ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor

Orit Aharonovitz, Nicolas Demaurex, Michael Woodside, and Sergio Grinstein

Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na+/H+ exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibition of the antiport. No evidence has been found for direct binding of nucleotide to exchangers or alteration in their state of phosphorylation, suggesting ancillary factors may be involved. This possibility was tested by comparing the activity of dog red blood cells (RBC) and their resealed ghosts. Immunoblotting experiments using isoform-specific polyclonal and monoclonal antibodies indicated RBC membranes express Na+/H+ exchanger isoform 1 (NHE1). In intact RBC, uptake of Na+ was greatly stimulated when the cytosol was acidified. The stimulated uptake was largely eliminated by amiloride and by submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also be elicited by acidification in resealed ghosts containing ATP, the absolute rate of transport was markedly diminished at comparable pH. Dissipation of the pH gradient was ruled out as the cause of diminished transport rate in ghosts. This was accomplished by a "pH clamping" procedure based on continued export of base equivalents by the endogenous anion exchanger. These observations suggest a critical factor required to maintain optimal Na+/H+ exchange activity is lost or inactivated during preparation of ghosts. Depletion of ATP, achieved by incubation with 2-deoxy-D-glucose, inhibited Na+/H+ exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate of Na+/H+ exchange in ATP-depleted but otherwise intact cells was similar to the transport rate of ghosts. Therefore, we tentatively conclude that full activation of NHE1 requires both ATP and an additional regulatory factor, which may mediate the action of the nucleotide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) that is inactivated or missing in ghosts.

pH regulation; amiloride; red blood cells; ghosts


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INTRODUCTION
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SODIUM/HYDROGEN EXCHANGE is detectable in the plasma membrane of virtually all mammalian cells, where it plays an important role in the regulation of intracellular pH (pHi) and cell volume. In addition, Na+/H+ exchange is essential for transepithelial movement of Na+ and acid equivalents in certain regions of the renal and gastrointestinal tracts. These specialized functions are mediated by distinct isoforms of the Na+/H+ exchanger (NHE), which have recently been identified and studied in isolation by heterologous expression in mutant cells that are devoid of endogenous exchangers (25, 28, 33, 41). To date, six different NHE isoforms have been described, and at least four of these appear to function at the plasma membrane (for review, see Ref. 34).

In all cases studied to date, the exchange reaction appears to be reversible and driven solely by the transmembrane chemical gradients of Na+ and H+. Transport is generally believed to be passive, not requiring expenditure of metabolic energy. Indeed, NHE activity was first detected in isolated membrane vesicles derived from epithelial brush borders, a system presumably devoid of energy sources (26, 32). Nevertheless, it was subsequently established that in intact cells depletion of ATP induces a marked depression of the rate of exchange (3, 4). The inhibitory effect was not caused by alteration of the ionic gradients but instead reflects a form of allosteric regulation of the exchanger by the nucleotide. All the plasmalemmal isoforms that have been studied by heterologous transfection are inhibited by depletion of ATP, with varying degrees of sensitivity (25, 28).

The mechanism whereby ATP modulates NHE remains obscure. The isoforms studied to date exist as phosphoproteins in untreated cells. In the case of NHE1, which has been studied most extensively, the extent of phosphorylation was not detectably altered when the cells were ATP depleted for the short periods required to induce profound inhibition of ion transport (15). Furthermore, the inhibitory effect of metabolic depletion persisted in truncated NHE1 mutants lacking all the putative phosphorylation sites (15, 40). Thus changes in the phosphorylation state of the exchanger itself are unlikely to mediate the effect of ATP.

ATP is also unlikely to regulate exchange by directly binding to the NHE. Analysis of the primary sequence of the known isoforms does not reveal the presence of consensus nucleotide binding sites, and direct association has not been documented experimentally. Therefore, it is more likely that regulation involves ancillary molecules, which could themselves be the primary targets of ATP. Such a mechanism has been invoked in the case of the Na+/Ca2+ exchanger, which is similarly activated by ATP (23). As in the case of NHE, neither direct phosphorylation nor nucleotide binding is likely to be responsible for the regulation of the Na+/Ca2+ exchanger (7). Instead, two types of associated molecules have been proposed to mediate regulation by ATP. In giant cardiac membrane patches, the level of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been suggested to be an important determinant of exchange activity. ATP is envisaged to modulate the Na+/Ca2+ exchanger by dictating the extent to which phosphatidylinositol becomes phosphorylated to yield PtdIns(4,5)P2 (21). In squid nerve fibers, modulation of Na+/Ca2+ exchange by ATP was also reported to depend on an ancillary factor (11). In this case, however, the regulatory component was found to be cytosolic.

