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Journal of Physiology (1991), 434, pp. 489-506 With 6 figures Printed in Great Britain

EFFECTS OF LOW IONIC STRENGTH MEDIA ON PASSIVE HUMAN RED CELL MONOVALENT CATION TRANSPORT

BY I. BERNHARDT*, A. C. HALL AND J. C. ELLORY From the University Laboratory of Physiology, Parks Road, Oxford OX1 3PT and the * Department of Biophysics, Section Biology, Humboldt University, 1040 Berlin, Germany

(Received 5 July 1990) SUMMARY

1. The effect of low ionic strength media on the residual, i.e. (ouabain + bumetanide + Ca2+)-insensitive, K+ influx was characterized in human red blood cells. 2. This K+ flux was enhanced significantly in isotonic solutions of low ionic strength using sucrose to maintain constant osmolarity. This effect was found for fresh red blood cells as well as for stored (bank) red blood cells. However, the absolute magnitude of K+ influx in solutions of low ionic strength was halved for stored red blood cells. 3. Anion replacement of Cl- by CH3SO4- did not affect residual K+ fluxes, showing that Cl-dependent transport pathways (e.g. the KCl co-transporter) are not involved in the low ionic strength effect. 4. The enhanced K+ influx in low ionic strength media was reversible when the cells were resuspended in a solution of physiological ionic strength. 5. K+ influx measured in light and dense fractions of erythrocytes (separated by centrifugation and corresponding to samples enriched with either 'young' or 'mature' red cells) showed that the low ionic strength effect does not change markedly with cell age. 6. Low ionic strength media elevated residual, i.e. (ouabain + bumetanide + Ca2+)insensitive, influx of both K+ and Na+ by about the same amount. In both cases the flux was linear with concentration in the range investigated (025-10 mM). No significant increase in the uptake of the cations Ca2+ and lysine in low ionic strength solutions could be found. 7. In CH3S04--containing solutions of physiological ionic strength the residual K+ influx was almost independent of cell volume, whereas this flux in CH3S04-containing solutions of low ionic strength declined as cell volume was increased. 8. K+ flux measurements in solutions of different external pH, where NaCl was replaced by sodium gluconate or sodium glucuronate, showed that the reduced ionic strength is of more importance for the enhanced residual K+ influx than the changed transmembrane potential or the changed intracellular pH. However, a small pH dependence could be found, the K+ flux passing through a minimum around pHi 7.3. 9. Hydrostatic pressure enhanced the residual K+ flux in media of low ionic MS 8628

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strength synergistically, so that very large fluxes (> 10 mmol (1 cells)-' h-1) were obtained at 40 MPa. The apparent activation volumes (AV*) for the pressuresensitive K+ flux were - 108 and -69 ml mol-' in low ionic strength or physiological ionic strength solutions respectively. 10. In solutions of physiological ionic strength the residual K+ influx did not vary greatly with temperature (0-37 °C). In low ionic strength solutions this K+ flux was elevated and showed a minimum at about 8 'C. The addition of 1 mM-LiCl inhibited the low temperature-induced K+ influx, the curve falling continuously with reducing temperature. 11. Taking into account the Goldman flux equation, one would expect a threefold decrease of the K+ influx in low ionic strength solution. However, a tenfold increase was observed which cannot be explained on the basis of classical electrodiffusion. 12. It is concluded that the low ionic strength-induced increase of the K+ influx is protein-mediated (which can be influenced by certain manoeuvres, i.e. temperature, pressure) or can occur through regions which allow ion translocation at instabilities between protein and lipid molecules. INTRODUCTION

It has been known for a long time that there is a dramatic increase in unidirectional tracer K+ fluxes as well as in net K+ efflux when human erythrocytes are suspended in isotonic sucrose or lactose solutions of low ionic strength, compared to high ionic strength media, (Davson, 1939; Wilbrandt, 1940; Wilbrandt & Schatzmann, 1960; Carolin & Maizels, 1965; LaCelle & Rothstein, 1966; Donlon & Rothstein, 1969; Bernhardt, Borning & Glaser, 1982; Bernhardt, Donath & Glaser, 1984; Jones & Knauf, 1985). When the Na+-K+ pump is inhibited with ouabain, an increase of the residual ouabain-insensitive sodium efflux in low ionic strength solution has also been demonstrated (Glaser, Bernhardt & Donath, 1980; Bernhardt & Glaser, 1982). These increases in K+ and Na+ efflux have been discussed in terms of a change in the membrane permeability induced by low ionic strength solutions. Using the Goldman flux equation (Goldman, 1943), Donlon & Rothstein (1969) calculated an increased permeability coefficient for K+ with the change in the transmembrane potential which occurred in sucrose medium. Bernhardt et al. (1984) used an extended version of the Goldman flux equation also taking into account the inner and outer surface potentials. Although it was possible to explain the increase in K+ efflux in an isotonic solution of low NaCl concentration without a change in the permeability coefficient, recently it has been suggested that this was unlikely to be a general explanation for this effect, since, under the same conditions which produce a large increase of K+ efflux in human erythrocytes, no significant increase in cation flux in bovine erythrocytes was measured (Bernhardt, Erdmann, Glaser, Reichmann & Bleiber, 1986; Bernhardt, Erdmann, Vogel & Glaser, 1987 b). This is in spite of the fact that by reducing the ionic strength of the media by lowering the extracellular NaCl concentration the transmembrane potential of both bovine and human erythrocytes changes by the same amount (Bernhardt et al. 1986). It is well known that there are a number of specific transport pathways for Na+ and K+ in the red cell membrane (summarized in Bernhardt, Hall & Ellory, 1988; see also e.g. Dunham, Stewart & Ellory, 1980; Ellory, Dunham, Logue & Stewart, 1982;

