Abnormal erythrocyte anion exchange in Alzheimer disease.

CONTEXT: Several abnormalities have been described in red blood cells of patients with Alzheimer disease (AD), but to date none of these has been confirmed by a second, independent study. Erythrocyte anion exchange has been reported to be abnormal in AD; we have developed a new technique for measuring anion exchange. OBJECTIVES: To confirm the abnormality of erythrocyte anion exchange in AD and to determine whether the phenomenon has potential for clinical utility. DESIGN: Comparison of patients with probable AD to age-matched controls. SETTING: University hospital and ambulatory clinic. METHODS: Chloride-bicarbonate exchange was measured in erythrocyte ghosts resealed with a fluorescent probe of chloride concentration. RESULTS: Erythrocyte anion exchange is abnormal in AD. This difference appears in citrate but not EDTA anticoagulant. Mahalanobis's generalized distance between the 2 populations is 1.7, and a discriminant function derived from our technique classifies 82% of the study population in accordance with the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria. Receiver operating characteristic analysis demonstrates the possibility of choosing cutoffs with high sensitivity and specificity. CONCLUSIONS: Measurement of red blood cell anion exchange may be useful in classifying patients with AD. The dependence of this phenomenon on anticoagulant suggests the involvement of platelet activation or complement fixation.

Kinetics of chloride-bicarbonate exchange across the human red blood cell membrane.

We use a fluorescent probe of [Cl-], 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), to study Cl-/HCO-3 exchange in human erythrocyte ghosts in a stopped-flow apparatus at 4 degrees C. The quench constant of SPQ in our Cl-/HCO-3/HPO=4 system at pH 7.4 is 0.065 +/- 0.005 mM-1. The time course of Cl-/HCO-3 exchange does not follow a single exponential function at 4 degrees C and we propose an extended ping-pong model in which slippage is explicitly considered in order to account for this phenomenon. The solution of the system of equations generated by our model is a double exponential function which fits the time course of Cl-/HCO-3 exchange. Our results confirm the predictions of the model concerning the functional dependence of the two rate constants. One rate constant (k1) is independent of medium composition; it is determined by the sum of the two slippage rate constants and its value is 1.04 +/- 0.14 sec-1. The other rate constant (k2) varies inversely with [Cl-]; the regression line is 1/k2 = 18.8 sec - 0.095 mM-1sec [Cl-].

Permeability and reflection coefficients of urea and small amides in the human red cell.

Measurement of the transport parameters that govern the passage of urea and amides across the red cell membrane leads to important questions about transport of water. It had initially been thought that small protein channels, permeable to water and small solutes, traversed the membrane (see Solomon, 1987). Recently, however, very strong evidence has been presented that the 28 kDa protein, CHIP28, found in the red cell membrane, is the locus of the water channel (see Agre et al., 1993). CHIP28 transports water very rapidly but does not transport small nonelectrolytes such as urea. The irreversible thermodynamic parameter, sigma i, the reflection coefficient, is a measure of the relationship between the permeability of the solute and that of water. If a solute permeates by dissolution in the membrane, sigma i = 1.0; if it permeates by passage through an aqueous channel, sigma i < 1.0. For urea, Goldstein and Solomon (1960) found that sigma urea = 0.62 +/- 0.03 which meant that urea crosses the red cell membrane in a water-filled channel. This result and many subsequent observations that showed that sigma urea < 1.0 are at variance with the observation that CHIP28 is impermeable to urea. In view of this problem, we have made a new series of measurements of sigma i for urea and other small solutes by a different method, which obviates many of the criticisms Macey and Karan (1993) have made of our earlier method. The new method (Chen et al., 1988), which relies upon fluorescence of the intracellular dye, fluorescein sulfonate, leads to the corrected value, sigma urea,corr = 0.64 +/- 0.03 for ghosts, in good agreement with earlier data for red cells. Thus, the conclusion on irreversible thermodynamic and other grounds that urea and water share a common channel is in disagreement with the view that CHIP28 provides the sole channel for water entrance into the cell.