There is precedent for the regulation of NHE activity by independent, associated factors. The apical isoform of the exchanger, NHE3, is inhibited by elevation of cAMP. Although some authors believe that direct phosphorylation of the antiporter by protein kinase A is involved (27), others have reported that an additional protein cofactor is required for the manifestation of the effect (42). Indeed, two such regulatory molecules, termed NHE regulatory factor (NHERF) and NHERF-2 (also known as E3KARP and TKA-1), have recently been identified and sequenced (43, 44). We therefore considered the possibility that ancillary factors could also be important for the ATP dependence of NHE. Red blood cells (RBC) were found to be particularly useful for this purpose because they can be lysed in mildly hypotonic solutions and then resealed upon restoration of the original osmolarity. By varying the extent of the initial dilution, different concentrations of cytosolic factors can be trapped in the resulting ghosts. For this study, we used canine RBC, which were shown by the pioneering work of Parker (36) to display considerable antiport activity. Herein we report that these RBC express NHE1 that is sensitive to ATP. The stimulatory effect of the nucleotide requires the presence of an additional regulatory factor that is lost or inactivated during preparation of RBC ghosts.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reagents and solutions. Amiloride and 2-deoxy-D-glucose were obtained from Sigma Chemical (St. Louis, MO). DIDS (disodium salt) was obtained from Molecular Probes (Eugene, OR). Enhanced chemiluminescence reagents were from Amersham International (Buckinghamshire, UK). The ATP assay kit was purchased from Calbiochem (San Diego, CA). 22Na+ was obtained from New England Nuclear Life Science Products (Boston, MA). All other chemicals were of analytical grade and were obtained from Aldrich Chemical (Milwaukee, WI). HOE-694 [(3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate] was kindly provided by Dr. A. Durckheimer (Hoechst, Frankfurt, Germany).

Polyclonal antibodies to NHE1 were raised by injecting rabbits with a fusion protein constructed with beta -galactosidase of Escherichia coli and the last 157 amino acids of the human antiporter. Polyclonal antibodies to the NHE3 isoform were generated against a glutathione S-transferase fusion protein with residues 565-690 of the rat NHE3. Polyclonal antibodies to the NHE4 isoform were generated against a glutathione S-transferase fusion protein with the last 40 amino acids of the rat NHE4. Antibodies were affinity purified as described (37). Monoclonal antibody to NHE1 was raised to the entire carboxy-terminal hydrophilic domain of pig NHE1 and was kindly provided by Dr. D. Biemesderfer (Yale University, New Haven, CT). Polyclonal antibodies to NHERF were raised by injecting recombinant full-length NHERF into rabbits and were kindly provided by Dr. R. A. Hall (Duke University, Durham, NC). Horseradish peroxidase-coupled goat anti-rabbit or anti-mouse antibodies were from Jackson ImmunoResearch Laboratories (Mississauga, ON, Canada).

PBS contained (in mM) 150 NaCl, 10 KCl, 8 sodium phosphate, and 2 potassium phosphate (pH 7.4). Laemmli sample buffer contained 0.8% SDS (wt/vol), 0.0015% bromphenol blue (wt/vol), 5% 2-mercaptoethanol (vol/vol), 8% glycerol (vol/vol), and 50 mM Tris · HCl (pH 6.8).

Preparation of RBC, ghosts, and membranes. Ghosts were prepared by a modification of the method of Parker (35). Briefly, RBC were obtained from heparinized blood of healthy dogs. After centrifugation, the plasma and buffy coat were discarded, and the RBC were washed three times with 2-3 volumes of 165 mM NaCl plus 1 mM HEPES (pH 7.3). The RBC were then diluted with 1 volume of this medium and chilled for 5 min on ice. This suspension was added to 10 volumes of an ice-cold lysing medium containing (in mM) 20 KCl, 4 MgSO4, 0.3 CaCl2, and 1.2 acetic acid. The suspension was brought to pH 5.8 while being stirred in an ice bath and incubated for 5 min. This suspension was then mixed with 1 volume (equal to the volume of the original cell suspension) of resealing solution containing (in mM) 1,458 NaCl, 162 KCl, 12 MgSO4, 30 Tris base, and 12 ATP. The mixture was titrated to pH 7.0 and, after 5 min on ice, incubated for 60 min at 37°C. The resealed ghosts were separated from the hemolysate by centrifugation at 13,000 g for 15 min and suspended in storage solution containing (in mM) 126 NaCl, 15 KCl, 1 MgCl2, 20 Tris-MES, 10 glucose, and 0.5 EGTA (pH 7.4). To prepare sodium gluconate or potassium gluconate-loaded ghosts, these salts were used to replace NaCl and KCl in the washing, lysing, resealing, and storage solutions. For cell volume and Na+ content measurements, ghosts were loaded with potassium gluconate. Counting and sizing of RBC and resealed ghosts were performed electronically using a model ZM Coulter counter and C1000 Channelyzer, as described earlier (16).