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Chipperfield. 1986; Escobales & Canessa, 1986). Previous work on the effects of low ionic strength did not take into account the possibility that the increased cation effluxes could be due to a stimulation of one or more of these specific transport pathways. It has been shown, for example, that the furosemide-sensitive component of the K+ efflux also increases in isotonic solutions of low ionic strength (Bernhardt et al. 1987b). This means that the ion fluxes measured in the absence of specific inhibitors, and the permeability coefficients calculated under these conditions in characterizing the low ionic strength effect, do not reflect the residual (leak) flux, but contain elements of carrier-mediated fluxes. It is therefore the aim of the present paper to report the cation fluxes in low ionic strength solutions under conditions where the principal identified mediated transport pathways have been inhibited. Cation uptake was measured both for convenience and to complement previous measurements of K+ efflux (Bernhardt et al. 1984, 1986). Additionally, by measuring K+ and Na+ tracer influxes at varying external Na+ and K' concentrations it was possible to investigate the kinetic behaviour of these fluxes. In an attempt to identify and characterize the low ionic strength effect on residual cation fluxes, it was useful to compare this treatment with other manoeuvres which increase residual fluxes. In this respect measurements of K+ flux in high and low ionic strength media under high hydrostatic pressure, and at low temperature, were performed (e.g. see Blackstock & Stewart, 1986; Hall & Ellory, 1986a). Reducing the ionic strength of the external solution by replacing external NaCl with sucrose will also change the transmembrane potential and the intracellular pH (at constant extracellular pH). To address the question of which of these factors might be relevant to the increase of cation transport in low ionic strength solutions, measurements have been made in sodium gluconate and sodium glucuronate solutions at different extracellular pH values. In this context, Chipperfield & Shennan (1986) described a pronounced increase of the K+ and Na+ tracer efflux in sodium gluconate solutions, in contrast with the findings of Bernhardt et al. (1987b) who could not find a significant increase of the K+ tracer efflux under the same conditions. Our present results identify the low ionic strength-induced component of residual cation (Na+ or K+) influx as separate from the previously identified mediated transport pathways of human red cells. The results are discussed in the context of surface charge and membrane properties. A preliminary account of some of this work has already been published (Bernhardt, Ellory & Hall, 1987a). METHODS

Blood Experiments on human erythrocytes from normal donors were usually carried out on fresh blood withdrawn by venipuncture into heparinized syringes. For comparison in some cases, stored bank blood (1 week old) was used. The blood was washed three times by centrifugation (1000 g, 5 min) in a medium of the following composition (mm): NaCl, 145; glucose, 10; 3-(N-morpholino)propanesulphonic acid (MOPS), 15; pH 7-4 at room temperature. Plasma and buffy coat were removed by aspiration. After this, the cells were washed once in a medium which had the same composition as the flux medium (except for radioisotopes and inhibitors (see individual figure legends for details)). In the experiments where Cl- was replaced by CH3SO4-, the cells were treated as described previously (Dunhamn et al. 1980) in a medium comprising (mM): NaCH3SO4, 165; glucose, 10; MOPS, 15; pH 7-4 at room temperature. This solution as well as the low ionic strength solution of composition (mM): sucrose. 250; glucose, 10; MOPS, 15; pH 7-4 has the same tonicity

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as the 145 mM-NaCl washing solution (300 mosM, measured with a vapour pressure osmometer). Haemoglobin content of red cell suspensions was determined as cyanmethaemoglobin using Drabkin's reagent (Hall & Ellory, 1985). In some experiments fractions of cells enriched in 'young' or 'mature' red cells were prepared by centrifugation by the method of Tucker & Young (1982).