Inhibition of red cell urea flux by anion exchange inhibitors.

When they studied the chemical properties of red cell anion exchange inhibitors such as DIDS (4,4'-diisothiocyanate-2,2'-stilbene disulfonate), Barzilay et al. (1979) Membr. Biochem. 2, 227-254 also examined the benzene sulfonates. These molecules are structurally similar to half a DIDS molecule and are also specific anion exchange inhibitors with ID50 values measured in mM, rather than microM, as for the stilbene disulfonates. We have studied several inhibitors of the benzene sulfonate (BS) class and found that they also inhibit red cell urea flux by up to 92% and stimulate water flux by up to 58%. The values of Kinhib,app for urea flux inhibition are the same as the ID50 values for anion flux inhibition; covalent DIDS completely suppresses the inhibition. These observations strongly suggest that the effect on urea flux is caused by BS binding at the stilbene site. Comparative studies on the short chain amides exclude lipid solubility and solute molar volume as factors that affect these BS actions. Kstim,app for water flux stimulation is also related to the anion exchange ID50 values; covalent DIDS suppresses the water flux stimulation. These observations on urea and water fluxes are consistent with a common driver, located at the stilbene site, which is responsible for the BS actions on urea, water and anion fluxes. The subsequent steps are independent with separate effectors to modulate each of the individual fluxes. These effectors are presumably located in different regions of the protein or proteins and carry out their separate processes by allosteric means.

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase.

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3-/Cl- exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3-. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.

Initial steps of alpha- and beta-D-glucose binding to intact red cell membrane.

The kinetics of the initial phases of D-glucose binding to the glucose transport protein (GLUT1) of the human red cell can be followed by stopped-flow measurements of the time course of tryptophan (trp) fluorescence enhancement. A number of control experiments have shown that the trp fluorescence kinetics are the result of conformational changes in GLUT1. One shows that nontransportable L-glucose has no kinetic response, in contrast to D-glucose kinetics. Other controls show that D-glucose binding is inhibited by cytochalasin B and by extracellular D-maltose. A typical time course for a transportable sugar, such as D-glucose, consists of a zero-time displacement, too fast for us to measure, followed by three rapid reactions whose exponential time courses have rate constants of 0.5-100 sec-1 at 20 degrees C. It is suggested that the zero-time displacement represents the initial bimolecular ligand/GLUT1 association. Exponential 1 appears to be located at, or near, the external membrane face where it is involved in discriminating among the sugars. Exponential 3 is apparently controlled by events at the cytosolic face. Trp kinetics distinguish the Kd of the epimer, D-galactose, from the Kd for D-glucose, with results in agreement with determinations by other methods. Trp kinetics distinguish between the binding of the alpha- and beta-D-glucose anomers. The exponential 1 activation energy of the beta-anomer, 13.6 +/- 1.4 kcal mol-1, is less than that of alpha-D-glucose, 18.4 +/- 0.8 kcal mol-1, and the two Arrhenius lines cross at approximately 23.5 degrees C. The temperature dependence of the kinetic response following alpha-D-glucose binding illustrates the interplay among the exponentials and the increasing dominance of exponential 2 as the temperature increases from 22.3 to 36.6 degrees C. The existence of these interrelations means that previously acceptable approximations in simplified reaction schemes for sugar transport will now have to be justified on a point-to-point basis.

Effect of pCMBS on anion transport in human red cell membranes.

The kinetics of binding of the mercurial sulfhydryl reagent, pCMBS (p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea transport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl- transport, we measured Cl-/NO3- exchange by fluorescence enhancement, using the dye SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase, correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl- binding to band 3 by the pCMBS anion. The ID50 of 2.0 +/- 0.1 mM agrees with other organic sulfonates, but is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracellular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with Ki = 5.5 +/- 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with Ki = 2.1 +/- 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to the cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.