RBC membranes were prepared for immunoblot analysis as follows. RBC were washed three times with 5 volumes of 150 mM NaCl plus 5 mM sodium phosphate (pH 8.0). After centrifugation, packed RBC (1 ml) were lysed by mixing with 40 ml of ice-cold 5 mM sodium phosphate (pH 8.0). The resulting membranes were sedimented at 30,000 g for 15 min. The clear red supernatant was removed by aspiration, and the pellet was washed twice with the lysis solution. Each 1-ml pellet of freshly prepared ghosts was resuspended in 40 ml of prewarmed 0.5 mM sodium phosphate (pH 9.0). The suspension was incubated at 37°C for 20 min. The resulting stripped membranes were sedimented at 30,000 g for 30 min and used for immunoblotting as described below. The pH 9.0 stripping procedure was also used to prepare membranes from resealed ghosts.

pHi manipulation and determination. In RBC, which are rich in Cl-/HCO-3 exchangers, the relationship between pHi and the extracellular pH is dictated by the transmembrane Cl- gradient. Because HCO-3 is a base equivalent, rapid anion exchange ensures that the ratio of internal H+ concentration to external H+ concentration equals the ratio of external Cl- concentration to internal Cl- concentration. We took advantage of this unique feature of RBC to set their pHi to the desired level by incubating them for 10 min at 37°C, in medium containing (in mM) 140 KCl, 40 sucrose, 0.15 MgCl2, 10 glucose, and 20 Tris-MES (pH 6.0-7.0). After this initial incubation, DIDS was added to a final concentration of 200 µM, and the cells were incubated for a further 30 min. Treatment with this anion exchange inhibitor enabled us to subsequently suspend the cells in media of different pH, without dissipation of the transmembrane pH gradient. The cells were finally sedimented and resuspended in the medium of choice.

Alternatively, we used the continuous exchange of extracellular Cl- for internal HCO-3 to induce a sustained acid loading of ghosts. Different pHi levels (6.4-6.9) were obtained by preincubating gluconate-containing ghosts for 10 min at 37°C in media containing various concentrations of Cl-. The media contained 0, 14, or 128 mM of the Cl- salt of N-methyl-D-glucammonium (NMDG), plus (in mM) 13 potassium gluconate, 2 NaCl, 1 MgCl2, 0.5 EGTA, 10 glucose, and 20 Tris-MES (pH 7.3) and were osmotically balanced using NMDG gluconate. The pHi attained by either method was determined individually in each experiment by sedimenting an aliquot of the suspension containing the equivalent of ~50 µl of packed RBC or ghosts and washing the pellet once in the same incubation medium devoid of buffer. The RBC or ghosts were next lysed in 1 ml of distilled water, followed by determination of the pH using a combination electrode.

ATP depletion and determination. For depletion of ATP, cells were incubated for 2 h at 37°C in PBS (pH 7.8) containing 10 mM 2-deoxy-D-glucose. Cellular ATP content was determined using the Calbiochem assay kit. Cells (~5 × 105) were extracted with 0.4 ml of 8% perchloric acid and placed on ice. The extract was then neutralized with 1 M K2CO3, debris were sedimented, and aliquots of the supernatant (10 µl) were mixed with the buffer and luciferin-luciferase mixture provided by the kit manufacturer. Sample luminescence was determined using a Beckman LS7000 counter and compared with ATP standards.

Isotope fluxes. NHE activity was measured as the rate of amiloride-inhibitable 22Na+ influx at 37°C. Uptake of 22Na+ was initiated by resuspension of a pellet of ~108 acid-loaded RBC or ghosts in 0.5 ml of medium consisting of (in mM) 135 NMDG-Cl, 2 NaCl, 2 MgCl2, 1 KCl, 0.5 EGTA, and 10 glucose with or without 1 mM amiloride, plus 0.5 µCi of 22Na+. Isotope uptake was terminated after 10 min by aspirating the radiolabeled medium and rapidly washing the cells or ghosts twice with ice-cold medium containing 128 mM NMDG-Cl and 13 mM potassium gluconate. Pellets were lysed with 1 ml of distilled water and counted in a LKB 1282 gamma counter. As necessary, e.g., when ghosts were acid loaded by imposing various inward Cl- gradients, the compositions of the radiolabeled and wash solutions were adjusted appropriately. 22Na+ uptake was linear for at least 15 min under all the experimental conditions used.

NHERF expression and purification. Hexahistidine-tagged full-length NHERF was produced by insertion of rabbit NHERF cDNA into pET30A (Novagen, Madison, WI) as described (19, 20). Fusion proteins were expressed according to the manufacturer's instructions and purified using nickel beads (Novagen).

Immunoblotting. For immunoblotting, samples were mixed with 0.25 volume of 5× concentrated Laemmli sample buffer and boiled for 5 min. The samples were subjected to SDS-PAGE and transferred to nitrocellulose. Blots were blocked with 5% nonfat dried milk and exposed to a 1:5,000 dilution of affinity-purified anti-NHE1, NHE3, or NHE4 polyclonal antibodies or to a 1:1,000 dilution of affinity-purified anti-NHE1 monoclonal antibody. The secondary antibodies, goat anti-rabbit or anti-mouse coupled to horseradish peroxidase, were used at a 1:5,000 dilution. Immunoreactive bands were visualized using enhanced chemiluminescence.