Influx measurements Washed red cells were suspended at about 5 % haematocrit in a total volume of 1 ml of flux medium contained in an Eppendorf 1-5 ml polypropylene microcentrifuge tube. In all experiments ouabain (0-1 mM), bumetanide (0-1 mM) and EGTA (0-1 mM) were present. The incubation temperature was 37 °C except where stated otherwise. The cell suspensions were equilibrated at the flux temperature for 5 min after which a small amount of isotonic [KCl or KCH3SO4 + 86RbCl] or [NaCl + 22NaCl] was added (with radioactivity to give about 50 kBq ml-'). When [K+]. or [Na+]. were varied up to 10 mm in the kinetic experiments, 86Rb (in KCl) or 22Na (in NaCl) was added to give the appropriate final concentration at the start of the flux. Previous experiments have established the validity of 86Rb as a tracer for K fluxes by comparing 86Rb and 42K fluxes in low ionic strength media (Bernhardt, 1981). For these experiments, media comprised [KCl + NaCl] at 145 mm for the physiological ionic strength solution and 10 mm for the low ionic strength solution. In all other experiments [86RbCl + KCl]. (or [86RbCl + KCH3S04]0) was 7-5 mm. The duration of exposure of cells to isotope (i.e. the flux time) at 37 °C was 30 min, whereas at lower temperatures 1-2 h was required for the cells to accumulate comparable radioactivity. The experiments under high hydrostatic pressure were carried out as described by Hall & Ellory (1986a). For the 45Ca uptake experiments, ATP depletion of the cells was necessary. This was achieved following the method of Lew (1971) by incubating red cells for 3 h in saline containing iodoacetamide (6 mM) and inosine (5 mM). Ouabain-sensitive K+ uptake, which was taken as a measure of ATP-driven Na+-K+ pump activity, was reduced to less than 5% of the pre-depletion value, showing the ATP concentration was at low micromolar levels. At the end of the flux period, cells were washed in the medium described below including EGTA (1 mM) to remove free and membrane-bound calcium. The isotope uptake was stopped by centrifugation at 15000 g (10 s), and the supernatants removed by aspiration. The cells were then washed free of extracellular radioactivity by four successive resuspensions and centrifugations (15000 g, 10 s) in ice-cold medium comprising (mM): MgCl2, 107; MOPS, 10 (pH 7 4). The cell pellet was lysed with 0 5 ml of 0-1 % (v/v) Triton X-100 and the protein precipitated by adding 0-5 ml of 5% (w/v) trichloroacetic acid followed by centrifugation at 15000 g for 5 min. The activity of 88Rb in the supernatant was determined by Cerenkov counting in a Packard Tri-Carb liquid scintillation analyser. 22Na activity was determined by liquid scintillation counting using Pico-fluor 40 scintillant (Packard Ltd). The specific activity of the 86Rb+, 22Na+, 45Ca or '4C lysine solutions were determined by counting a suitable sample of the radioactive stock solution. 14C-l-lysine uptake (10 mM; 1 #Ci ml-') was measured as described for 86Rb influx (see above), except that Pico-fluor 40 scintillant was used.

Reagents Inorganic salts, glucose and sucrose were Analytical Reagent Grade, and purchased either from BDH, Fisons or Sigma. Sodium methylsulphate (NaCH3SO4) and potassium methylsulphate (KCH3SO4) were obtained from Hopkins & Williams or Kodak Limited. Ouabain, MOPS, EGTA and sodium glucuronate were obtained from Sigma and sodium gluconate from BDH and Sigma. Bumetanide was a generous gift from Leo Laboratories Ltd. 86Rb, 45Ca, '4C-l-lysine and 22Na were obtained from Amersham International. Statistical treatment of results Each experimental manoeuvre was repeated at least 3 times on blood from different donors. The results are presented either as the mean + S.E.M. of three experiments, or representative data from one of three experiments, in which case the errors represent the S.E.M. of triplicate samples. When errors are not shown they were smaller than the symbols. RESULTS

The effects of low ionic strength medium and anions on passive K+ and Na+ uptake When human red cells are placed in media of low ionic strength (NaCl replaced by sucrose, same osmolarity), there is a marked enhancement of the residual, i.e.

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Fig. 1. Effect of reducing extracellular NaCI concentration (sucrose replacement, constant osmolarity) on residual K+ influx of human red blood cells. K+ influx was measured in the presence of inhibitors (0 1 mM-ouabain, bumetanide, EGTA) as described in Methods. The incubation and flux solution contained 10 mM-glucose and 15 mM-MOPS, pH 7-4. Results shown are for one representative experiment; for pooled data see Table 1. TABLE 1. The effect of the ionic strength of the medium on residual K+ influx in stored and fresh red cells, in the presence of Cl- or CH3SO4-. K+ influx was measured as described (see Methods) in media of physiological or low ionic strength. The solutions also included inhibitors (041 mmouabain, bumetanide, EGTA), 10 mM-glucose, 15 mM-MOPS buffer and were adjusted to pH 7-4 and 300 mosm (see Methods). Results are means + S.E.M. with the number of independent experiments shown in parentheses Stored cells Fresh cells Incubation conditions 0-075+0-013 (10) 04148+0-028 (11) 145 mM-NaCl+7-5 mM-KCl 0-827 + 0-211 (10) 1-452 + 0-262 (9) 250 mM-sucrose + 7-5 mM-KCl 0-084+0 005 (3) 0 165+0 044 (6) 165 mM-NaCH3SO4+ 7-5 mM-KCH3SO4 0-712+0-260 (3) 1-837+0-460 (4) 250 mM-sucrose + 75 mM-KCH3SO4

(ouabain, bumetanide and Ca2+)-insensitive, K+ uptake (Fig. 1). Over the higher range of ionic strength, corresponding to 20-145 mm-NaCl, there was only a small change in K+ transport, but below 20 mm-NaCl there was a dramatic increase in K+ uptake. Figure 1 shows a representative experiment and pooled data on this effect are presented in Table 1, including a comparison between fresh and stored red cells including the effect of anion (Cl-) replacement. It can be seen that for both fresh and stored red blood cells there is about a 10-fold increase in K+ uptake in the low ionic strength solution. However, the absolute magnitude of K+ uptake in both media is halved for stored red blood cells. We have therefore always used fresh blood (less than 4 h old) in subsequent experiments. It is also clear from Table 1, that replacement of Cl- by CH3SO4- (see Methods) had no effect on the measured fluxes. This result eliminates a role for Cl--dependent transport processes in the low ionic strength response. The elevated K+ flux observed in low ionic strength media was reversible when the cells were resuspended in a solution of high ionic strength. Thus, the residual K+ uptake in cells suspended in high and low ionic strength media was initially

