Permeability studies on red cell membranes of dog, cat, and beef

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm⁴/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.     

The volume dependence of the erythrocyte water diffusion permeability

The product of the water diffusion permeability and the membrane area of a human erythrocyte has been found to be nearly independent of the cell volume. The product was measured by an NMR technique. This result conflicts with previous flow tube determinations but is in accord with recent measurements of the hydraulic permeability and various solute permeabilities. The results are consistent with the major part of the water fllux traversing the membrane through a fixed number of pores. There may also be a minor non-pore flux. It appears to be practicable to follow volume changes in the red blood cell by an NMR technique.     

Protonophore anion permeability of the human red cell membrane determined in the presence of valinomycin

Summary A transport model for translocation of the protonophore CCCP across the red cell membrane has been established and cellular CCCP binding parameters have been determined. The time course of the CCCP redistribution across the red cell membrane, following a jump in membrane potential induced by valinomycin addition, has been characterized by fitting values of preequilibrium extracellular pHvs. time to the transport model. It is demonstrated, that even in the presence of valinomycin, the CCCP-anion is well behaved, in that the translocation can be described by simple electrodiffusion. The translocation kinetics conform to an Eyring transport model, with a single activation energy barrier, contrary to translocation across lipid bilayers, that is reported to follow a transport model with a plateau in the activation energy barrier. The CCCP anion permeability across the red cell membrane has been calculated to be close to 2.0×10^–4 cm/sec at 37°C with small variations between donors. Thus the permeability of CCCP in the human red cell membrane deviates from that found in black lipid membranes, in which the permeability is found to be a factor of 10 higher.     

The Measurement of Permeability to water in Disks of Storage Tissues4

1. Experiments have been performed to examine methods to determinehydraulic conductivity and diffusional permeability in disksof red beet (Beta vulgaris) and Jerusalem artichoke (Helianthustuberosus). 2. The half-time for the weight change caused by transferringdisks between pure water and a sucrose solution, or vice-versa,has been shown to be linearly dependent on the square of thedisk thickness. 3. The efflux kinetics of sucrose from these disks are alsocharacterized by a half-time linearly dependent on the squareof the disk thickness. 4. It is concluded that extracellular solute diffusion, andnot hydraulic conductivity, is the principal rate-controllingfactor in experiments of this type. 5. Efflux studies with tritiated water also show a characteristichalf-time which is linearly dependent on the square of the diskthickness. The cell membrane does not constitute a sufficientbarrier to diffusion fot its diffusional permeability to bedetermined.     

The State of Water in Human and Dog Red Cell Membranes

The apparent activation energy for the water diffusion permeability coefficient, P_d, across the red cell membrane has been found to be 4.9 ± 0.3 kcal/mole in the dog and 6.0 ± 0.2 kcal/mole in the human being over the temperature range, 7° to 37°C. The apparent activation energy for the hydraulic conductivity, L_p, in dog red cells has been found to be 3.7 ± 0.4 kcal/mole and in human red cells, 3.3 ± 0.4 kcal/mole over the same temperature range. The product of L_p and the bulk viscosity of water, η, was independent of temperature for both dog and man which indicates that the geometry of the red cell membrane is not temperature-sensitive over our experimental temperature range in either species. In the case of the dog, the apparent activation energy for diffusion is the same as that for self-diffusion of water, 4.6–4.8 kcal/mole, which indicates that the process of water diffusion across the dog red cell membrane is the same as that in free solution. The slightly, but significantly, higher activation energy for water diffusion in human red cells is consonant with water-membrane interaction in the narrower equivalent pores characteristic of these cells. The observation that the apparent activation energy for hydraulic conductivity is less than that for water diffusion across the red cell membrane is characteristic of viscous flow and suggests that the flow of water across the membranes of these red cells under an osmotic pressure gradient is a viscous process.     