Conformational changes in human red cell membrane proteins induced by sugar binding.

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

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.

High proton flux through membranes containing antidiuretic hormone water channels.

Antidiuretic hormone (ADH) stimulation of toad urinary bladder granular cells causes simultaneous increases in transepithelial water and H+ permeabilities (PF and PH+, respectively), suggesting that ADH-elicited water channels inserted into granular cell apical membranes might be permeable to both water and H+. We have previously used self-quenching fluorophores entrapped within endocytic vesicles selectively retrieved from water-permeable apical membranes to measure vesicle PF. The membranes of these vesicles possess an extremely high PF such that our measurements provide only minimum estimates of vesicle PF and have limited our ability to quantitate the properties of ADH water channels. We therefore quantitated vesicle PH+ using similar rapid mixing techniques. Vesicle PH+ was 5.1 +/- 0.5 x 10(-3) cm/s. Activation energy of this process was 3.6 +/- 0.6 kcal/mol, indicative of H+ flux through an aqueous channel. The mercurial reagent, para-chloromercuribenzenesulfonate (PCMBS), which inhibits ADH-stimulated transepithelial PF in intact bladders by 50-60%, inhibited vesicle PH+ by 55%. N-Ethylmaleimide and phloretin, which do not alter ADH-stimulated PF, did not affect vesicle PH+. We conclude that membranes containing ADH water channels possess substantial PH+ that likely reflects proton flux through water channels. The apparent high PH+ of the ADH water channel may have important implications for intracellular trafficking of these water channels in ADH-responsive epithelial cells.

Transport parameters in the human red cell membrane: solute-membrane interactions of hydrophilic alcohols and their effect on permeation.

A systematic study has been made of the three coefficients that describe the human red cell membrane transport of a series of short straight-chain hydrophilic alcohols: the permeability coefficient, omega i, the reflection coefficient, sigma i, and the hydraulic conductivity, Lp. Ethylene glycol transport is saturable with Km = 220 +/- 50 mM; there is a second, low-affinity, ethylene glycol site which inhibits water transport (K = 570 +/- 140 mM, max. inhib. = 90 +/- 10%). sigma eth gly = 0.71 +/- 0.04 which is significantly less than 1 (n = 6, P less than 0.001), as are sigma i for six other alcohols (n = 23), thus providing strong thermodynamic evidence that water and these alcohols cross the red cell membrane, at least in part, in an aqueous channel. The solute/membrane frictional coefficient, fsm, for all seven alcohols has been determined and found to decrease monotonically as membrane permeability increases. The red cell membrane has been perturbed by treatments with phenylglyoxal and BS3 (bis(succinimidyl suberate]; these treatments are accompanied by correlated modulation of both ethylene glycol and urea permeability. In one set of experiments in control cells, urea permeability is correlated with water permeability; and, in another set, ethylene glycol permeability is correlated with water permeability. All of these observations support the proposition that the urea class of solutes, the ethylene glycol class of solutes and water all cross the membrane through the same aqueous pore. A schematic model of the red cell pore, consistent with the experimental observations, is presented.

Interaction among anion, cation and glucose transport proteins in the human red cell.

The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.

Sites of p-chloromercuribenzenesulfonate inhibition of red cell urea and water transport.

The mercurial sulfhydryl reagent, p-chloromercuribenzene sulfonate (pCMBS), inhibits water and urea fluxes across the human red blood cell membrane. The kinetics and affinities for pCMBS binding to separate water transport and urea transport inhibition sites were previously determined by Toon and Solomon ((1986) Biochim. Biophys. Acta 860, 361-375) in red cells that had been treated with N-ethyl-maleimide (NEM) to block five of the six sulfhydryls on the red cell anion exchange protein, band 3. We have used autoradiographs of gels from NEM-treated cells, labeled with 203Hg-pCMBS, to localize these water and urea transport inhibition binding sites separately and find that both are on band 3. Each site is saturable and the time course of each uptake can be fitted to the equation for a bimolecular association (with negligible dissociation) with time constants in agreement with those of Toon and Solomon. Determination of the binding stoichiometry shows one urea inhibition site and three water inhibition sites for every four band 3 molecules. These results indicate that band 3 plays a role in both urea and water transport and suggest that the functional unit may be a tetramer.