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0-130+0-010 and 2-020+0-58 mmol (1 cells)-1 h-1 respectively. The cells in low ionic strength media were then centrifuged and resuspended in the high ionic strength medium and incubated at 37 °C for 30 min. Residual K+ uptake was then determined on a sample of these cells and had decreased to 0-241+0-072 mmol (1 cells)-1 h-',

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5 10 5 External [NaCIl (mM) External [KCI] (mM) Fig. 2. Dependence on the extracellular cation concentration of the residual Na+ and K+ influxes of human red blood cells in solutions of physiological and low ionic strength (see Methods). A, effect of [Na']i on residual Na+ influx. 0, physiological ionic strength; low ionic strength. B, effect of [K+]. on residual K+ influx. 0, physiological ionic strength; *, low ionic strength. The solutions contained 0-1 mM-ouabain, bumetanide, EGTA, and 10 mM-glucose, 15 mM-MOPS, pH 7-4. Results shown are one representative experiment of three.

10

0,

whereas K+ uptake in control cells (i.e. those suspended in high ionic strength media) 0-123 + 0-005 mmol (1 cells)-' h-1. After a further 30 min incubation, K+ uptake in the control cells was unchanged (0-117+0-011), but the K+ uptake of the cells exposed initially to the low ionic strength medium had decreased further (0-181+0-019 mmol (1 cells)-1 h-1; results are means+S.E.M. from one of three similar experiments). K+ uptake was also measured in light and dense fractions of erythrocytes separated by prolonged centrifugation. These fractions correspond to samples enriched with either 'young ' or 'mature' red cells. These experiments were performed in CH3SO4--containing media to suppress the volume-sensitive KCl was

PASSIVE RED CELL CATION TRANSPORT 495 transporter found in young cells (Hall & Ellory, 1986b). In the 'young' cell-enriched fraction K+ uptake was 0 187 + 0-001 in NaCH3SO4 and 2-544 + 0014 mmol (1 cells)-' h-' in the sucrose medium. Corresponding values for K+ fluxes in cells from the dense fraction ('mature' cells) were 0 134 + 0003 and 1P674 + 0043 mmol

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200 250 300 External [sucrose] (mM) Fig. 3. Effect of varying the sucrose concentration in low ionic strength solution (see Methods) on the residual K+ influx of human red blood cells. The solutions contained 0-1 mM-ouabain, bumetanide, EGTA, and 10 mM-glucose, 15 mM-MOPS, pH 74. The experiments were carried out in CH3SO4--containing solutions to prevent Cl--dependent KCl co-transport (see Methods). Results shown are one representative experiment of three.

(1 cells)-' h-1. The stimulation of K+ flux induced by the low ionic strength medium is similar (13-6 x vs. 12-5 x ) in both fractions showing that the effect of low ionic strength does not change markedly with cell age. Characterization of the low ionic strength effect The specificity of the low ionic strength-induced flux was investigated by comparing K+, Na+, Ca2+ and lysine uptake in normal (physiological ionic strength) and low ionic strength media. Additionally, the concentration dependence of the Na+ and K+ flux was measured over the range 0 25-10 mm (Fig. 2A and B). It can be seen that the low ionic strength media elevated Na+ and K+ transport by about the same amount, and in both cases the flux was linear with concentration, showing no signs of saturation over the range studied. This supports the notion that other pathways (e.g. Na+-K+ pump, Na+-K+ co-transport) which would show a non-linear dependence over this range of cation concentrations are not involved in the effect. When Ca2+ uptake at 6-7 mm was determined in ATP-depleted cells, the control flux decreased from 0 473 + 0 010 to 0-302 + 0-002 mmol (1 cells)-' h-' in low ionic strength solution. Lysine uptake (10 mM) was 1-23 + 0-022 mmol (1 cells)-' h-' in physiological saline and increased slightly to 1-35 + 0-018 mmol (1 cells)-' h- (P < 0-05) in low ionic strength solution. These two cations are therefore excluded from the low ionic strength-induced flux pathway, only the two monovalent alkali metal cations showing the large stimulation (about x 10) of transport.

