An NMR study of the temperature dependence of the coefficient of water self-diffusion through lipid bilayer membranes

The temperature dependence of the coefficient of water self-diffusion through plane-parallel lipid multilayers of the phospholipid dioleoylphosphatidylcholine oriented on a glass support has been studied in the temperature range of 20-60 degrees C by the method of NMR with magnetic field pulse gradient. The values of the coefficients of transbilayer water diffusion are by four orders of magnitude less than for bulky water and ten times less than the coefficients of lateral diffusion of the lipid under the same conditions. The temperature dependence of the coefficient of water diffusion is described by the Arrhenius law with an apparent activation energy of about 41 kJ/mol, which far exceeds the activation energy for the diffusion of bulky water (18 kJ/mol). The experimental data were analyzed using a "dissolving-diffusion" model, by simulating the passage of water through membrane channels, and by analyzing the exchange of water molecules in states with different modes of translation mobility, including pore channels and bilayer "defects". Each of the approaches used made it possible to take the significance of bilayer permeability for the apparent energy of activation of water diffusion into account and estimate the energies of activation of water diffusion in the hydrophobic moiety of the bilayer, which were found to be close to the values for bulky water. The coefficients of water diffusion in the system under examination and the coefficients of permeation of water through the bilayer were estimated, and the effect of bilayer "defects" on the coefficients of water diffusion along and across bilayers was studied.     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.     

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.     

Red Cell Membrane Permeability Deduced from Bulk Diffusion Coefficients

The permeability coefficients of dog red cell membrane to tritiated water and to a series of[¹⁴C]amides have been deduced from bulk diffusion measurements through a "tissue" composed of packed red cells. Red cells were packed by centrifugation inside polyethylene tubing. The red cell column was pulsed at one end with radiolabeled solute and diffusion was allowed to proceed for several hours. The distribution of radioactivity along the red cell column was measured by sequential slicing and counting, and the diffusion coefficient was determined by a simple plotting technique, assuming a one-dimensional diffusional model. In order to derive the red cell membrane permeability coefficient from the bulk diffusion coefficient, the red cells were assumed to be packed in a regular manner approximating closely spaced parallelopipeds. The local steady-state diffusional flux was idealized as a one-dimensional intracellular pathway in parallel with a one-dimensional extracellular pathway with solute exchange occurring within the series pathway and between the pathways. The diffusion coefficients in the intracellular and extracellular pathways were estimated from bulk diffusion measurements through concentrated hemoglobin solutions and plasma, respectively; while the volume of the extracellular pathway was determined using radiolabeled sucrose. The membrane permeability coefficients were in satisfactory agreement with the data of Sha'afi, R. I., C. M. Gary-Bobo, and A. K. Solomon (1971. J. Gen. Physiol. 58:238) obtained by a rapid-reaction technique. The method is simple and particularly well suited for rapidly permeating solutes.     

On the theoretical aspects of tracer exchange in red blood cells

An analytical solution is presented for isotopic exchange between red blood cells and a finite surrounding medium where the intracellular diffusion is taken into account for two models of the red blood cell, the rectangular and the spherical. The solution is applied on experimental results from the literature with exchange of water, methanol, formamide, urea and ethylene glycol. It is seen that if the diffusion coefficient for those substances is less than 10^?7 cm²/sec, then the previous assumption about exchange between two well-stirred compartments becomes doubtful.     

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 inside human erythrocytes was found to be 17 ms at 24°C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

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.     

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.     

Sheep Erythrocyte Membrane Binding and Transfer of Long-Chain Fatty Acids

We have studied binding and membrane transfer rates of unsaturated long-chain fatty acids in sheep red cells, as previously done for human red cells, in order to elucidate the transport mechanism. Observed differences must be assigned to the different composition of the membrane in the two species. Equal surface areas of the membranes of the two species have similar binding capacities and affinities for palmitic-, linoleic-, oleic- and arachidonic acid at 37°C. The competitive bindings of linoleic- and arachidonic acid as well as the distribution of bound arachidonic acid on the two sides of the membrane are not different in the two species. However, the rate constants for membrane transfer in sheep are less than half of those measured previously for human ghosts. This finding is confirmed by the exchange efflux kinetics of ghosts containing albumin-bound fatty acid. Studies of sheep ghost membranes with oleic-, arachidonic- and linoleic acid reveal a proportionality between the membrane transfer rate constants and the number of fatty acid double bonds, as found previously for human ghost membrane, and the effect of double bonds is in harmony with a large negative activation entropy for diffusion through the membrane. The established replacement of lecithin by sphingomyelin with a low unsaturation fatty acid index in sheep membranes probably causes a lower transversal lipid phase fluidity. Double bonds diminish the flexibility of hydrocarbon chains and thus the large negative activation entropy of diffusion across the membrane. The smaller transfer rate constants of the three unsaturated fatty acids in sheep membranes support the hypothesis that the transfer is diffusion in protein defined annular lipid domains and not carrier mediated. Received: 24 February 1999/Revised: 10 June 1999     

