THE EFFECTS OF CHOLESTEROL DEPLETION ON THE SODIUM PUMP IN HUMAN RED CELLS

Experimental Physiology (1991), 76, 437-443 Printed in Great Britain THE EFFECTS OF CHOLESTEROL DEPLETION ON THE SODIUM PUMP IN HUMAN RED CELLS F. J. LUCIO*, B. M. HENDRYt AND J. C. ELLORY* * University Laboratory of Physiology, Parks Road, Oxford OX] 3PT and t Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU (MANUSCRIPT RECEIVED 8 NOVEMBER 1990, ACCEPTED II JANUARY 1991) SUMMARY Sodium pump function has been studied in human erythrocytes depleted of membrane cholesterol by incubation with phosphatidylcholine liposomes. The cells were sodium loaded by incubation in alkaline sodium phosphate and sodium pump activity was assessed by measurements of ouabain-sensitive 86Rb uptake at 37 'C. Cholesterol depletion had a biphasic effect; depletion by 5-25 % increased sodium pump activity by a mean of 16-1 % (S.D. 3-2%), whereas depletion by 35-50% decreased sodium pump activity by a mean of 148% (S.D. 3-8 %). Cholesterol depletion had no reproducible effect on the ouabain-insensitive uptake of Rb. These results support the hypothesis that there may be an optimum membrane cholesterol content for sodium pump function. INTRODUCTION Alterations in membrane lipid composition are known to modulate membrane transport processes (Spector & Yorek, 1985; Deuticke & Haest, 1987; Yeagle, 1989), but the precise physiological role of this form of interaction is not clear. Changes in membrane cholesterol content have been studied in most detail, partly because of interest in the relationship between cholesterol and cardiovascular disease (Grundy, 1988). The sodium pump (Na+, K+-ATPase) is an important membrane transport system which plays an essential role in cellular function and is affected by changes in membrane composition. The relationship between membrane cholesterol content and sodium pump function is not fully understood; the enzyme activity of purified Na+, K+-ATPase appears to be relatively insensitive to cholesterol content (De Pont, Peters & Bonting, 1983), while pump function in membrane vesicles and intact cells appears to be modulated by membrane cholesterol (Yeagle, 1983; Yeagle, Young & Rice, 1988; Yeagle, 1989). Some studies have demonstrated pump inhibition with increasing cholesterol content (Kroes & Ostwald, 1971; Yeagle, 1983), while other work suggests that, at low levels, cholesterol acts to stimulate pump activity (Yeagle et al. 1988; Yeagle, 1989). These latter experiments give rise to the concept of an optimum membrane cholesterol level for Na+, K+-ATPase function. One difficulty with sodium pump measurements made on intact cells is the maintenance of high intracellular concentrations of Na and ATP so that pump activity is measured at maximum velocity Vmax and does not change due to variations in intracellular substrate. In this context only one group has systematically approached this problem by Na loading of erythrocytes before determination of pump fluxes (Giraud, Claret & Garay, 1976; Claret, Garay & Giraud, 1978; Giraud, Claret, Bruckdorfer & Chailley, 1981). Their results differed from other work in showing a large activation (five- to ten-fold) in Vmax for pump activity with cholesterol depletion, with modified kinetics, with no evidence for an optimum membrane cholesterol for pump Vma.. In the present study we have investigated the effects of cholesterol depletions of between 5 and 50% on sodium pump activity in human

438 F. J. LUCIO, B. M. HENDRY AND J. C. ELLORY erythrocytes previously Na-loaded to establish conditions close to Vma. for internal Na. Our results indicate a biphasic behaviour on cholesterol depletion; removal of 5-25 % of the membrane cholesterol stimulated the pump by 15-20%; depletion of 30-50 % of membrane cholesterol reversed this effect, giving a consistent inhibition of about the same magnitude. These results indicate a smaller effect of changing cholesterol than those of Claret et al. (1978). The sequential stimulation followed by inhibition of the Na+ pump on progressive depletion of cholesterol suggested that there was an optimum membrane cholesterol for maximal sodium pump V.,ax which was lower than the native erythrocyte cholesterol content in the subjects studied. METHODS Preparation of erythrocytes depleted in