Other methods. The concentration of Na+ in the hemolysates was determined by flame photometry using Li+ as an internal standard. All experiments were performed on at least three separate occasions, each with triplicate determinations. Data are presented as means ± SE of the number of experiments specified.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Identification of NHE1 in dog RBC. Isoform-specific antibodies, directed to unique sequences in the cytosolic domains of NHE1, NHE3, or NHE4, were used to establish which isoform(s) of the antiporter is present in canine RBC. No antibodies to the remaining isoforms were available to us at the time of this study. Anti-NHE3 and NHE4 antibodies failed to react with NHE in dog RBC (data not shown). In contrast, a polypeptide of ~98 kDa reacted positively with both the monoclonal and polyclonal anti-NHE1 antibodies (Fig. 1). The immunoreactive band was somewhat smaller and migrated more sharply than the human platelet NHE1, which, as shown earlier (1), migrates as a wide band of ~110 kDa in human platelets. The width of the NHE1 band in SDS-PAGE has been attributed to carbohydrate heterogeneity, since impairment of glycosylation yields a sharp band of 85 kDa (8). Therefore, our results suggest that dog RBC express a form of NHE1 with reduced and more homogeneous glycosylation.


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Fig. 1.   Detection of Na+/H+ exchanger (NHE) isoform 1 by immunoblotting. Membranes from dog red blood cells (RBC) and from human platelets, prepared as described under MATERIALS AND METHODS, were subjected to electrophoresis in SDS, followed by blotting onto nitrocellulose. Blots were probed with either monoclonal or polyclonal NHE1 antibodies, as indicated, and visualized by enhanced chemiluminescence using appropriate peroxidase-conjugated secondary antibodies. Position of molecular mass markers is indicated at left. Blot is representative of 3 similar experiments.

The presence of other isoforms in RBC could not be ruled out by immunologic means. Not only were antibodies to some of the isoforms unavailable, but those tested may have failed to react due to species specificity. To determine the possible presence of other isoforms and to assess the contribution of NHE1 to the net transport activity, we used the inhibitor HOE-694. This compound inhibits NHE1 competitively with a comparatively high affinity (IC50 0.16 µM). The potency of this compound toward NHE1 is considerably greater than it is for NHE2 and NHE3 (IC50 5 and 650 µM, respectively), thus providing means of discerning between the isoforms (9). The effect of HOE-694 on the exchange activity of RBC, estimated from the rate of 22Na+ uptake in acid-loaded intact cells, is illustrated in Fig. 2. Dissipation of the transmembrane pH gradient during the course of the measurements was precluded by treatment of the cells with DIDS to inhibit anion exchange (see pHi manipulation and determination). NHE activity could be effectively and virtually completely inhibited by very low concentrations of HOE-694 (IC50 0.09 µM), similar to those required to inhibit NHE1 in nucleated cells. The shape of the concentration dependence curve is consistent with the existence of a single form of the exchanger. Together, the immunochemical and functional findings indicate that the Na+/H+ exchange activity in acid-loaded dog RBC can be attributed to the NHE1 isoform.


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Fig. 2.   Inhibition of Na+/H+ exchange by HOE-694. Intact RBC were acid loaded by preequilibration in medium of pH 6.0 and then treated with DIDS to prevent dissipation of pH gradient upon resuspension in flux assay medium. Next, 22Na+ uptake was measured in medium of pH 7.4, in presence of increasing concentrations of HOE-694, as indicated. Results are means ± SE of 4 experiments. Where absent, error bars are smaller than symbol.

ATP depletion inhibits Na+/H+ exchange in dog RBC. In native systems, as well as when it is heterologously expressed by transfection, NHE1 is sensitive to the concentration of cytosolic ATP (3, 4, 25, 28). To test whether NHE1 is similarly sensitive in dog RBC, ATP was depleted by omission of glucose from the medium and addition of 2-deoxy-D-glucose. Under these conditions, >70% of the ATP was depleted after 2 h, as determined using luciferase [from (6.83 ± 0.22) × 10-11 mol/106 cells to (2.00 ± 0.18) × 10-11 mol/106 cells]. We undertook a detailed analysis of the pHi dependence of the activity of the exchanger in ATP-depleted and ATP-replete cells (Fig. 3). As shown earlier for other cells (41), NHE1 activity increases progressively with decreasing cytosolic pH. It is noteworthy that depletion of ATP inhibited the amiloride-sensitive flux at all pHi levels studied. In the range studied, the fractional inhibition was greater at more alkaline pHi values. The pHi was measured individually in each experiment at the end of the transport determination. Therefore, the effect of ATP depletion is due to inhibition of the exchanger and not to a spurious effect on pHi.


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Fig. 3.   Effect of ATP depletion on NHE activity in intact RBC. Dog RBC were pretreated with (open circle ) or without () a glucose-free medium containing 10 mM 2-deoxy-D-glucose for 2 h at 37°C to deplete ATP. Intracellular pH (pHi) was next manipulated by variation of external pH and clamped with DIDS, and 22Na+ uptake was measured as in Fig. 2. In all cases, the small amiloride-resistant component of flux was determined separately and subtracted. pHi was determined individually immediately before onset of flux assay, as described in MATERIALS AND METHODS. Results are presented as percent of maximal value for control RBC and grouped within 0.1-pH unit intervals. Data are means ± SE of at least 4 experiments. Where absent, error bars are smaller than symbol.