BERNHARDT, A. C. HALL AND J. C. ELLORY The volume dependence of the low ionic strength effect was investigated by varying the sucrose concentration of the external medium between 150 and 300 mm. These experiments were carried out in CH3S04--containing media to prevent volume-sensitive KCl co-transport (Hall & Ellory, 1986b). Results for one of three 496

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Fig. 4. Effect of extracellular pH on the residual K+ influx of human red blood cells in solutions of physiological ionic strength (NaCl), low ionic strength (NaCl replacement by sucrose), or sodium gluconate or sodium glucuronate solutions of physiological ionic strength (NaCl replacement) (see Methods). The solutions contained 0.1 mM-ouabain, bumetanide, EGTA, and 10 mM-glucose, 15 mM-MOPS. The open columns represent a pHo of 7'4, and the hatched columns a pH, of 6-1. The values given for intracellular pH (pH,) and transmembrane potential (AVf) are calculated according to Glaser (1979). Results (means+ s.E.M.) are pooled data from three independent experiments.

similar experiments are presented in Fig. 3. The K+ influx in low ionic strength medium declined as cell volume increased. In physiological ionic strength solution the residual K+ influx was independent of cell volume (0 097 + 0 003 mmol (1 cells)-1 h-1 in shrunken cells; 0 103 ± 0-004 mmol (1 cells)-1 h-1 in swollen cells) which is in agreement with earlier findings (Hall & Ellory, 1986a).

Influence of pH and membrane potential The effects of suspending cells in sucrose media include changes in transmembrane potential, ionic strength and intracellular pH. In Fig. 4 we compare fluxes measured at two external pH values (6-1 and 7 4) and in either a physiological medium, sucrose or impermeant anion solutions. In each case we have included in Fig. 4 the calculated intracellular pH and membrane potential values. At either external pH, sucrose medium gives a large stimulation of residual K+ uptake. In contrast, fluxes measured

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in sodium gluconate or sodium glucuronate were increased only modestly at pH. 7-4 and decreased at pH, 6 1, conditions which give the same changes in transmembrane potential and pH, as the sucrose media. We can conclude therefore that sucrose medium is not acting by altering these parameters, but rather by lowering ionic strength. In these experiments it was important to use pure sodium gluconate. With sodium gluconate from Sigma Chemical Company we found increased fluxes and some lysis (data not shown), which may explain the discrepancy between the results in Fig. 4 and those of Chipperfield & Shennan (1986). Chipperfield & Shennan (1986) also reported that sodium gluconate from the same source increased passive cation fluxes in human red cells, in contrast to our results (Fig. 4). Sodium glucuronate from Sigma or sodium gluconate from BDH did not produce these anomalous effects (Fig. 4). When the intracellular pH dependence of K+ influx at physiological ionic strength was studied the flux passed through a minimum around pH 7 3, since at both lower (6 2) and higher (8 3) pHi values the flux was about 2-fold greater (Fig. 4).

Pressure dependence High hydrostatic pressure has two major effects on cation transport in human red cells. It activates the latent KCl co-transport pathway and also stimulates an anionindependent monovalent cation (e.g. K+ or Na+) pathway in these cells (Hall & Ellory, 1986a). In the present experiments we have eliminated the first component by measuring transport in red cells suspended in CH3S04--containing media. The results presented so far show that the characteristics of the low ionic strengthinduced flux are similar to those previously described as a high hydrostatic pressureinduced passive flux in human red cells (Hall & Ellory, 1986a), e.g. linear concentration dependence, no Na+-K+ selectivity, Cl- independence and reduction on cell swelling. Experiments were therefore carried out changing both parameters (pressure, ionic strength) simultaneously (Fig. 5). Hydrostatic pressure enhanced the fluxes measured in low ionic strength media synergistically, so that very large (> 10 mmol (1 cells)-' h-1) fluxes were obtained at 40 MPa in the low ionic strength solution. Under these conditions the K+ influx was about two orders of magnitude higher than the residual flux in physiological saline medium at normal pressure. No haemolysis occurred under these conditions. The inset in Fig. 5 presents pooled data for experiments on different donors (n = 4), plotted as the natural logarithm of K+ influx against pressure. The results fit well to a straight line and can be used to obtain the apparent activation volume (AV*) for transport (see Hall & Ellory, 1986a). It can be seen that the slopes for K+ uptake in either low ionic strength or physiological ionic strength solution are significantly different (P < 0-01), yielding values of -108 + 8 and -69 + 4 ml mol-1 respectively. The latter value is in good agreement with the value of about -75 ml mo-1 for residual K+ uptake in NaCH3SO4 medium previously reported (Hall & Ellory, 1986a).

Temperature dependence and influence of monovalent cations Since it has been demonstrated that the residual K+ flux in human red cells suspended in physiological ionic strength media increases with a decrease in temperature from 12 to 0 TC, (Stewart, Ellory & Klein, 1980), we have measured the flux in low ionic strength medium over the temperature range 0-37 °C to determine if there are any similarities between the two stimulatory manoeuvres. Figure 6

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compares K+ influx in sucrose medium with that in physiological NaCl medium as a function of temperature. In addition, data on the effect of 1 mM-LiCl in sucrose medium are given, since it has been shown that Li' is a potent inhibitor of the paradoxical temperature effect (Blackstock & Stewart, 1986). 10 3 2

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Fig. 5. Effect of hydrostatic pressure on the residual K+ influx of human red blood cells as a function of the extracellular NaCH3SO4 concentration (sucrose replacement, constant osmolarity). The solutions contained 01 mM-ouabain, bumetanide, EGTA, and 10 mmglucose, 15 mM-MOPS, pH 7-4. The experiments were carried out in CH3SO4--containing solutions to prevent pressure-stimulated KCI co-transport (see text). Symbols represent conditions as follows: C, 1 MPa; *, 20 MPa; 0, 30 MPa; 0, 40 MPa. The inset shows the natural logarithm of the flux as a function of hydrostatic pressure in physiological ionic strength (0) and low ioniC strength (@) solutions. The linear regressions of the slopes describing the data are 0-027 + 0 005 and 0-042 + 0 003 MPa-1 for K+ influx in physiological and low ionic strength solutions respectively. The corresponding values for the activation volumes are given in the text. Data for the figure are from a single experiment, representative of four; the inset shows pooled data from four independent experiments (mean + S.E.M.).