cholesterol The methods for alteration of erythrocyte membrane cholesterol were similar to those described in the literature (Claret et al. 1978; Shinitzky, 1978). Erythrocytes were obtained immediately before the experiments from fresh blood drawn into heparinized syringes by venepuncture from healthy donors. The blood samples were centrifuged at 4 C (3000 g) for 10 min and plasma and buffy coats removed. Erythrocytes were washed twice at 4 C with 10 volumes of buffered 150 mmol 1-1 NaCl and resuspended at 15% haematocrit either in incubating medium (140 mmol 1-1 NaCI, 10 mmol 1-1 KCI, 1-0 mmol 1-1 MgCI2, 10 mmol 1-1 MOPS, 10 mmol 1-1 glucose, 17 mg 1-1 penicillin, 17 mg 1-1 streptomycin, ph set at 7-4 by addition of HCI) or in incubating medium containing phosphatidylcholine (PC) liposomes. PC was type V-EA obtained from Sigma Chemical Co. PC liposomes were made by evaporating PC in chloroform to dryness under N2, and by dispersion of the lipid in the incubating medium (100 mg PC (ml incubating medium)-1). Finally, this mixture was sonicated for 120 min with an Astell Scientific sonicator under N2. Cells were incubated in saline with or without PC at 37 C, under continuous rotary mixing, for different periods of time, in order to achieve cholesterol depletions of up to 50% of the original cell cholesterol content. Incubation with PC liposomes promotes cholesterol depletion, but does not modify the total phospholipid content of the cell nor induce PC uptake by the erythrocytes (Claret et al. 1978). Coulter counter measurements of mean cell volume were made to establish that cell volume was constant to within + 2% on cholesterol depletion. It was found that up to 50% depletion of cholesterol could be achieved without significant haemolysis, but haemolysis did occur if the incubating medium did not contain magnesium ions. 86Rb influx experiments Erythrocytes were loaded with sodium by incubation in alkaline sodium phosphate solution as described by Garay (1987) and Rosati, Meyer & Garay (1988). Sodium loading by alkaline sodium phosphate stimulates sodium pump activity in a manner consistent with the presence of three independent internal Na binding sites per pump. This method avoids the use of lipophilic agents, such as p-chloromercuribenzenesulphonate (PCMBS) or monensin, which may themselves alter the organization of membrane lipid. After incubation to alter cholesterol erythrocytes were washed three times at 4 C (3000 g) with 20 volumes of unbuffered phosphate saline (100 mmol 1-I Na2HPO4; 75 mmol 1-1 sucrose, ph 8-5-995), resuspended in this medium (10% haematocrit) and incubated for 180 min at 37 'C. During the last 90 min glucose, adenine and inosine (5, 2 and 10 mmol 1-1 final concentrations, respectively) were added to the incubating suspensions to replenish the energy stores of the cells. This procedure caused significant sodium loading. At the end of this period, suspensions of both types of erythrocytes (controls and those incubated with PC liposomes) were divided into two aliquots and washed three times with saline at 4 'C (150 mmol 1-1 NaCl, 10 mmol 1-1 MOPS, 5 mmol 1-1 glucose, ph set at 7-4 by addition of HCI). One of the aliquots was resuspended in this medium whereas the other one was resuspended in the same medium plus 0 1 mmol 1-1 ouabain. Both suspensions (4-8 % haematocrit) were incubated at 37 'C for 20 min. This period of incubation with ouabain was sufficient to allow ouabain binding and to establish complete inhibition of sodium pump activity. Experiments were performed to confirm this using variable incubation times. Aliquots (1-0 ml) of cell suspension were saved to measure the intracellular sodium and potassium as well as the erythrocyte cholesterol content (see below). 86Rb

