Water Permeability Of Cells Research Articles

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.

Effects of PCMBS on the water and small solute permeabilities in frog urinary bladder

It has been reported that PCMBS (p-chloromercuribenzene sulfonate) blocks the water permeability of red cells and of the tubular kidney membranes. In this study we compare the effects of this mercurial compound on the permeability of water and other small solutes in the frog urinary bladder. We observed that: (i) 5 mM PCMBS applied at pH 5.0 to the mucosal side inhibited the net and unidirectional water fluxes induced by oxytocin without changing the delta Pf/delta Pd ratio. (ii) The oxytocin-induced urea and Na+ influxes were also inhibited by PCMBS. (iii) The unidirectional Cl- movement was first reduced and then increased during the course of PCMBS treatment. (iv) The short-circuit measured at low mucosal Na+ concentration (10 mM), diminished continuously, whereas the transepithelial resistance first increased and then diminished. (v) Mannitol, raffinose, alpha-methyl-glucose, antipyrine, caffeine and Rb+ movements were not changed significantly during the first 26 min of the water permeability inhibition. (i) The ADH-sensitive water, urea and Na+ transport systems were inhibited by PCMBS, (ii) PCMBS did not induce a nonspecific and general effect on the permeability of the membrane during the development of the water permeability inhibition, and (iii) in terms of water channels, the inhibition of water transport with the maintenance of a high Pf/Pd ratio suggests that PCMBS closes the water channels in an all or none manner, reducing their operative number in the apical border of frog bladder.

Water and urea transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum

The permeability properties of the human red cell membrane to various solutes are altered by malarial infection. In the present work we show that the permeability of the red cell membrane to water is also affected by the intraerythrocytic growth of the malaria parasite Plasmodium falciparum, whereas urea permeability appears unchanged. The data from infected cells show decreases in membrane surface area, cell volume, the osmotically active water fraction ( W eff), and osmotic water permeability ( P f) as measured by stopped-flow spectroscopy. On the other hand, the data suggest an increase in diffusive water permeability ( P d) in infected cells with no change in urea permeability when measured by the continuous flow method. The decreased P f P d ratio of infected cell membranes and its implications in the geometry of the red cell membrane water channel or pore are discussed.

Evidence that the glucose transporter serves as a water channel in J774 macrophages.

Water transport across plasma membranes is a universal property of cells, but the route of such transport is unclear. In this study, volume changes of cells of the J774 murine macrophage-like cell line were monitored by recording the intensity of light scattered by the cells. We investigated the effects of several inhibitors of glucose transport on cell membrane osmotic water permeability as calculated from the rates of cell volume change. Cytochalasin B (2.5 micrograms/ml), phloretin (20 microM), and tomatine (3 microM) reversibly blocked glucose uptake into these cells. All three inhibitors reversibly decreased the osmotic water permeability of J774 cells from 89.6 +/- 3.2 to 27.2 +/- 1.4 microns/sec. We conclude that a major component of the osmotic water flow across the plasma membranes of these cells is accounted for by water traversing their glucose transporters.

Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes

A quantitative description of transmembrane water transport requires specification of osmotic (Pf) and diffusional (Pd) water permeability coefficients. Methodology has been developed to measure Pf and Pd simultaneously on the basis of the sensitivity and rapid response of the fluorophore aminonaphthalenetrisulfonic acid (ANTS) to solution H2O/D2O content. Cells loaded with ANTS in an H2O buffer were subjected to an inward osmotic gradient with a D2O buffer in a stopped-flow apparatus. The time courses of cell volume (giving Pf) and H2O/D2O content (giving Pd) were recorded with dual photomultiplier detection of scattered light intensity and ANTS fluorescence, respectively. The method was validated by using sealed red cell ghosts and artificial liposomes reconstituted with the pore-forming agent gramicidin D. At 25 degrees C, red cell ghost Pf was 0.021 cm/s with Pd 0.005 cm/s (H2O/D2O exchange time 7.9 ms). Pf and Pd were inhibited by 90% and 45% upon addition of 0.5 mM HgCl2. The activation energy for Pd increased from 5.1 kcal/mol to 10 kcal/mol with addition of HgCl2 (18-35 degrees C). In 90% phosphatidylcholine (PC)/10% cholesterol liposomes prepared by bath sonication and exclusion chromatography, Pf and Pd were 5.1 X 10(-4) and 6.3 X 10(-4) cm/s, respectively (23 degrees C). Addition of gramicidin D (0.1 micrograms/mg of PC) resulted in a further increment in Pf and Pd of 7 X 10(-4) and 3 X 10(-4) cm/s, respectively. These results validate the new methodology and demonstrate its utility for rapid determination of Pf/Pd in biological membranes and in liposomes reconstituted with water channels.

Permeability studies of lipid vesicles from alkalophilic Bacillus firmus showing opposing effects of membrane isoprenoid and diacylglycrol fractions and suggesting a possible basis for obligate alkalophily

Previous studies of the membrane lipids of extremely alkalophilic bacilli had indicated that both facultative and obligate alkalophiles contained a substantial fraction of isoprenoid lipid as well as high concentrations of cardiolipin. Facultative alkalophiles differed from obligate strains in having a phospholipid fatty acid composition that would be expected to result in a more ordered membrane structure. Current studies of ion permeability in vesicles prepared from lipids from obligately alkalophilic Bacillus firmus RAB and its facultatively alkalophilic strain, OF4, support the suggestion that membranes of the latter strain form a tighter barrier structure, with the difference especially pronounced at near neutral pH values. The water permeability of whole cells and the reflection coefficients for acetamide in vesicles were also consistent with a tighter membrane in the facultatively alkalophilic strain than in the obligately alkalophilic strain. The permeability properties of vesicles prepared from phospholipids from these organisms were studied as a function of the addition of either homologous membrane isoprenoid or diacylglycerol. For each permeability parameter that was assayed, in lipids from both strains, the isoprenoid fraction decreased the permeability, whereas the diacylglycerol fraction increased the permeability of the vesicles to solute.

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder.

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone.

Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al., J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.

Water permeability of human hematopoietic stem cells

It is currently impossible to isolate or identify human hematopoietic progenitor cells from the bone marrow, yet the biophysical properties of these cells are important for the development of techniques to isolate and preserve stem cells for transplantation. Osmotic permeability properties of human bone marrow stem cells were estimated from the kinetics of cell damage in a hypotonic solution measured using in vitro colony assays for multipotential (CFU-GEMM) and committed (BFU-E, CFU-GM) progenitor cells. Cells exposed to a hypotonic solution swell as a result of water influx, and the rate of change of volume is proportional to the hydraulic conductivity of the plasma membrane. Cell damage occurs when the cell volume exceeds the maximum tolerable volume, so the hydraulic conductivity can be estimated from the kinetics of cell damage. For all the progenitor cells studied, the mean value of the hydraulic conductivity was 0.283 μm 3/μm 2/min/atm at 20 °C, with an Arrhenius activation energy of 6.41 kcal/mole. No significant differences were observed in the osmotic properties of the various progenitor cells. These data were used to predict the osmotic responses of human bone marrow stem cells at subzero temperatures during freezing.