NHE activity in RBC is known to be exquisitely sensitive to changes in cell volume. The changes in activity reported in Fig. 3, however, are not secondary to ATP depletion-induced swelling of the cells because the volumes of control and ATP-depleted RBC were not significantly different (49.4 ± 4.9 and 53.3 ± 2.3 fl/cell, respectively).

Na+/H+ exchange in ghosts. Before this report, NHE activity in dog RBC had been elicited primarily by osmotic shrinkage (12, 36). This response was lost when the cells were lysed and the ghosts resealed by conventional means (i.e., restoring the surface-to-volume ratio of the original cells; see Ref. 6). It was therefore of interest to determine whether acid-induced NHE activity would persist in the ghosts. Ghosts were prepared as described under Preparation of RBC, ghosts, and membranes and were acid loaded by equilibration with an external acidic medium, followed by treatment with DIDS to preserve the pH gradient. A comparison of the rates of 22Na+ uptake in acid-loaded RBC and ghosts is presented in Fig. 4, which summarizes results of seven individual preparations. As in Fig. 3, which illustrates a different set of experiments over a narrower pH range, the RBC displayed a robust, pH-dependent uptake. The ghosts, which were obtained from the same cells illustrated in Fig. 4, also displayed amiloride-sensitive NHE activity. However, at comparable pHi values, the rates of 22Na+ uptake were lower than those of RBC. When the pHi approached neutrality, the activity of the ghosts became insignificant. The reduced activity of the ghosts was not due to proteolytic cleavage of NHE1 during the ghosting and resealing procedures. This was confirmed by immunoblotting the resealed ghost membranes, as shown in Fig. 4, inset.


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Fig. 4.   pHi dependence of NHE activity in intact RBC and ghosts. Intact RBC and resealed ghosts prepared from RBC were compared in parallel experiments. pHi was set to indicated values as described for Fig. 3, and 22Na+ uptake was measured. In all cases, small amiloride-resistant component of flux was determined separately and subtracted. Data from 7 individual experiments are presented. Each point is mean of 2 determinations. Inset: stripped membranes from dog RBC (M-RBC), NHE1-transfected AP-1 cells, and resealed ghosts (M-Ghosts) were subjected to electrophoresis in SDS, followed by blotting onto nitrocellulose. Blots were probed with monoclonal anti-NHE1 antibody and visualized by enhanced chemiluminescence using peroxidase-conjugated secondary antibodies.

The pHi of the ghosts was verified at the beginning of the transport assay and is reported in the abscissa of Fig. 4. It was conceivable that, if ghosts were more leaky to H+ (equivalents) than the RBC, substantial dissipation of the pH gradient would occur during the course of the uptake assay, which lasted 10 min. We therefore compared the pHi of intact cells and ghosts at the beginning and end of this period. The original pHi remained essentially unaltered in RBC, but a significant drift occurred in the ghosts over the 10-min period. For instance, when the initial pHi was 6.52 ± 0.02, the final pHi increased to 6.77 ± 0.02. Such a change may have contributed, in part, to the reduced rate of uptake in the ghosts.

A method for the imposition of a sustained acid load in ghosts. To establish definitively whether the process of lysis and resealing reduces the intrinsic activity of NHE1, we required a procedure whereby a sustained acidification could be imposed. We took advantage of the large anion exchange activity of the cells and ghosts to generate and maintain an acid load of predictable magnitude. Ghosts were resealed in medium containing gluconate as the predominant anion. Subsequent resuspension of such ghosts in Cl--rich media promoted the exchange of external Cl- for intracellular HCO-3, generated by diffusion and hydration of CO2 and subsequent dissociation of H2CO3. Exchange of Cl- for cytosolic OH- may have also contributed to the acidification. Figure 5A illustrates the effectiveness of this approach: gluconate-loaded ghosts suspended in high-Cl- medium have a pHi that is 0.4-0.5 pH units more acidic than their counterparts suspended in low-Cl- medium. We found that the change in pH (Delta pH) imposed using this method was more stable than that created using stilbene derivatives (a change of 0.014 ± 0.001 vs. 0.024 ± 0.001 pH units/min, respectively, at a comparable starting pHi). Over the course of 10 min, the duration of the transport assay, such a rate of dissipation altered the pHi only marginally (from 6.53 ± 0.01 to 6.67 ± 0.01).



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Fig. 5.   Use of a Cl- gradient to establish a transmembrane Delta pH. A: ghosts resealed in gluconate-rich (low-Cl-) solution were incubated for 10 min in medium containing either 4 or 132 mM extracellular Cl- concentration ([Cl]o). Next, ghosts were washed in a solution of same ionic composition and pH but with low buffering power, and pellet was lysed in distilled water, followed by determination of pH. Results are means ± SE of at least 4 experiments. B: Na+/H+ exchange-induced swelling of ghosts. Ghosts loaded with potassium gluconate were suspended in medium containing 128 mM NaCl with (open circle ) or without () 1 mM amiloride or in medium containing 128 mM sodium gluconate (black-triangle) at 37°C. Volume of ghosts was measured at indicated intervals using a ZM Coulter counter and C1000 Channelyzer. Delta Cell volume, change in cell volume. Data are representative of 5 experiments.