The Effect of the Unstirred Layer on Human Red Cell Water Permeability

A study has been made of water entry into human red blood cells under an osmotic pressure gradient. The measurements were made using a rapid reaction stop flow apparatus, whose construction, calibration, and performance are described in detail. Red cell volume changes were determined from 90° scattered light. The permeability coefficient for water entry under a relative isosmolar concentration of 1 to 1.5 was found to be 0.22 ± 0.01 cm⁴/sec osmol, which agrees well with our previously published value. The experiments were also designed to measure the thickness of the unstirred layer around the6 red cells. This was found to be 5.5 ± 0.8 µ under the present experimental conditions. It is concluded that our previously measured permeability coefficient for water entrance under a diffusion gradient does not require correction on account of the unstirred layer.     

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.     

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

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, 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 $\text{T}{}_1$ 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 $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ 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.     

Reflection coefficients of permeant molecules in human red cell suspensions

The Staverman reflection coefficient, sigma for several permeant molecules was determined in human red cell suspensions with a Durrum stopped-flow spectrophotometer. This procedure was first used with dog, cat, and beef red cells and with human red cells. The stopped-flow technique used was similar to the rapid-flow method used by those who originally reported sigma measurements in human red cells for molecules which rapidly penetrate the red cell membrane. The sigma values we obtained agreed with those previously reported for most of the slow penetrants, except malonamide, but disagreed with all the sigma values previously reported for the rapid penetrants. We were unable to calculate an "equivalent pore radius" with our sigma data. The advantages of our equipment and our experimental procedure are discussed. Our sigma data suggest that sigma is indirectly proportional to the log of the nonelectrolyte permeability coefficient, omega. Since a similar trend has been previously shown for log omega and molar volume of the permeant molecules, a correlatioo was shown between sigma and molar volume suggesting the membrane acts as a sieve.     

An experimental study of some indigenous drugs with special reference to hydraulic permeability

The effect of commonly used indigenous drugs for hepatic disorders i.e. Tinospora cordifolia, (Guduchi/Amrita), Andrographis paniculata (Kalmegha), Picrorhiza kurroa (Kutki), Phyllantnus niruri (Bhoomyamalaki) and Berberis aristata (Daruharidra) was tested on the hydraulic permeability of water in the presence of bile salt through a transport cell model. The data on hydraulic permeability were calculated as t (time). JV = Lp x AP, where Lp = hydraulic conductivity and AP is the pressure difference. It was observed that the value of controlled hydraulic permeability (0.49 x 10(-8) M3 S(-1) N(-1)) decreased in the presence of indigenous drugs and bile salt. The results suggest that these drugs might have the cell membrane stabilizing property which may lead to prevention of the toxic effect of bile salts in various hepatic disorders.     

Simulations of NMR-detected diffusion in suspensions of red cells: the effects of variation in membrane permeability and observation time

Monte Carlo random-walk simulations of diffusion in virtual lattices of cells have been used to study and characterize diffusion-coherence phenomena that arise when pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiments are conducted on human red blood cell (RBC; erythrocytes) suspensions. These coherence effects are manifest as diffraction-like patterns when the normalized PGSE signal intensities are plotted as a function of the spatial wave vector q in so-called q-space plots. q-Space analysis is sensitive to small changes in cell morphology, cell size, membrane transport rates, hematocrit, and packing arrangement. In the present study we used simulations to predict the effect of varying the time over which diffusion is measured (the "observation time" or "diffusion time") and the permeability of the membrane on the form of q-space plots. Thus we predict that inhibiting water exchange across the human RBC membrane, such that the value of the permeability coefficient is reduced by approximately an order of magnitude below the normal physiological value, will effectively render the membrane impermeable on the timescale of the PGSE NMR experiment; further inhibition will therefore result in negligible reduction in the measured root-mean-square displacement (r.m.s.d.) of diffusing water as a function of the observation time. The work also underscores the importance of using an appropriate experimental observation time if q-space data are to be used to estimate compartment dimensions and interbarrier spacing, and illustrates an expeditious method for determining this value.     

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.     

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.     