Effects of UV‐C and UV‐B on cytomorphology and water permeability of inner epidermal cells of Allium cepa

Changes of cytomorphology and water permeability of inner epidermal cells of Allium cepa L. cvs Spartan and Keep Well were investigated with the light microscope after irradiation with UV‐C (254 nm) and UV‐B (280 and 310 nm). The sequence of the investigated changes of viscosity, protoplasmic streaming, organelle shape and water permeability was the same with 254 as with 280 nm. although a higher dose of 280 nm was needed to produce the same biologically equivalent effect. However, when calculated as a percentage of the lethal dose, the initial doses that produced the effects were the same for 254 and 280 nm. No changes could he observed at the cellular level after the cells were irradiated with 310 nm. The lethal dose depended upon the time between irradiation and observation. At 254 nm it was 1.1 kJ m−2 up to 1 h after irradiation and dropped to 860 J m−2 when measured 24 and 48 h later. At 280 nm a dose of 2.8 kJ m−2 killed the cells within I h while the dose needed after 24 and 48S h was 1.99 kJ m−2. The minimum doses which caused the different cytomorphological effects did not depend upon the observation time. Normal cell structure and functions that were altered immediately alter irradiation did not recover. Doses that were not immediately effective alter irradiation caused no later damage. Doses which increased water permeability were much higher than doses which influenced cytomorphological parameters of the cell.

ADH or theophylline-induced changes in intracellular free and membrane-bound calcium.

Ca2+ is thought to play a role in the enhancement of water permeability of toad urinary bladder epithelial cells by antidiuretic hormone (ADH) or theophylline. This study examined the effects of ADH and theophylline on intracellular free Ca2+ ([Ca2+]i) and total cellular exchangeable Ca2+ in isolated toad bladder epithelial cells. ADH or theophylline enhanced water permeability maximally by 15-25 min after a 4-min lag. 45Ca2+ efflux, a probe for total cellular exchangeable (plasma membrane plus intracellular) Ca2+, was enhanced by ADH within 2 min and returned to control by 8 min. Chlortetracycline fluorescence, a probe for intracellular Ca2+ only, was not affected, suggesting that ADH released only plasma membrane-bound Ca2+. Theophylline enhanced 45Ca2+ efflux and decreased chlortetracycline fluorescence, suggesting release of Ca2+ from intracellular sources. Both agents decreased [Ca2+]i as assessed by quin-2 fluorescence with a time course similar to the enhancement in water permeability. The results suggest that the changes in membrane-bound Ca2+ and [Ca2+]i induced by ADH and theophylline may play a role in the enhanced permeability to water in response to these agents.

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, p- chloromercuribenzene sulfonate , osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton T 1 NMR and obtained a value of 4.0 · 10 −3 cm · s −1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of N- phenyl-1- naphthylamine by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.

Thermodynamics of all-or-none water channel closure in red cells.

The relation of osmotic to diffusional water permeability of human red blood cells was compared after treating the cells with different concentrations of PCMBS (p-chloromercuribenzene sulfonate). After subtracting the PCMBS-insensitive permeability (presumably the water permeability of the lipid bilayer) from each, the ratio of osmotic to diffusional permeability remains invariant (approximately equal to 11) as more and more water channels are inhibited by increasing concentrations of PCMBS. This result implies that the channels close in an all-or-none way and suggests a two-state model. Analysis of the dependence of osmotic water permeability on PCMBS concentration in terms of the model reveals a 1:1 stoichiometry and a dissociation constant for the PCMBS/membrane receptor complex of about 0.019 mM at 37 degrees C. Temperature dependence studies show that the reaction is entropically driven (delta H degrees approximately equal to 25 kcal/mol, delta S degrees approximately equal to 100 cal/mol-deg) and suggest the involvement of hydrophobic interactions.

Auxin increases the water permeability of Rhoeo and Allium epidermal cells

The ability of IAA to increase the permeability of plant cells to water was investigated in epidermal tissue of Rhoeo and Allium. At 10 −7 M, indole-3-acetic acid (IAA) enhanced the rate of deplasmolysis, measured visually, of Rhoeo epidermal cells and the stimulation was detectable as early as 1 min after IAA application. Indole-3-carboxylic acid (ICA), an inactive auxin analog, failed to promote deplasmolysis under similar conditions: 10 −4 M IAA was less stimulatory than 10 −7 M. A microphotometric method was used to monitor osmotically-induced swelling and shrinking of Allium epidermal cell protoplasts. IAA (10 −7 M) increased both the swelling and shrinking rates of Allium protoplasts within seconds of hormone addition. CaCl 2 (10 mM) antagonized auxin-stimulated swelling but had no effect on auxin-induced shrinking. The results suggest a rapid, direct enhancement of the water permeability of the plasma membrane in Rhoeo and Allium epidermal cells.