Effective activation energy of enzymatic and nonenzymatic reactions. Evolution-imposed requirements to enzyme structure

The difference of the activation energies in a protein globule and water has been treated in terms of the theory of an elementary act of charge transfer reaction with regards to the energy spent on the transfer of charged reactants from water into the protein. The protein was treated as a structureless dielectric with a given optical and static dielectric constants surrounded by the aqueous phase. Reactions of different types (charge exchange between reactants, charge separation, neutralization, etc.) have been analyzed both under prevalence of purely electrostatic effects and under considerable nonelectrostatic contributions to the activation energies. It is shown that for all one-electron and most multi-electron reactions involving two reaction centres the energy spent for charged reactant transfer from water into protein is greater than the concomitant activation energy gain. The same effect takes place in a number of cases for multi-centre processes as well. To overcome the entropy hindrances, the reactants and catalysts must combine into multiparticle complexes, i.e. form microscopic regions of low dielectric constant. This results in increased effective activation energy as compared to reactions in water. It has been hypothesized that in order to make up for this loss the evolution has selected the proteins which are characterized by considerable intraglobular permanent electric fields. The presence in proteins of high concentrations of strongly polar peptide groups renders them advantageous in this respect over other polymers that are less polar.     

Temperature dependence of water self-diffusion through lipid bilayers assessed by NMR

The temperature dependence of the coefficient of water self-diffusion across plane-parallel multib-ilayers of dioleoylphosphatidylcholine oriented on a glass support was studied in the 20–60°C range by pulsed field gradient NMR. The coefficient for transbilayer diffusion of water proved almost four orders of magnitude smaller than for bulk water, and 10 times smaller than that for lateral diffusion of lipid under the same conditions. The temperature dependence obeyed the Arrhenius law with apparent activation energy of 41 kJ/mol, much higher than that for bulk water (18 kJ/mol). The experimental data were analyzed using the “dissolution-diffusion” model, by simulating water passage through membrane channels, and by examining water exchange in states with different modes of translational mobility, including pore channels and bilayer defects. Each approach could take into account the role of bilayer permeability and assess the apparent activation energy for water diffusion in the hydrophobic part of the bilayer, which proved close to the value for bulk water. Estimates were obtained for water diffusion coefficients in the system, coefficients of bilayer permeability for water, and the influence of bilayer defects on the lateral and transverse diffusion coefficients.     

Mechanism of anion transport in red blood cells: role of membrane proteins.

A number of anionic chemical probes that inhibit anion permeability of red blood cells are localized in a membrane protein of about 100,000 daltons, known as band 3. The inhibitory site has been explored using a series of disulfonic stilbene compounds. It apparently contains three positive charges, probably amino groups. Two probes, pyridoxal phosphate and N-(4-azido-2-nitropheyny)-2-amino ethyl sulfonate, are transported by the anion system but can be fixed in an irreversible bond under specified conditions (reduction with NaBH4 or exposure to light, respectively). Data obtained with these compounds indicate that the inhibitory site in band 3 is the transport site itself. Band 3 protein is exposed in part on the outside of the cell but it is also hydrophobically associated with membrane lipid. A model is proposed in which the band 3 protein acts as an anion permeation channel through the lipid bilayer. Near the outer aspect of the channel an anion binding site can undergo a local conformational change allowing a one-for-one anion exchange across a diffusion barrier.     

Characterization of oxalate transport by the human erythrocyte band 3 protein

This paper describes characteristics of the transport of oxalate across the human erythrocyte membrane. Treatment of cells with low concentrations of H2DIDS (4,4'-diisothiocyanatostilbene-2,2'- disulfonate) inhibits Cl(-)-Cl- and oxalate-oxalate exchange to the same extent, suggesting that band 3 is the major transport pathway for oxalate. The kinetics of oxalate and Cl- self-exchange fluxes indicate that the two ions compete for a common transport site; the apparent Cl- affinity is two to three times higher than that of oxalate. The net exchange of oxalate for Cl-, in either direction, is accompanied by a flux of H+ with oxalate, as is also true of net Cl(-)-SO4(2-) exchange. The transport of oxalate, however, is much faster than that of SO4(2-) or other divalent anions. Oxalate influx into Cl(-)-containing cells has an extracellular pH optimum of approximately 5.5 at 0 degrees C. At extracellular pH below 5.5 (neutral intracellular pH), net Cl(-)- oxalate exchange is nearly as fast as Cl(-)-Cl- exchange. The rapid Cl(- )-oxalate exchange at acid extracellular pH is not likely to be a consequence of Cl- exchange for monovalent oxalate (HOOC-COO-; pKa = 4.2) because monocarboxylates of similar structure exchange for Cl- much more slowly than does oxalate. The activation energy of Cl(-)- oxalate exchange is about 35 kCal/mol at temperatures between 0 and 15 degrees C; the rapid oxalate influx is therefore not a consequence of a low activation energy. The protein phosphatase inhibitor okadaic acid has no detectable effect on oxalate self-exchange, in contrast to a recent finding in another laboratory (Baggio, B., L. Bordin, G. Clari, G. Gambaro, and V. Moret. 1993. Biochim. Biophys. Acta. 1148:157-160.); our data provide no evidence for physiological regulation of anion exchange in red cells.     