In NaCl medium the K+ influx does not vary greatly with temperature over the range of 0-25 °C, although the flux at 0 °C is higher than at 8 °C in accordance with previous results (Stewart et al. 1980). This K+ flux in NaCl medium is not sensitive to 1 mM-LiCl addition (data not shown). In sucrose medium, the K+ flux is much larger and shows a more pronounced

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paradoxical effect of lowering temperature, with a clear minimum about 8 'C. In this case the addition of 1 mm-LiCl led to a substantial but not complete inhibition of this low temperature-induced K+ flux, the curve falling continuously with reducing temperature in the presence of LiCl. In four separate experiments, where the flux

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Fig. 6. Effect of reducing temperature on the residual K+ influx of human red blood cells. The solutions contained 0-1 mM-ouabain. bumetanide. EGTA, and 10 mm-glucose, 15 mMMOPS. pH 7 4. Symbols are as follows: 0. physiological ionic strength solution; *, low ionic strength solution; C, low ionic strength solution + 1 mM-LiCl (see MIethods). Results shown are one representative experiment of three.

was measured at 0 °C, K+ influx was 0-055+0-010 mmol (1 cells)- h-1 in NaCl, 0 954 + 0-012 in sucrose and 0 407 + 0 064 in sucrose + 1 mM-Li+. Thus, there is a 61 % reduction of the low ionic strength-induced flux after the addition of 1 mM-LiCl. In a control experiment at 0 °C, where 1 mM-NaCl was added to assess the contribution of ionic strength per se, the inhibition of K+ influx in low ionic strength solution was 15%. Therefore Li+ addition reduces the K+ influx by slightly raising the ionic strength but principally by a more specific inhibitory action. At 37 °C LiCl slightly reduced K+ influx in sucrose medium (Fig. 6) whereas NaCl (1 mM) addition inhibits the low ionic strength effect to the same degree. This indicates that at the higher temperature the reduction of K+ influx by LiCl is due solely to the change in the ionic strength of the medium.

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The present results confirm and extend to influx measurements, previous efflux studies which showed a dramatic increase in K+ transport when human red cells are suspended in low ionic strength media (LaCelle & Rothstein, 1966; Donlon & Rothstein, 1969; Bernhardt et al. 1982, 1984, 1986; Jones & Knauf, 1985). Unlike these earlier studies, we have used selective inhibitors and ion substitution to eliminate the possibility that the Na+-K+ pump, Na+-K+ co-transport, Ca2+activated K+ channel and KCl co-transporter are involved in this effect. The measured fluxes in low ionic strength solutions are relatively large and reversible. One result which emerged in the present work was the difference between fluxes in fresh and stored red cells. Although the stimulation by low ionic strength was about one order of magnitude, for both cell types, K+ influx was halved in stored cells. Several studies have shown differences in transport parameters between fresh and stored red blood cells, e.g. glucose and nucleoside transport (Jarvis, Hammond, Paterson & Clanachan, 1982; Weiser, Razin & Stein, 1983; Plagemann & Wohlhueter, 1984). In those cases, it has been suggested that accumulation of an intracellular inhibitor may be responsible for the effect (Stein, 1986). In the present work we have not pursued this further, but have always used fresh cells. Although, as mentioned above, the use of specific inhibitors can eliminate a role for the Na+-K' pump, Na+-K+ co-transport and Ca2+-activated K+ channels, the case against KCl co-transport being involved in the low ionic strength effect should be made in more detail, since no specific inhibitor was used in this study. The KCl cotransport system in human red cells is dependent on Cl-, i.e. CH3504- replacement suppresses the K+ flux via the KCl co-transport system (Hall & Ellory, 1986b). Table 1 shows, however, that the low ionic strength-induced flux is independent of Cl-. The KCl co-transport is also normally expressed in 'young' human red blood cells (Hall & Ellory, 1986b), although some manoeuvres (NEM (n-ethyl maleimide) treatment, hydrostatic pressure, cell swelling, acid pH) can induce it even in 'mature' cells (Ellory, Hall & Amess, 1987). Volume sensitivity, i.e. activation by cell swelling is an important characteristic of KCl co-transport (Ellory & Hall, 1988). The low ionic strength effect, however, decreases slightly on cell swelling (Fig. 3; cf. pressure effects (Hall & Ellory, 1986a)). Further, the KCl system excludes Na+ whereas the low ionic effect is shown equally for Na+ fluxes (Fig. 2A). The low ionic strength-induced fluxes reported here cannot be identified with any of these criteria and we therefore conclude that KCl co-transport is not involved in the present effect of low ionic

strength. In addition, it is known that human red blood cells suspended in sucrose solutions with the same osmolarity as the physiological NaCl solutions have a smaller volume (Glaser et al. 1980). This shrinkage is.,not responsible for the elevated K+ influx observed in low ionic strength solutions. Thus Fig. 3 shows that in sucrose medium changing the osmolarity by up to 200 mosm only slightly influences the K+ influx. Previous attempts have been made to explain the increased K+ efflux on the basis of electrodiffusion, by applying the Goldman equation (Donlon & Rothstein, 1969). These authors proposed that there was a\ change in membrane permeability with changing transmembrane potential. There seems no a priori justification for this

