circular cylindrical pores when the solute and the pore walls are electrically charged, and the suspending fluid is an electrolytic solution containing small cations and anions. Under the condition in which the radius of the pores and that of the solute molecules greatly exceed those of the solvent as well as the ions, a fluid mechanical and electrostatic theory is introduced to describe the osmotic flow in the presence of electric charge. The interaction energy, including the electrostatic interaction between the solute and the pore wall, plays a key role in determining the osmotic flow. We examine the electrostatic effect on the osmotic flow and discuss the difference in the interaction energy determined from the nonlinear Poisson-Boltzmann equation and from its linearized equation (the Debye-Hückel equation).     

MEMBRANE CHARACTERISTICS AND OSMOTIC BEHAVIOR OF ISOLATED ROD OUTER SEGMENTS

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na⁺ influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na⁺ influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na⁺ permeability constant of about 2.8 x 10^-6 cm/s and an osmotic permeability coefficient of greater than 2 x 10^-3 cm/s.     

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.     

Osmosis and solute—Solvent drag

In 1903, George Hulett explained how solute alters water in an aqueous solution to lower the vapor pressure of its water. Hulett also explained how the same altered water causes osmosis and osmotic pressure when the solution is separated from liquid water by a membrane permeable to the water only. Hulett recognized that the solute molecules diffuse toward all boundaries of the solution containing the solute. Solute diffusion is stopped at all boundaries, at an open-unopposed surface of the solution, at a semipermeable membrane, at a container wall, or at the boundary of a solid or gaseous inclusion surrounded by solution but not dissolved in it. At each boundary of the solution, the solute molecules are reflected, they change momentum, and the change of momentum of all reflected molecules is a pressure, a solute pressure (i.e., a force on a unit area of reflecting boundary). When a boundary of the solution is open and unopposed, the solute pressure alters the internal tension in the force bonding the water in its liquid phase, namely, the hydrogen bond. All altered properties of the water in the solution are explained by the altered internal tension of the water in the solution. We acclaim Hulett's explanation of osmosis, osmotic pressure, and lowering of the vapor pressure of water in an aqueous solution. His explanation is self-evident. It is the necessary, sufficient, and inescapable explanation of all altered properties of the water in the solution relative to the same property of pure liquid water at the same externally applied pressure and the same temperature. We extend Hulett's explanation of osmosis to include the osmotic effects of solute diffusing through solvent and dragging on the solvent through which it diffuses. Therein lies the explanations of (1) the extravasation from and return of interstitial fluid to capillaries, (2) the return of luminal fluid in the proximal and distal convoluted tubules of a kidney nephron to their peritubular capillaries, (3) the return of interstitial fluid to the vasa recta, (4) return of aqueous humor to the episcleral veins, and (5) flow of phloem from source to sink in higher plants and many more examples of fluid transport and fluid exchange in animal and plant physiology. When a membrane is permeable to water only and when it separates differing aqueous solutions, the flow of water is from the solution with the lower osmotic pressure to the solution with the higher osmotic pressure.     

Permeability of the E. coli cell membrane for thiourea, dimethylsulfoxide and glycerol

Permeability of cell envelope of Escherichia coli was investigated by the method of osmotic shock for thiourea, dimethylsulfoxide and glycerol. Characteristic times were obtained for passive permeation of these reagents. Variation of r in consequence dimethylsulfoxide less than thiourea less than glycerol was determined. The dependence of tau on the concentration of permeant reagents was observed and it was found that tau was decreased with increasing concentration of the permeant solute which was connected with the modification of the cytoplasmic membrane.     

Epithelial cell volume modulation and regulation

Summary Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.