The Delta pH generated by the anion exchanger in the presence of an inward Cl- gradient is sufficient to activate NHE in the ghosts. This was confirmed both isotopically and by measuring the volume of the cells. As shown in Fig. 5B, gluconate-containing ghosts incubated in low-Cl- solution gain little volume over the course of 1 h. In contrast, when acid loaded by the presence of external Cl-, the ghosts nearly double their size within this period. That this volume gain is mediated by NHE (in parallel with anion exchange) is indicated by two findings. First, swelling occurs only when Na+ is present extracellularly (not shown), and, second, the volume gain is greatly inhibited by amiloride (Fig. 5B).

Having established that NHE is activated by the pH gradient generated by anion exchange and that the acidification is sustained throughout the assay, we compared the magnitude of the fluxes in intact cells and ghosts at the same pHi. In six determinations, the uptake of 22Na+ in the RBC averaged 4.19 ± 0.49 pmol · 106 cells-1 · min-1. Under comparable conditions, the rate in ghosts averaged 0.96 ± 0.41 pmol · 106 cells-1 · min-1. The rates of uptake of the isotope were linear over the period analyzed in both RBC and ghosts, implying that back flux was negligible in both instances. We nevertheless considered the possibility that imperfect sealing of the ghosts may have resulted in "leakage" of trapped 22Na+ during the uptake or wash periods. Cells and ghosts were allowed to accumulate 22Na+ over 10 min, a period equivalent to that used for the uptake assay. After one rapid wash to remove extracellular isotope, efflux was started by resuspension of the packed RBC or ghosts in uptake medium devoid of radioisotope. As expected, 22Na+ efflux from RBC was negligible (0.038 pmol · 106 cells-1 · min-1). The rate of 22Na+ efflux was somewhat higher for ghosts (0.053 pmol · 106 cells-1 · min-1). Nevertheless, this value is a small fraction of the rate of influx determined under comparable conditions. Moreover, the rate of efflux measured represents a maximal estimate, as it was determined at the end of the 10-min period. At earlier times, the rate must have been lower, since less isotope would have accumulated in the ghosts. Therefore, the leakiness of the ghosts cannot account for the lower rates of NHE in ghosts than in the corresponding intact cells. We conclude that loss or inactivation of a cofactor during lysis or resealing reduces the activity of NHE1.

Effect of ATP depletion on Na+/H+ exchange in ghosts. The reduced NHE activity of the ghosts superficially resembles the inhibition induced in the intact cells by ATP depletion. It was therefore conceivable that the content of ATP of the ghosts was reduced compared with that of cells. However, direct measurements using luciferin-luciferase indicated that the ATP content of RBC and resealed ghosts is similar [(6.83 ± 0.22) × 10-11 mol/106 cells and (6.56 ± 0.20) × 10-11 mol/106 ghosts, respectively]. It therefore appears that the transport rate of ghosts is reduced, despite the presence of normal ATP levels.

Inasmuch as NHE1 does not display any recognizable nucleotide binding motifs, it is likely that the effects of ATP require the participation of additional molecules. Such cofactors may have been lost during the lysis and resealing process, resulting in reduced NHE activity. If this were the case, we would predict that the residual activity of the ghosts would be independent of ATP and therefore insensitive to its depletion. This premise was tested experimentally (Fig. 6) in ghosts resealed with and without added ATP. To ensure thorough depletion of ATP, intact RBC were treated with a glucose-free medium containing 10 mM 2-deoxy-D-glucose for 2 h at 37°C before lysis and resealing in medium devoid of the nucleotide. Under these conditions, the ATP trapped in the depleted, resealed ghosts averaged (0.74 ± 0.01) × 10-11 mol/106 ghosts (~10% of the content of control resealed ghosts). The volumes of control and ATP-depleted ghosts were not significantly different (36.4 ± 1.0 and 36.9 ± 1.4 fl/ghost, respectively). Three different methods were used to compare NHE activity in the ATP-depleted and ATP-replete ghosts. First, we monitored the rate of volume increase in cells that were acid loaded using the anion gradient method detailed above. As shown in Fig. 6A, the rate of volume gain was indistinguishable in ghosts with or without ATP. In both cases, swelling was mediated by the antiport and associated with a net gain in Na+. As shown in Fig. 6A, the increases in cytosolic Na+ content determined by photometry were similar in ATP-depleted and ATP-replete cells. Note that canine RBC do not have a functional Na+-K+-ATPase (5), so that depletion of the nucleotide is not expected to directly affect Na+ efflux.


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Fig. 6.   Effect of ATP depletion on NHE activity in ghosts. A: gluconate-loaded ghosts were prepared from control RBC or ATP-depleted RBC. ATP was omitted from resealing solution when ghosts were made from depleted cells. Ghosts were next suspended in Cl--rich solution (128 mM NaCl) in presence and absence of amiloride. Rate of swelling was then measured electronically as in Fig. 5, and amiloride-sensitive component is plotted (left ordinate). Parallel samples were used to measure Na+ content by flame photometry. Amiloride-inhibitable component of Na+ taken up by control or ATP-depleted ghosts is shown on right ordinate. B: 22Na+ uptake in control or ATP-depleted ghosts that were acidified to indicated values. Acid loading was accomplished by either equilibration and DIDS procedure or by gluconate/Cl- gradient method. Because results were indistinguishable, they were pooled. Data are means ± SE of at least 4 determinations. Where absent, error bars were smaller than symbol.

The conclusion reached by measuring cell volume and Na+ content was confirmed and extended by measuring antiport activity as unidirectional 22Na+ uptake. For these experiments, pHi was set either by resealing the ghosts with Cl-, lowering external pH, and finally treating with stilbene derivatives or by exposing gluconate-loaded ghosts to buffered media with various Cl- concentrations. Because similar results were obtained in both instances, the results obtained using both methods are presented jointly in Fig. 6B. Contrary to the observations in intact cells, the rate of amiloride-sensitive 22Na+ uptake was not depressed in the ghosts by depletion of ATP at most of the pHi studied. These findings are in good agreement with the volumetric and Na+ content determinations of Fig. 6A.

NHERF, a protein associated with NHE3, has been shown to reconstitute cAMP-dependent inhibition of this isoform of the exchanger in renal brush-border vesicles in vitro (42) and in cultured cells (44). It was therefore of interest to consider whether NHERF could also modulate the activity of NHE1, perhaps accounting for its dependence on ATP. We therefore undertook studies to determine whether NHERF could restore the ATP sensitivity of resealed ghosts. We initially determined the intracellular concentration of NHERF in Chinese hamster ovary cells by calibration of immunoblots with recombinant NHERF. Next, we resealed comparable concentrations of recombinant NHERF in ghosts (200 µg/ml) and assessed its effects on the activity and ATP dependence of Na+/H+ exchange, using equivalent concentrations of albumin as a control. In ATP-containing ghosts, the amiloride-sensitive rate of 22Na+ uptake was not increased by trapping of NHERF. In fact, the rate of exchange was slightly higher (21 ± 4%; n = 8) in cells containing albumin than in those containing NHERF. We therefore concluded that NHERF is not the ancillary factor lost from resealed ghosts that is necessary to confer ATP dependence on NHE1. This conclusion was reinforced by the observation that only negligible levels of NHERF were detected by immunoblotting in RBC (not illustrated).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NHE1 in RBC. Our results indicate that NHE1 is present in canine RBC and that it mediates most or all of the Na+/H+ exchange in these cells. Briefly, we found that RBC membranes contain an ~95-kDa polypeptide that is immunoreactive with both polyclonal and monoclonal anti-NHE1 antibodies (Fig. 1) but not with antibodies to other isoforms. In addition, RT-PCR analysis of RNA isolated from dog reticulocytes failed to detect NHE2, NHE3, or NHE4 (unpublished observations). Although suggestive, the latter observations are not conclusive because oligonucleotides based on the rat sequences were used, since the sequences of the canine exchangers are not known. On the other hand, the exclusive presence of NHE1 was also indicated by pharmacological experiments in which >95% of the exchange activity of RBC was inhibited by <= 5 µM HOE-694. At this dose of the drug, only NHE1 is significantly inhibited (9).

ATP dependence of intact cells and ghosts. Inhibition of Na+/H+ exchange activity by depletion of ATP has been consistently found in a variety of cellular systems naturally expressing NHE1, as well as in antiport-deficient cells stably transfected with this isoform. A similar observation is reported here in canine RBC. The universality of the phenomenon would seem to suggest that ATP dependence is an intrinsic property of the exchanger. However, we also found that, whereas ghosts display considerable exchange activity, they were unaffected by the presence or absence of the nucleotide. Interestingly, the residual NHE activity observed in ATP-depleted but otherwise intact cells is similar in absolute magnitude to the activity recorded in ghosts. We therefore surmise that a cooperative effect of ATP and a second factor is required for optimal activity of NHE1. Omission of either component results in suboptimal exchange activity. Because the second factor is lost or inactivated during the course of RBC lysis or resealing, the basal transport rate of ghosts is lower and depletion of ATP has little additional effect.

Earlier work using nucleated cells patched in the whole cell configuration (i.e., under conditions in which the cytosol is continuous with the filling solution of the pipette) found that the ATP sensitivity of NHE1 was preserved for many minutes (10). This observation is not necessarily in conflict with the present findings in RBC. First, dialysis of cellular components through the comparatively small opening of the pipette is slow, limiting the loss of macromolecules. Second, lysis of the ghosts at low ionic strength may have dislodged loosely bound proteins, which would otherwise remain attached and would not diffuse in the case of patch-clamped cells.