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3.

A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682-1688, 1986) found that C1-/HCO-3 exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4'-diisothiocyano-2,2'-disulfonic stilbene), with a K1 similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4'-dibenzamido-2,2'-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (Ks1 = 93 +/- 24 nM) and a lower affinity site (Ks2 = 430 +/- 260 nM), which are closely similar to values for the red cell of 110 +/- 51 nM for the high-affinity site and 980 +/- 200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). When Cl- replaces citrate in the buffer, the two sites collapse into a single one with Ks1 = 1500 +/- 400 nM, similar to the single Ks1 = 1200 +/- 200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon, J. Membrane Biol. 89:211-223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 microM-1 are characterized by a fast process, tau = 0.14 +/- 0.03 sec, similar to tau = 0.12 +/- 0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell 'band 3' closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.

Modulation of water transport in human red cells: effect of urea.

We have studied the effect of urea on water flux in the human red cell and have found that 500 mosmolal urea inhibits osmotic water transport by 39%. The Ki for urea inhibition of water flux is 550 +/- 80 mosmolal, higher than, but comparable with, the Km of urea transport into the red cell of 220-330 mM given by Mayrand and Levitt (J. Gen. Physiol. 55 (1983) 427) and Brahm (J. Gen. Physiol. 82 (1983) 1). Other amides, such as propionamide and valeramide, as well as methyl-substituted ureas, have similar effects, although an indifferent molecule, such as 0.5 M creatinine, has no effect. Urea can be washed off the inhibition site with buffer, and the effects of urea concentrations as high as 1.2 osmolal are entirely reversible. 500 mosmolal urea also significantly increases the reflection coefficient for ethylene glycol, sigma eth gly, from 0.71 +/- 0.03 in control experiments to 0.86 +/- 0.04 (P less than 0.0005, t-test), and propionamide has a similar effect on sigma eth gly. These results show that urea can modulate ethylene glycol transport through the red cell membrane, and are consistent with, but not proof of, the presence of a single class of aqueous channels through which both ethylene glycol and urea enter the red cell. It is suggested that the physiological purpose of these low-affinity urea sites is to modulate water flow out of the red cell during passage through the regions of 0.5-0.6 M urea in the kidney.

Interrelation of ethylene glycol, urea and water transport in the red cell.

The reflection coefficient, sigma j, which measures the coupling between the jth solute and water transport across a semipermeable membrane, varies between 0 and 1.0. Values of sigma j significantly less than 1.0 provide irreversible thermodynamic proof that there is coupling between the transport of solute and solvent and thus that they share a common pathway. We have developed an improved method for measuring sigma and have used it to determine that sigma ethylene glycol = 0.71 +/- 0.03 and sigma urea = 0.65 +/- 0.03, in agreement with many, but not all, previous determinations. Since both of these values are significantly lower than 1.0, they show that there is a common ethylene glycol/water pathway and a common urea/water pathway. Addition of first one and then two methyl groups to urea increases sigma to 0.89 +/- 0.04 for methylurea and 0.98 +/- 0.4 for 1,3-dimethylurea, consistent with passage through an aqueous pore with a sharp cutoff in the 6-7 A region.

Modulation of water and urea transport in human red cells: effects of pH and phloretin.

It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta 211:104-106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol. 67:381-397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with a KI of 25 +/- 8 microM in reasonable agreement with the KD of 54 +/- 5 microM for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport.