Osmotic equilibrium and elastic properties of eel intestinal vesicles.

Changes in volume of intestinal brush border membrane vesicles of the European eel Anguilla anguilla were measured as vesicles were exposed to media with different osmotic pressures. Preparing the vesicles in media of low osmotic pressure allowed the effects of a small hydrostatic pressure to become a significant factor in the osmotic equilibration. By applying LaPlace's law to relate pressure and volume and assuming a linear relation between membrane tension and area expansion, we estimate an initial membrane tension at 4.02 x 10(-5) N cm(-1) and an area compressibility elastic modulus at 0. 87 x 10(-3) N cm(-1). The elastic modulus estimate falls in the low range of values reported for membranes from other tissues in other species. This lower modulus quantitatively accounts for why eel intestinal vesicles show measurable changes in volume in hypotonic media while rabbit kidney vesicles do not.

Kinetics of water transport in eel intestinal vesicles.

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P(f) for eel vesicles was not affected by pCMBS and was measured at 1.6 x 10(-3) cm sec(-1) at 23 degrees C, a value lower than 3.6 x 10(-3) cm sec(-1) exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E(a) of 14.7 Kcal mol(-1) for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol(-1) determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E(a), as well as the low P(f) for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P(f) dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 x 10(-3) N cm(-1), compares favorably with the corresponding value, 0. 87 x 10(-3) N cm(-1), estimated from measurements at osmotic equilibrium.

Mechanical properties of brush border membrane vesicles from kidney proximal tubule.

The mechanical properties of brush border membrane vesicles, BBMV, from rabbit kidney proximal tubule cells, were studied by measuring the initial and final equilibrium volumes of vesicles subjected to different osmotic shocks, using cellobiose as the impermeant solute in the preparation buffer. An elevated intracellular hydrostatic pressure was inferred from osmotic balance requirements in dilute solutions. For vesicles prepared in 18 and 85 mosM solutions, these pressures are close to 17 mosM (290 mm Hg). The corresponding membrane surface tension is 6.0 x 10(-5) N cm-1 while the membrane surface area is expanded by at least 2.2%. When these vesicles are exposed to very dilute solutions the internal hydrostatic pressure rises to an estimated 84 mosM (1444 mm Hg) just prior to lysis. The corresponding maximal surface tension (pre-lysis) is 18.7 x 10(-5) N cm-1, and the maximal expansion of membrane area is 6.8%. The calculated area compressibility elastic modulus was 2.8 x 10(-3) N cm-1.

Water permeability of brush border membrane vesicles from kidney proximal tubule.

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability Pf evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. Pf values were of the order of 4 x 10(-3) cm sec-1 independent of the osmolarity of the preparation buffer. Arrhenius plots of Pf vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol-1. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of Pf caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule.

Membrane stress causes inhibition of water channels in brush border membrane vesicles from kidney proximal tubule.

Brush border membrane vesicles (BBMV) from rabbit kidney proximal tubule cells, prepared with different internal solute concentrations (cellobiose buffer 13, 18 or 85 mosM) developed an hydrostatic pressure difference across the membrane of 18.7 mosM, that causes a membrane tension close to 5 x 10(-5) N cm-1. When subjected to several hypertonic osmotic shocks an initial delay of osmotic shrinkage (a lag time), corresponding to a very small change in initial volume was apparent. This initial osmotic response, which is significantly retarded, was correlated with the initial period of elevated membrane tension, suggesting that the water permeability coefficient is inhibited by membrane stress. We speculate that this inhibition may serve to regulate cell volume in the proximal tubule.

Independence of water and solute pathways in human RBCs.

We have investigated the permeability of the human red blood cell to four di-hydroxy alcohols, 1,2PD (1,2 propanediol), 1,3PD (1.3 propanediol), 1,4BD (1,4 butanediol), and 2,3BD (2,3 butanediol), and to water by using a recently developed ESR stopped-flow method which is free from artifacts found in light scattering methods. Numerical solutions of the Kedem-Katchalsky equations fit to experimental data yielded the following permeability coefficients: P1,2PD = 3.17 x 10(-5) cm sec-1, P1,3PD = 1.75 x 10(-5) cm sec-1, P1,4BD = 2.05 x 10(-5) cm sec-1, P2,3BD = 7.32 x 10(-5) cm sec-1. Reflection coefficients (sigma) were evaluated by comparing data fit with assumed values of sigma = 0.6, 0.8 and 1.0. In all four cases the best fit was obtained with sigma = 1.0. Treatment of cells with PCMBS (para-chloro mercuri-benzene-sulfonate) was followed by a large (> 10-fold) decrease in water permeability with virtually no change in alcohol permeability. We conclude that these alcohols do not permeate the water channels to any significant extent, and discuss some of the problems in light scattering measurements of reflection coefficients that could lead to erroneous values for sigma.

Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial.

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that < or = 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22 degrees C is calculated suggesting a channel mechanism.

Hemoglobin polymerization in sickle cells studied by circular polarized light scattering.

We have studied intracellular polymerization of hemoglobin S in suspensions of small populations of sickle cells using circular polarized light scattering. We argue that the preferential scattering of right circular polarized light (as expressed by measurements of the S14 Mueller scattering matrix element) directly reflects the amount of polymer inside cells. This technique has made it possible to investigate the effect of oxygen tension, cell density and osmotic stress on intracellular hemoglobin polymerization. Using S14 to determine hemoglobin polymer, we show that the polymer increases with deoxyhemoglobin concentration, that cells containing higher hemoglobin concentrations show significantly more polymer than cells containing less hemoglobin, and that polymerization occurs in sickle-trait cells in hypertonic solutions as the oxygen tension in the suspension is reduced. We also present kinetic measurements of polymerization, including that induced by osmotic shock. Finally, we demonstrate that the total light scattered (S11 Mueller scattering matrix element) that is routinely measured simultaneously with S14 can be used to estimate the percent of reduced (deoxy) Hb in the sample. These experiments demonstrate the potential of this technique to monitor hemoglobin polymerization simultaneously with oxygen dissociation under a wide variety of physiological conditions.

Urea transport deficiency in Jk(a-b-) erythrocytes.

The activity of the urea transporter was determined in human erythrocytes of the Kidd blood type Jk(a-b-) by measuring unidirectional urea and thiourea fluxes in tracer flux experiments and urea net fluxes in light-scattering experiments. When compared with control cells, Jk(a-b-) cells exhibited diminished urea and thiourea fluxes and lacked the kinetic characteristics of mediated transport, suggesting that in these cells urea and thiourea moved only by simple diffusion through the lipid bilayer. Control experiments showed that anion, water, and ethylene glycol permeabilities were the same in Jk(a-b-) and control cells. These experiments thus demonstrate that Jk(a-b-) cells lack mediated urea transport and strongly support the notion that urea transport function is separate from the transport for anions, water, and ethylene glycol, probably because different proteins are responsible for these transport functions.

Temperature- and concentration-dependence of urea permeability of human erythrocytes determined by NMR.

The temperature- and concentration-dependence of [13C]urea self-exchange across the human red cell membrane has been determined by NMR measurements of T1 (spin-lattice) relaxation times. T1 for intracellular label is 17 s, which is much longer than the urea exchange time across the cell membrane (about 0.5 s). T1 for urea in extracellular solution is quenched with 17 mM of impermeable Mn2+ in less than 2 ms. Hence the observed T1 (corrected for intracellular decay) is a measure of urea exchange across the cell membrane. The method is tested by showing both PCMBS and increasing concentrations of urea lengthen T1. Urea exchange permeability, defined as Purea = flux/conc, can be described by Purea = Vmax/(K1/2 + conc). Studies of temperature-dependence showed that activation energies were strongly dependent on both temperature and concentration. However, this apparently anomalous behavior was resolved into two well-behaved functions, K1/2 and Vmax, with linear Arrhenius plots and apparent 'activation energies' of 15.5 and 12.4 kcal/mol, respectively. These were used to construct an equation for calculating Purea at any concentration and temperature. Assuming a simple channel model with single binding, K1/2 becomes the dissociation equilibrium constant for the site with delta H degree = 15.5 kcal/mol and delta S degree = 51.8 cal/(mol.deg); dissociation is entropically driven.

ESR measurement of time-dependent and equilibrium volumes in red cells.

Red cell water volumes were measured using ESR methods during transient osmotic perturbation, and under equilibrium conditions. Cell water contents were determined using the spin label Tempone (2,2,6,6-tetramethyl piperidine-N-oxyl) and the membrane impermeable quencher potassium chromium oxalate. With appropriate corrections for intracellular viscosity and changes in cavity sensitivity, equilibrium cell water measured both by electron spin resonance (ESR) and wet minus dry weight methods gave excellent agreement in solutions from 243-907 mOsm. Intracellular viscosities determined from the Tempone correlation times in the same cells gave values ranging from 9-47 centipoise at 21 degrees C. Osmotically induced transient volume changes were measured using Tempone and an ESR stopped-flow configuration. The Tempone response time was estimated at 17 msec compared to 250-350 msec for normal water relaxations. Nonlinear least square solutions to the Kedem-Katchalsky equations including a correction for the finite Tempone permeability gave 0.029 and 0.030 cm/sec for the osmotic permeability of RBCs in swell and shrink experiments, respectively. In stopped-flow experiments accurate water flux data are obtained very soon after challenging cells and do not require baseline subtractions. These results represent significant improvements over conventional light scattering techniques which necessitate corrections for long lasting optical artifacts (200-300 msec), and baseline drifts.

A method to distinguish between pore and carrier kinetics applied to urea transport across the erythrocyte membrane.

Permeability coefficients (P) measured at various penetrant concentrations (C) by the perturbation method can be plotted to distinguish simple diffusion, simple pore kinetics and simple carrier kinetics as follows: for simple diffusion, 1/P = constant; for a simple pore, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C; for a simple carrier, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C + 1/Po[1/(K3K4)] C2 where Po is the maximal permeability at zero penetrant concentration and the K's are combinations of kinetic constants defining each of the transport steps. (Kin and Ko are the half-saturation constant for zero-trans efflux and influx, respectively; K3 is the half-saturation constant for equilibrium exchange, and K4 is related to the mobility of the free carrier). In human erythrocytes, permeability coefficients for diethylene glycol were constant suggesting simple diffusion. For glucose, a plot of 1/P versus concentration was nonlinear indicating carrier kinetics. Plots of 1/P versus penetrant concentrations gave straight lines with positive slopes for urea in human and bovine erythrocytes and for methylurea in human red cells, indicating these penetrants follow simple pore kinetics or simple carrier kinetics in which K4 is very large.

Amine and carboxylate spin probe permeability in red cells.

Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation.

Amine spin probe permeability in sonicated liposomes.

Permeabilities for an homologous series of amine nitroxide spin probes were measured in liposomes of varying composition by an electron paramagnetic resonance (EPR) method. Results show that the rate-limiting step in permeation is not adsorption/desorption at the aqueous/membrane interface for two probes in phosphatidylcholine/phosphatidic acid liposomes and for one probe in phosphatidylcholine/cholesterol/phosphatidic acid liposomes. Accordingly, we interpret observed selectivity patterns for the entire series of probes in liposomes and red cells in terms of the properties of the bilayer interior. Results are inconsistent with simple applications of either free volume or hydrocarbon sheet models of nonelectrolyte permeation. In the former case, it was found that liposomes do not select against these probes on the basis of molecular volume. In the latter case, probe permeabilities are all much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. Together with the results of previous studies of spin-labeled solutes in membranes, as well as studies of lipid dynamics in membranes, these latter results suggest that the rate-limiting region in nonelectrolyte permeation is not in the center of the bilayer, but in the relatively ordered acyl chain segments near the glycerol backbone.

Modification of the erythrocyte membrane dielectric constant by alcohols.

Aliphatic alcohols are found to stimulate the transmembrane fluxes of a hydrophobic cation (tetraphenylarsonium, TPA) and anion (AN-12) 5-20 times in red blood cells. The results are analyzed using the Born-Parsegian equation (Parsegian, A., 1969, Nature (London) 221:844-846), together with the Clausius-Mossotti equation to calculate membrane dielectric energy barriers. Using established literature values of membrane thickness, native membrane dielectric constant, TPA ionic radius, and alcohol properties (partition coefficient, molar volume, dielectric constant), the TPA permeability data is predicted remarkably well by theory. If the radius of AN-12 is taken as 1.9 A, its permeability in the presence of butanol is also described by our analysis. Further, the theory quantitatively accounts for the data of Gutknecht and Tosteson (Gutknecht, J., Tosteson, D.C., 1970, J. Gen. Physiol. 55:359-374) covering alcohol-induced conductivity changes of 3 orders of magnitude in artificial bilayers. Other explanations including perturbations of membrane fluidity, surface charge, membrane thickness, and dipole potential are discussed. However, the large magnitude of the stimulation, the more pronounced effect on smaller ions, and the acceleration of both anions and cations suggest membrane dielectric constant change as the primary basis of alcohol effects.

Osmotic stability of red cells in renal circulation requires rapid urea transport.

Urea transport by the human erythrocyte occurs via an asymmetric-facilitated diffusion system with high Michaelis constants and high maximal velocities; the equivalent permeability in the limit of zero urea concentration is approximately 10(-3) cm/s (J. Gen. Physiol. 81: 221-237, 239-253, 1983). A physiological role for this system is revealed by numerical integration of the appropriate equations that show that rapid urea transport is essential for red cell stability in passing through the renal medulla. The calculation compares two cells. Cell A transports urea with permeability characteristics of normal red cells; cell B has urea permeability similar to lipid bilayers. On entering the hypertonic medulla, both cells shrink, but only B swells on leaving the medulla. The osmotic stress for cell B is greater than for A. Cell B is close to hypertonic hemolysis in the medulla and to hypotonic hemolysis in the cortex. Cell B remains swollen for some time after its exit; the resulting decreased deformability presents a hazard if B reenters the microcirculation. Furthermore, cell B removes a significant fraction of the filtered load of urea and compromises the osmotic gradients in the medulla.

Calcium-induced transient potassium efflux in human red blood cells.

Human red blood cells pretreated with low-ionic-strength solutions and resuspended in saline respond biphasically to extracellular Ca. At first, addition of Ca causes a large transient K efflux of as much as 600 mM . liter cell H2O-1 . h-1; this is followed by a decrease in K flux below control levels. The first phase (phase I) resembles the Gardos effect in several respects. It is inhibited by oligomycin, by external K, and by increased exposure time to Ca. Further, the K permeability of phase I is similar to that of the Gardos effect (5 X 10(-8)-9 X 10(-8) cm/s), and the cells hyperpolarize in a low-K medium when Ca2+ is added. However, phase I is not identical to the Gardos phenomenon. For example, La, which prevents the Gardos response, is ineffective on phase I. Moreover, external Ba prevents the development of phase I but not the Gardos response, whereas internal Ba prevents the Gardos response. Attempts to demonstrate a Ca leak or pump failure during phase I have failed; passive Ca movements of both treated and normal cells are similar. The results suggest that low-ionic-strength solution exposes Ca-sensitive sites to the external medium; these sites are maintained when the cells are returned to saline.

Transport of hydrophobic ions in erythrocyte membrane: I. Zero membrane potential properties.

The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism.

Apparent activation volumes of hydrophobic ions and carriers in planar lipid bilayers.

A gas-free high-pressure cell has been developed to measure planar bilayer conductances induced by hydrophobic ions and ionophores as a function of hydrostatic pressure. Plots of log conductance versus pressure for valinomycin and nonactin-mediated potassium transport in egg phosphatidyl cholinedecane membranes are essentially linear over a pressure range of 1 to 818 atm. Calculated activation volumes give similar results for both nonactin and valinomycin yielding values of + 48 and + 42 cc/mole, respectively. The valinomycin activation volume agrees reasonably well with the results obtained by Johnson and Miller (Biochim. Biophys. Acta 375:286-291, 1975) for K+-valinomycin transport in liposomes. In contrast to the activation volumes for nonactin and valinomycin, relaxation measurements of tetraphenyl boron (TPB) and dipicrylamine (DPA) give very small values of less than 5 cc/mole for the translocation rate constant, ki. Similarly, steady-state conductance measurements on tetraphenyl arsonium (TPA) and carbonylcyanide m-chlorophenylhydrazone (CCCP), give small values of 6 and 7 cc/mole, respectively. These low figures do not support transport theories based on the formation of bilayer holes or kinks (H. Träuble, J. Membrane Biol. 4:193-208, 1971). The low values for TPB and TPA are especially interesting because their cross-sectional areas are not much different than those of valinomycin and nonactin. Pressure-induced changes in membrane dielectric constant and thickness which lower the bilayer electrostatic barrier could explain the low values for the hydrophobic ions. Additionally, larger activation volumes might be expected for carriers such as nonactin and valinomycin that undergo significant rearrangement and change in hydration during surface complexation of cations.