The nature of the putative cofactor required for ATP to exert its stimulatory action remains unknown. We attempted to regenerate the ATP sensitivity of the ghosts by resealing within them concentrated cytosol. (The supernatant of a lysed suspension was freeze dried, resuspended in a volume of water equal to the original volume of the cells, and used as the resealing solution.) These attempts were unsuccessful, suggesting that the factor is labile. In this regard, our results differ from those of Colclasure and Parker (6), who could restore the volume-induced activation of NHE by resealing RBC cytosol or concentrated protein solutions into ghosts with decreased volume-to-surface ratios. Therefore, although macromolecular crowding can appropriately explain the osmotic sensitivity of the exchanger, it is unlikely to contribute to the metabolic dependence of NHE1. Accordingly, the inhibitory effects of ATP depletion occur without discernible volume changes.

Although the detailed mechanism of regulation of ATP remains unresolved, several possibilities can be contemplated. First, it is conceivable that the lysis or resealing procedures may have altered the conformation of NHE1 itself. Second, an ATP-dependent reversible association with a soluble or integral membrane (phospho)protein may regulate the exchanger. An analogous mechanism operates in the case of the neuronal Na+/Ca2+ exchanger, which is similarly ATP sensitive. In this case, a soluble cytoplasmic protein isolated from squid axoplasm or brain reconstitutes the ATP stimulation of the exchanger (11). At least three distinct proteins have been reported thus far to interact with NHE1: calmodulin, 70-kDa heat shock protein (HSP-70), and a calcineurin homologue protein (CHP) (2, 29, 38). A 24-kDa polypeptide also shown to bind to NHE1 in vivo (14) may be identical or related to CHP. Of these, only HSP-70 interacts with the exchanger in an ATP-dependent manner: the nucleotide induces dissociation of the chaperone with NHE1 (38). The possible role of HSP-70 in the regulation of NHE1 activity, however, remains to be demonstrated directly.

As has been reported for a variety of other transporters and channels (30, 31, 39), it is alternatively possible that NHE1 may be associated with and regulated by the cytoskeleton. This interaction could, in turn, be modulated by ATP. Association with the cytoskeleton has been suggested by the modification of NHE activity by changes in cell size and shape (17, 25) and by the uneven distribution of NHE1 on the surfaces of some cells (18, 38). Moreover, the actin cytoskeleton rearranges upon ATP depletion (13). Yet, to our knowledge, direct evidence of an interaction between the skeleton and NHE1 is still lacking. The proteins reported thus far to interact with NHE1 (see above) are not known cytoskeletal components.

Finally, it is possible that the regulatory component is not a protein but a (phospho)lipid. In this regard, a possible role of acidic phospholipid asymmetry in the control of NHE1 was considered earlier (10). This hypothesis was entertained in view of results suggesting control of Na+/Ca2+ exchange by phosphatidylserine and ethanolamine (22). However, this notion was not borne out by direct experimental analysis (10). Instead, it is possible that, as also suggested for a variety of K+ channels and for the cardiac Na+/Ca2+ exchanger (21, 24), NHE1 may be modulated by polyphosphoinositides. Depletion of ATP is accompanied by a concomitant reduction of the polyphosphoinositide pool, due to the dynamic equilibrium between inositide kinases and phosphatases. If, as is the case for other transporters (21, 24), PtdIns(4,5)P2 is essential for optimal NHE1 function, the inhibitory effect of ATP depletion may be mediated by depletion of this lipid.

Any of the above hypotheses could be easily reconciled with the requirement for both ATP and a second, soluble and/or labile cofactor. The inhibition noted in ghosts may have resulted from loss or inactivation of a regulatory protein that associates directly with NHE1. Such a protein could be a soluble or cytoskeletal polypeptide. Alternatively, a kinase responsible for protein or lipid phosphorylation may be the elusive factor. In any event, loss of such a component would clearly result in inhibition of the exchanger despite the presence of ATP.

In conclusion, NHE1 can apparently exist in two distinct functional states: an optimal conformation that transports cations effectively and requires both ATP and an as yet unidentified cofactor and a suboptimal conformation that predominates when either ATP or the cofactor is lacking. The factor, which likely mediates the action of the nucleotide on the exchanger, confers on NHE1 a higher sensitivity to pHi.


    ACKNOWLEDGEMENTS

We thank Marcella Prasad for help during the initial phases of this study.


    FOOTNOTES

This research was supported by the Canadian Cystic Fibrosis Foundation (CCFF) and the Medical Research Council of Canada. O. Aharonovitz is the recipient of a CCFF Postdoctoral Fellowship.

S. Grinstein is cross-appointed to the Department of Biochemistry of the University of Toronto, is an International Scholar of the Howard Hughes Medical Institute, and is the current holder of the Pitblado Chair in Cell Biology.

Present address of N. Demaurex: Dept. of Physiology, University of Geneva Medical Center 1, Michel-Servet, CH-1211 Geneva 4, Switzerland.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Grinstein, Div. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8 (E-mail: sga{at}sickkids.on.ca).

Received 30 September 1998; accepted in final form 24 February 1999.


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DISCUSSION
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