Nonelectrolyte diffusion through lecithin-water lamellar phases and red-cell membranes

The longitudinal diffusion of a homologous series of monoamides through lecithin-water lamellar phases with aqueous channel widths of 16–27 Å has been studied. The diffusion coefficients relative to water of the hydrophilic amides, formamide and acetamide, depend logarithmically on solute molar volume, as previously demonstrated in human red cells. Aqueous diffusion of amides in red-cell membranes is similar to that in a lecithin-water phase of aqueous channel width less than 16 Å, the smallest channel width used. Partition coefficients of the lipophilic amides, valeramide and isovaleramide, between lecithin vesicles and water are 1.64 and 1.15 at 20 °C. These data enabled us to compute a valeramide diffusion coefficient of 6.5 · 10⁻⁷cm² · s⁻¹ at 20 °C in the lipid region of a lamellar phase containing 30% water about one order of magnitude greater than the diffusion coefficient of spin-labelled analogs of phosphatidylcholine. The discrimination between the permeability coefficients of valeramide and isovaleramide is more than twice as great in the human red cell as between lipid diffusion coefficients in a phase containing 8% water. This suggests that the lipid region of the human red cell is more highly organized than lipid in the lecithin-water lamellar phase.     

Water permeability and lipid composition of toad urinary bladder: The influence of temperature

Summary The water diffusional permeability, its activation energy and the lipid composition were studied in urinary bladders from toads adapted to different temperatures. It was observed that the unidirectional water flux greatly depends on the temperature at which the experiments are performed. This dependence is greater in the animals adapted to higher temperatures. Toads adapted to cold show strong reduction in the activation energy for water diffusion permeability (from 11.4±1.9 kcal·mol^–1 to 4.4±1.1 kcal·mol^–1) and an increase of 30% in the amount of total lipids from bladder epithelial cells. There were no significant changes in the phospholipid/cholesterol ratio, composition of the paraffinic chains or protein concentration between toads adapted to both temperatures. The possibility that water translocates through the mucosal border of the toad bladder by partitioning in the polar zone and diffusioning between the hydrocarbon chains of the membrane lipids and that cold adaptation would induce a stronger packing of lipids in the membrane is discussed.     

Modifications of ouabain inhibition of xenon accumulation in red blood cells. Implications regarding cell water structuring

Ouabain is known to depress uptake of xenon by red blood cells, but it is now found that the depressive effects of phenolsulfonphthalein (PSP) or heavy water are even more marked. Ouabain partly reverses those effects in a manner suggesting it exerts a constant action on xenon exchange regardless of the presence or absence of PSP or D2O. Processes analogous to exchange diffusion between certain anesthetic substances and xenon were also observed. The theoretical consequences of the findings are discussed.     

Fluorescence depolarization studies of red cell membrane fluidity. The effect of exposure to 1.0-GHz microwave radiation

The internal viscosity of human red blood cell membranes was investigated during exposure to continuous wave 1.0-GHz microwave radiation using fluorescence measurements of a lipid seeking molecular probe, diphenylhexatriene. Samples were exposed in a Crawford cell arranged so that fluorescence was measured during microwave exposure; specific absorption rates calculated from electrical measurements were approximately 0.6, 2 and 15 W/kg. Measurements were obtained at selected temperatures between 15 °C and 40 °C and as a function of the duration of exposure at 23 °C. Arrhenius-type plots of the temperature profile data were linear and showed no difference between exposed and control samples. The exposure duration data also showed no difference between exposed and control samples except for a small effect of elevated temperature at the highest exposure. The activation energy for motion of the fluorescent probe in its environment within the membrane lipid was not affected by the application of the microwave energy and no evidence for a lipid phase transition was found. These results indicate that the increased cation efflux from red cells, observed by others at certain transition temperatures during microwave exposure, was more likely to have been caused by alteration of the membrane bound protein than by changes in the lipid constituents of the red cell membrane.