PASSIVE RED CELL CATION TRANSPORT 501 assumption, since flux is necessarily related linearly to driving force for electrodiffusion. A further refinement took into account the inner and outer surface potential and provided an adequate explanation of the K+ efflux data without a need for membrane permeability to vary with transmembrane potential (Bernhardt et al. 1984). However, applying the Goldman flux equation in either its classical or extended form to the present influx results would not be satisfactory, since a decreased K+ influx in solution of low ionic strength should occur under conditions where the transmembrane potential is positive (low external chloride conditions) unless unreasonable large positive surface potential values are assumed. This point was explored further by altering external anion concentration and adding the impermeant anions gluconate or glucuronate. Under these conditions, in marked contrast to sucrose, there was only a small increase in K+ influx, although transmembrane potential and pHi were equivalent in both cases. Recently Bernhardt et al. (1986), using the Cl- distribution ratio, have measured transmembrane potential more directly under these ionic conditions, obtaining values of -5.7 + 47 mV in normal physiological medium, and + 45-1 + 3-7 mV in low ionic strength medium, confirming the theoretical values presented in Fig. 4. Our conclusions from these experiments must be that the effect of sucrose is due neither to the change in the transmembrane potential nor pH. There is some indication that the induced flux is sensitive to internal pH, since from Fig. 4 at pHi of either 6-2 or 8-3 the residual K+ uptake is higher than at 7 3, but this effect is small relative to the 10-fold effect of low ionic strength at pHo = 7*4 (Fig. 1). Recently, Chipperfield & Shennan (1986) and Zade-Oppen, Tosteson & Adragna (1988) have demonstrated a small pH dependence for residual K+ transport in human red cells. The obvious difference inherent in the sucrose solutions used presently is the low medium ionic strength which has dramatic effects on surface charge and the conformation of membrane components (see Bernhardt et al. 1988). Earlier findings by Bolingbroke & Maizels (1959), who described a significant reduction of cation fluxes induced by lactose solutions (i.e. low ionic strength) after the addition of small amounts of Ca2+, are in agreement with such an explanation. Kracke & Dunham (1987) and Halperin, Brugnara, Tosteson, Van Ha & Tosteson (1989) have reported that DIDS and methazolamide-treated human red blood cells exhibit voltage-dependent changes in passive cation fluxes. These fluxes (Na+, K+, Ca2+) which are activated by increasing the transmembrane potential show different characteristics from those reported here and by others (Jones & Knauf, 1985). A comparison between the fluxes induced by low ionic strength and those seen with other manoeuvres is worthwhile since this may give clues as to the site of action of low ionic strength on the residual cation fluxes. In this respect low temperature, high hydrostatic pressure and SH-reactive reagents could be of some interest. There is a significant literature on cation fluxes induced by SH-reactive reagents including PCMBS, diamide and periodate (Knauf & Rothstein, 1971; Deuticke, Poser, Liitkemeier & Haest, 1983; Heller, Poser, Haest & Deuticke, 1984; Haas & Schmidt, 1985). However, a substantial portion of the PCMBS-induced cation fluxes shows a specific anion requirement for Cl- or Br- (Haas & Schmidt, 1985) which is different from the low ionic strength-stimulated cation fluxes. Diamide- or periodate-treated