CHOLESTEROL AND THE SODIUM PUMP influx was measured in triplicate with 1 0 ml aliquots of cell suspension incubated with or without ouabain. 86Rb (35,1, 30,tCi ml-' in 150 mmol 1-1 KCl) was added to each tube (final KCl concentration 5-3 mmol 1-1). Flux measurements were performed by incubation at 37 C for 10 min and were started and stopped on ice. After the incubation the cell suspensions were centrifuged and a 20,tl aliquot taken from each supernatant for measurement of extracellular 86Rb. Following this the cells were washed free of extracellular label by four rapid centrifugation washes in ice-cold MgCl2 solution (107 mmol 1-1 MgCl2, 10 mmol 1-1 MOPS, ph 7 4). The cells were then lysed by addition of 05 ml of 01 % (v/v) Triton X-100 and 05 ml of 5% (w/v) trichloroacetic acid was added to precipitate the protein. The 86Rb content of the erythrocytes was measured by Cerenkov counting in water in a Packard Tri-Carb 2000CA scintillation counter. Haemoglobin was determined spectrophotometrically at 540 nm in triplicate using Drabkins reagent; packed cell volumes were then calculated on the basis of OD (optical density) packed cells = 247. Finally, the ouabain-sensitive 86Rb influx was calculated as the difference between influx measured with or without 0-1 mm-ouabain. Results for ouabain-sensitive Rb uptake are expressed as a percentage of the control values measured in the non-cholesterol-depleted cells. The data are also presented as K fluxes on the assumption that Rb is a valid congener for K. Determination of intracellular sodium and potassium and cell cholesterol content Six 1-0 ml aliquots of cell suspension were washed four times in ice-cold MgCl2 solution (composition as above). After the final centrifugation, Triton X-100 and trichloroacetic acid were added as above to the cell pellet and the sodium and potassium concentrations were measured in the supernatant on an IL943 flame photometer using caesium as internal standard. Results were expressed as mmoles of cation per litre of packed cells. Cell pellets from a further three saved aliquots were lysed with 300,1u of water and erythrocyte membrane lipids were extracted with chloroform-isopropanol (7:11) (Yandrasitz, Berry & Segal, 1981). The lipid extracts were evaporated under N2 and redissolved in 3 ml of hexane. The cholesterol content was assayed by normal phase HPLC using a modification of the method of Yandrasitz et al. (1981). In brief, 100,tl of sample were injected through a Dynamax Microsorb 80-125-C5 column (250 x 4-6 mm, 5,tm pore diameter, Rainin Inst Co) and cholesterol was eluted isocratically with hexane-isopropanol 97:3 pumped at 1 5 ml min-'. Retention time for cholesterol was about 5 min and the sterol was detected spectrophotometrically at 205 nm. The cholesterol content of the samples was calculated by interpolation from a calibration curve of pure cholesterol (Sigma Chemical Co.) and the final result was calculated as,umoles of cholesterol per millilitre packed cells. The results are presented as the percentage of cholesterol present in the depleted cells compared with the matched controls cells incubated in PC-free saline. Measurements were made to establish that the control incubation, including the sodium loading procedure, did not alter cell cholesterol by more than 3%. 439 RESULTS The mean cholesterol content of the control erythrocytes in six subjects was 3 31 (S.D. 0-27),tmol (ml cells)-1. The mean ouabain-sensitive K influx in control erythrocytes was 2-61 (S.D. 0-21) mmol (1 cells)-1 h-1. Both of these results are consistent with previously reported values (Owen & McIntyre, 1978; Fervenza, Hendry & Ellory, 1989). The results of cholesterol depletion experiments on the six subjects are presented in Fig. 1. In most experiments samples from a donor were depleted of cholesterol by incubating with liposomes for several different times, so that one donor yielded three to six points in the figure. Certain donors were used more than once, yielding consistent results on each occasion. Figure 1A is an illustration of the change in sodium pump activity measured as ouabain-sensitive Rb flux as a function of the percentage cholesterol depletion. Progressive depletion in each subject gave both activation and then inhibition as depletion increased beyond 25 %. To eliminate the possibility that depletion time was a significant factor, certain samples were depleted rapidly using small (highly sonicated) liposomes which lower the cholesterol content 2-3 times faster. Results for samples from three donors treated in this way were consistent with the other data. All samples were analysed for intracellular Na

440 F. J. LUCIO, B. M. HENDRY AND J. C. ELLORY A 40 - CZ20 0 20 B,;~~~1-10 - _ =X10-40 0 3.S -100 20 z Low High Amount of cholesterol depletion Fig. 1. The relationship between sodium pump flux (measured as the ouabain-sensitive influx of 86Rb) and the percentage depletion of erythrocyte cholesterol. A shows the individual results; each point is the mean of three measurements of pump flux for a given donor at a single value for cholesterol depletion. The flux results are expressed as a percentage of the flux measured in control cells from the same donor incubated for identical periods in PC-free saline. The different symbols each represent a single donor and the lines link points from the same donor. B shows the results of grouping the data in cholesterol depletion ranges of 10-25 % and 35-50 %. The mean percentage change in sodium pump flux is illustrated. Error bars are S.D.. Comparison of the mean results for each donor for the low and high depletion states showed that the two states were significantly different (P < 001, Student's paired t test; P < 0-02, Wilcoxon rank sum test). giving mean values of 21-6 (S.D. 5 3) mmol (1 cells)-' for the control erythrocytes, and 25-8 (S.D. 6 8) mmol (1 cells)- cells for the cholesterol-depleted erythrocytes. Corresponding values for intracellular K were 89 5 (S.D. 9-3) and 84-4 (S.D. 10-7) mmol (1 cells)-'. These differences were not significant. Figure 1 B presents the data of Fig. 1 A collected into two ranges of cholesterol depletion, 10-25 % (low depletion) and 35-50 % (high depletion). These values for cholesterol were chosen from Fig. 1 A where it is obvious that the effect of depletion reverses in the range 25-35 %. The mean values for percentage change in pump flux were + 16-1 (S.D. 3-2) % in the lower depletion state and -14-8 (S.D. 3-8) % in the higher range. The mean value for ouabain-sensitive K influx in the lower depletion state was 3-1 1 (S.D. 0-22) mmol (1 cells)-1 h-1,

CHOLESTEROL AND THE SODIUM PUMP 441 U 40 C~~~~~ 0 e 20- E 0O 0 x 0EX EX ax o 0 m -20- M 0 E 0 0~~~~~~~ 40 0 10 20 30 40 50 Depletion of cholesterol (%) Fig. 2. The lack of correlation between erythrocyte cholesterol depletion and the percentage change in ouabaininsensitive 86Rb influx with respect to control cells. The points represent mean data from the same experiments used to measure the ouabain-sensitive fluxes shown in Fig. 1. compared with 2-17 (S.D. 0-18) mmol (1 cells)-1 h-' in the higher depletion state. The ouabain-insensitive Rb influx for all samples in Fig. 1 is plotted as a function of cholesterol depletion in Fig. 2. The results are variable but indicate a small (4-4 %) increase in mean flux, which did not correlate with the level of cholesterol depletion, in contrast with the data for Na+-K+ pump fluxes. The mean ouabain-insensitive K flux in control erythrocytes was 0-861 (S.D. 0-21) mmol (1 cells)-1 h-1 compared with 0-899 (S.D. 0-27) in cholesterol-depleted cells. DISCUSSION The results presented here confirm the general observation that the pump activity of the membrane-bound Na+, K+-ATPase is modulated by the cholesterol content of the membrane. The observation that moderate reductions in membrane cholesterol (10-25 %) activate the erythrocyte Na+ pump in cells with high intracellular Na concentration is consistent with other studies, although the magnitude of the stimulation observed in these experiments was somewhat smaller than the effects reported by other authors (Giraud et al. 1976; Claret et al. 1978; Giraud et al. 1981). This activation of the sodium pump by moderate cholesterol depletion is also consistent with studies of the sodium pump in other cell types, including membrane vesicles isolated from rabbit renal medulla (Spector & Yorek, 1985; Yeagle et al. 1988; Yeagle, 1989). Activation of membrane transport by depletion of cholesterol has also been reported in studies of erythrocyte sulphate transport via the anion exchange protein (Grunze, Forst & Deuticke, 1980). On the other hand cholesterol depletion is reported to inhibit the membrane transport of L-lactose and L- arabinose in human erythrocytes (Grunze et al. 1980). In these experiments the activity of the sodium pump showed a biphasic response to progressive cholesterol depletion, moderate depletions in erythrocyte cholesterol causing pump stimulation and larger depletions associated with pump inhibition. The cross-over between these two responses occurred at 25-35 % depletion, while the maximal stimulation of pump activity was seen at depletions of 15-20 %. This biphasic response to altered membrane cholesterol has been reported for the Na+, K+-ATPase enzyme activity in rabbit renal medulla membrane vesicles (Yeagle et al. 1988) and has been observed in studies of