Diffusion and permeation of cations in human and dog erythrocytes

The kinetics of movement of tracer Na into human and dog red cells have been studied. The time courses of these processes and of K transfer were compared with the theoretical time course for saturation of a flat sheet having a resistive surface. The theoretical and the experimental curves when plotted against t^½ have a considerable portion which is linear; on the basis of this resemblance the results are interpreted in terms of a permeability constant and an internal diffusion constant. It is supposed that selective adsorption acts to bring about concentration of K in the human cell and that the bulk of the Na of that cell is present in a thin outer region, while the K is in the interior. The action of strophanthin is to remove the usual limit to the Na capacity of the cell and it is proposed that the Na region increases in thickness at the expense of the K region. Omission of K from the medium has a similar result. Na uptake into poisoned cells measured either with tracer or as a net gain has a linear dependence upon t^½ after a delay. The permeability of the dog cell to Na is reduced when K is added to the medium; this may be due to the formation of an outer K-rich region which imposes a resistance to Na movement.     

Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells

The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration K_i = ; 3 ± 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = ; 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.     

An Adenosinetriphosphate-Activated Hemolytic System : III. Effect of rabbit hemolytic factor on the membrane permeability of human red cells

Lysis of human red cells in vitro by an enzyme obtained from rabbit red cell hemolysates and the inhibition of this lytic activity by human stroma have been shown to require Mg⁺⁺ and ATP, and ATP utilization has been demonstrated in both reactions. We find that sodium or potassium ions are also required for the lytic phenomenon and that they enhance the inhibition. The rate of hemolysis is not affected by the internal concentrations of these ions but depends only on the external concentration. The rate of influx and efflux of Na²²Cl and K⁴²Cl in surviving red cells is greatly enhanced both during and after treatment with rabbit hemolytic factor whereas the entry of C²⁴-sucrose, a small foreign molecule, is mediated only in the presence of hemolytic factor. Glycolysis neither protects against lysis nor enhances the activity of this system, and cardiac glycosides which are known inhibitors of active transport of ions also have no effect. It appears that lysis in this system is not brought about by increased active transport of ions into the cell but that the rabbit factor degrades or combines with some membrane component, altering permeability and resulting in increased diffusion, first of sodium and potassium ions and other small molecules, and finally of large molecules (hemoglobin) out of the cell.     

Interaction between phloretin and the red blood cell membrane

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.     

Determination of Solute Permeability in Chara Internodes by a Turgor Minimum Method : Effects of External pH

The analysis of Sha'afi et al. (Sha'afi, Rich, Mickulecky, Solomon 1970 J Gen Physiol 55: 427-450) for determining solute permeability in red blood cells has been modified and applied to turgid plant cells. Following the addition of permeating solute to the external medium, a biphasic response of cell turgor can be measured with the pressure probe in isolated internodes of Chara corallina. After an initial decrease in turgor due to water flow (water phase), turgor increases due to the uptake of the solute (solute phase) until the original turgor is reattained. From the pressure/time course in the neighborhood of the minimum turgor, the permeability of the osmotic solute can be determined. The data obtained by the minimum method for rapidly permeating (ethanol, methanol) and slowly permeating (formamide, dimethylformamide) solutes are similar to those calculated from the half-time of pressure changes during the solute phase and to data obtained by other workers using radioactive tracers. The methods employing the pressure probe were applied to examine the effect of high pH (up to pH 11) on the membrane permeability. There appeared to be no effect of high pH on the permeability coefficients, reflection coefficients, and hydraulic conductivity.     

An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II).

Nucleated cells (Ehrlich ascites tumor cells or L strain cells) and human red blood cells (RBC)-ghosts were mixed and fused by ultraviolet-inactivated HVJ (Sendai virus). The cell mixture was stained with FITC conjugated anti-RBC ghost antiserum and then applied to FACS II apparatus. The apparatus sorted mononuclear cells fused with RBC-ghosts from the cell mixture on the basis of both the light scattering and fluorescence profiles. When the same procedure was carried out on a mixture containing cells and intact human RBC, the cells sorted by this method were cells into which hemoglobin had been injected. The sorted cells were capable of forming colonies in culture. This sorting method may be useful for collecting cells in which macromolecules have been injected artificially by fusion of RBC-ghosts enclosing macromolecules.     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique.

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn2+, and is used in vivo. The mean lifetime of water insed human erythrocytes was found to be 17 ms at 24 degrees C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.