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cells show some similar characteristics with the low ionic strength effect in inducing cation fluxes, but at the present stage of investigation it is not possible to favour a common mechanism. In the present work there were two reasons for investigating the effects of temperature on low ionic strength-induced cation transport. Firstly, Stewart et al. (1980) and Blackstock & Stewart (1986) have shown that low temperature induces a cation flux which increases when temperature is lowered (below 8 °C) with properties reminiscent of the low ionic strength effect. There is therefore the possibility that the present low ionic strength effect and the temperature effect are manifestations of the same phenomenon. We have taken advantage of the observation that low concentrations of Li' abolish the paradoxical temperature effect to resolve this problem. Thus from Fig. 6 it can be seen that in the presence of Li' at low temperature there is indeed a reduction of the K+ uptake in low ionic strength. However at 37 °C, Li+ had no effect on the low ionic strength-stimulated flux. We therefore conclude that the flux in a solution of low ionic strength at low temperature can be resolved into two independent components: (i) a low temperature-induced component which is Li+ sensitive (Blackstock & Stewart, 1986) and (ii) a low ionic strength-induced component which is not Li+ sensitive. Secondly, if the present phenomenon resembles free diffusion, i.e. a weak interaction with a water-filled 'pore' (cf. Hall & Willis, 1986), a low temperature dependence would be expected. In fact, this was not the case (Fig. 6) since K+ uptake was significantly temperature dependent. We estimated an activation energy of 50-60 kJ mol-P in low ionic strength solution containing 1 mM-LiCl, which is much higher than the expected value for electrodiffusion through a water-filled 'pore' (Hall & Willis, 1986; about 20 kJ mol-'). Ultimately, it is necessary to try and identify mechanisms by which the low ionic strength could induce residual cation fluxes. This seems to be of importance since a role for the known ion transport pathways has been excluded (see above). However, as we stated above it is also not possible to explain the residual cation fluxes solely on the basis of electrodiffusion. The observed characteristics of the low ionic strength-induced fluxes (increased by hydrostatic pressure, high temperature dependence, not selective for Na+ vs. K+) do not allow us to determine whether this transport pathway is a previously undescribed channel or carrier. One key criterion frequently used to distinguish between these two options is the concentration dependence of the flux. The present data show that the flux is linear in the range 0-25-10 mm (Fig. 2A and B). However if high Km values are a property of the flux, saturation will only be apparent at higher substrate concentrations. In fact Canessa, Brugnara, Cusi & Tosteson (1986) have described that residual K+ as well as Na+ effluxes into K+- and Na+-free solutions saturates when the intracellular K+ or Na+ concentration is increased. There are several possible explanations for the effect of low ionic strength on residual cation transport. The simplest explanation would be to assume an alteration of the physical state of the phospholipids in the membrane producing a defect in membrane structure. It is known that changes to a variety of parameters including temperature, pH and hydrostatic pressure, alter the ordering of the phospholipids (Boggs, 1980; Macdonald, 1984). However, it can be seen from Fig. 5 that the

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measured influxes increased under pressure in both physiological and low ionic strength solution (i.e. negative activation volumes). Since it is known that pressure inhibits K+ efflux in liposomes (i.e. a positive activation volume (Hall & Ellory, 1987)), we have to assume that proteins play an important role in the low ionic strength effect. This proposal is supported by comparative physiology, since our experiments on red cells of several species (HK (high potassium) sheep, LK (low potassium) and HK cows, camel) indicate that the low ionic strength-induced residual K+ influx is lacking from these animals whilst LK sheep red cells do show a significant low ionic strength effect (data not shown). Lowering the ionic strength might result in a conformational change of proteins involved in residual cation fluxes and/or in a changed lipid-protein interaction, leading to regions able to allow transmembrane ion movement at instabilities between protein and lipid molecules. When K+ influx was measured in low ionic strength medium at high hydrostatic pressure (up to 40 MPa) there was a synergism such that very large fluxes were obtained. This facilitation of the low ionic strength effect by high hydrostatic pressure suggests that a change in the lipid-protein interaction rather than a direct protein conformational change is involved in this process. This is because in general, lipids are more compressible than proteins (e.g. Macdonald, 1984), therefore the defects in membrane structure at the lipid-protein interface caused by low ionic strength could be exacerbated by hydrostatic pressure. The calculated activation volumes for fluxes measured under pressure in physiological and low ionic strength solution were similar in sign, but slightly different in magnitude (Fig. 5). This is not surprising since the specific interactions between membrane proteins and lipids will not be affected identically by high hydrostatic pressure and low ionic strength. A final possibility is that there is a specific membrane protein involved in the low ionic strength-stimulated K+ and Na+ uptake. At this stage of the investigation it is not possible to answer this question. However, it is worthwhile to note that DIDS inhibits the low ionic strength effect by about 70 % (Jones & Knauf, 1985) which suggests a role for the anion transport protein Band 3. Also Solomon, Chasan, Dix, Lukacovic, Toon & Verkman (1983) have already speculated that the anion transport protein can be involved in cation transport. It is possible of course that the DIDS effect could be due to a more non-specific DIDS-membrane protein interaction. An exclusive role for Band 3 (possibly by a conformational change) seems unlikely since it is known that hydrostatic pressure in the range used in our experiments decreases anion equilibrium exchange via Band 3 in human red blood cells (Canfield & Macey, 1984) as well as zero-trans exchange via other transporters (Hall & Ellory, 1987), although it could be possible that the 'slippage' process via Band 3 is increased under pressure. In addition, if the low ionic strength effect was due only to Band 3 then the phenomenon should be present in red blood cells from all mammalian species. However, this is not the case (see above for discussion on LK vs. HK sheep red blood cells). In conclusion, it is obvious that many possible factors can influence the residual cation transport through biological membranes. We have considered that both protein conformation and the physical state of membrane lipids (influencing the lipid-protein interaction) may be important. These effects are not necessarily

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independent, but may be interactive, a reasonable assumption since both proteins and lipids can be influenced by a variety of different factors, including ionic strength, temperature, pressure, pH and SH-group reagents. Over the last three decades the original pump-leak concept (Tosteson & Hoffman, 1960) has been modified substantially to take account of the wide range of cation transport pathways. In contrast, the residual cation transport (leak) of biological membranes has not received such attention, and the present work shows that this fundamental process is complex and deserves further investigation. This work was made possible by travel grants to Dr Bernhardt from the Wellcome Trust and the British Council. J. C. E. and A. C. H. acknowledge support from the Wellcome Trust. REFERENCES

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