442 F. J. LUCIO, B. M. HENDRY AND J. C. ELLORY glucose transport in human erythrocytes and 3T3 mouse fibroblasts (Yuli, Wilbrandt & Shinitzky, 1981). The biphasic response supports the idea of an 'optimum' membrane cholesterol for maximal activity of a membrane transport system. This optimum membrane cholesterol content may vary from tissue to tissue and among different transport systems. For example, the present data suggest that the optimum erythrocyte cholesterol for sodium pump Vmax is lower than that found in unmanipulated cells from normal donors, whereas the human erythrocyte glucose transporter appears to exhibit maximum turnover rates at cholesterol levels above those found in fresh unaltered cells (Yuli et al. 1981). The present results differ in two respects from the study reported by Claret et al. (1978). First, the magnitude of the stimulation of sodium pump activity by moderate cholesterol depletion was considerably smaller in these experiments (10-30%) than that reported by Claret et al. (5- to 10-fold). Secondly, the results of this work demonstrate inhibition of sodium pump activity at higher values of cholesterol depletion, an effect which has not been described previously in studies of erythrocytes. The explanation for these differences is not clear. One possibility is that even when using sodium-loaded cells variations in intracellular sodium concentration are important, especially as in the present work no attempt has been made to study the precise relationship between intracellular sodium and pump activity. The intracellular concentrations measured in this study (20-30 mmol (1 cells)-') are similar to the highest concentrations achieved in the study of Claret et al. (1978). If the affinity of the pump for intracellular sodium is unaltered by cholesterol depletion then these sodium concentrations are sufficient to activate the pump at 70-80% of Vmax. It is possible that some of the effects of cholesterol depletion observed were due to alterations in the affinity of the pump for intracellular sodium, as suggested by Claret et al. (1978). Nevertheless, at similar values of intracellular sodium Claret et al. reported 5-fold stimulation of sodium pump flux, but we did not observe similar effects. A further possible source of difference between these studies is the different methods used to load erythrocytes with sodium. Claret et al. (1978) used PCMBS treatment of erythrocytes while incubation of cells in alkaline sodium phosphate was employed in this work. Experiments were attempted to examine the sodium pump after cholesterol depletion of erythrocytes without any sodium loading. Unfortunately these results were very difficult to interpret as the cholesterol loading procedure itself caused a significant and variable increase in intracellular sodium which did not occur in the control cells. There is evidence that PCMBS itself alters both the Vm.. of the sodium pump and its affinity for intracellular sodium (Giraud et al. 1976). Incubation with alkaline sodium phosphate does not appear to have these effects (Rosati et al. 1988). The physiological or pathological importance of the regulation of sodium pump activity by membrane lipid composition is difficult to assess. The effects observed here are considerably smaller than the modulation by cholesterol reported by Claret et al. (1978). Nevertheless, alterations in pump activity of + 25 % could significantly affect cell function. There are numerous clinical syndromes in which plasma cholesterol and lipid composition are altered, but less is known about membrane lipid composition in disease. In renal failure erythrocyte cholesterol content is raised, and this alteration could account in part for the lowered sodium pump activity seen in this syndrome (Fervenza et al. 1989). Whether altered lipid membrane composition is a factor in pathogenesis in hypercholesterolaemia and related syndromes remains unknown. This work was supported by the National Kidney Research Fund.

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