Diffusional water permeability of human erythrocytes and their ghosts.

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)-sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.

Osmotic water permeability of human red cells.

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.

The permeability of the human red cell to deuterium oxide (heavy water).

Using a stopped-flow device, the osmotic water permeability of human red cells to D2O and H2O was studied as a function of temperature and under the influence of the sulfhydryl reagent paracholoromercuribenzene sulfonic acid (PCMBS), an inhibitor of water transport. The ratio, permeability (D2O)/permeability (H2O) at each temperature can be predicted simply by assuming that permeability varies inversely with macroscopic viscosity. When water permebility is inhibited with PCMBS, this dependency on viscosity vanishes; the inhibited permeabilities in D2O and H2O are indistinguishable.

Water permeability of mammalian cells as a function of temperature in the presence of dimethylsulfoxide: correlation with the state of the membrane lipids.

The water permeability of V-79 Chinese hamster lung fibroblasts was determined by measuring the rate of cell shrinkage in hypertonic medium using a cell sizer. The water permeability appears to follow Arrhenius kinetics as a function of temperature with a sharp discontinuity at 21 degrees C. An activation energy of 7.0 +/- 1.6 kcal/mole was found below 21 degrees C and 22.8 +/- 3.1 kcal/mole above 21 degrees C. The correlation time of rotation of the spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide was measured as a function of temperature in the cellular membranes, and shows a break at 20 degrees C. A discontinuity was also found in the membrane to water partitioning of the spin label 2,2-dimethyl-5-pentyl-5-butyloxazolidine-N-oxide near 20 degrees C. These breaks may correspond to a membrane lipid phase transition. Dimethylsulfoxide, in the concentration range of 0.2--0.5 M, decreases the water permeability by a factor of two.

Water permeability changes studied by 17O nuclear magnetic resonance during differentiation of Friend leukemia cells.

Water permeability of Friend leukemia cells was studied by 17O nuclear magnetic resonance during differentiation induced by dimethyl sulfoxide (Me2SO). While in noninduced cells water permeability was essentially constant during the growth period, in the Me2SO-induced cells there were two distinct periods at which the water permeability was increased by at least an order of magnitude. These periods correspond to approximately one doubling time and 6 days of growth. This change in water permeability is not due to direct interaction of Me2SO with the membrane but must be ascribed to structural changes in the membrane during the course of differentiation.

Water permeability of yeast cells at sub-zero temperatures

A combined cryomicroscopic-multiple nonlinear regression analysis technique has been used to determine the water permeability of the yeast cell Saccharomyces cerevisiae during freezing. The time rate of change in volume of "supercooled" yeast cells was photographically monitored using a "cryomicroscope" which is capable of controlling in a programmable manner both the temperature and the time rate of change in temperature of the cell suspension being studied. Multiple nonlinear regression analysis together with a thermodynamic model of cell water transport during freezing was then used to statistically deduce the subzero temperature dependence of the cell water permeability. The water permeability process for S. cerevisiae being cooled at subzero temperatures was found to be rate-limited by the passage of water through either the plasmalemma, the cell wall, or a combination of these two permeability barriers. The hydraulic water permeability coefficient for yeast at 20 degrees C is approximately 1--2 x 10(-13) cm3/dyne sec, if extrapolation from subzero temperatures to room temperature is permissible, while the apparent activation energy governing the permeability process at subzero temperatures is approximately 45--68 kJ/mol (11--16 kcal/mol).