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J Gen Physiol. May 1, 2000; 115(5): 599–608.
PMCID: PMC2217220

Polarization of Na+/H+ and Cl/Hco 3 Exchangers in Migrating Renal Epithelial Cells

Abstract

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na+/H+ exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pHi. However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pHi. Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na+. When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.

Keywords: migration, Na+/H+ exchanger, Cl/HCO3 exchanger, cell volume, pH

INTRODUCTION

Cell migration plays a central role for such diverse physiological and pathophysiological processes as embryogenesis, wound healing, immune defense, and tumor metastases. A typical feature of migrating cells is their polarization within the plane of movement into lamellipodium (front) and cell body (rear) reflecting underlying cytoskeletal “polarization.” Distinct cytoskeletal mechanisms underlie the protrusion of the lamellipodium and the retraction of the trailing end of migrating cells (for review, see Condeelis 1993; Lauffenburger and Horwitz 1996; Mitchison and Cramer 1996).

Whereas the role of the cytoskeleton in cell migration has been studied in great detail, limited information is available on the role of ion transporters and ion channels in cell migration and on their distribution in crawling cells. In neutrophil granulocytes, chemotaxis is regulated by activation of the Na+/H+ exchanger (NHE) (Rosengren et al. 1994; Simchowitz and Cragoe 1986). At least two explanations can account for the permissive effect of NHE activity on chemotaxis: regulation of intracellular pH (pHi) or of cell volume. Accordingly, hypotonic swelling of granulocytes mimics the stimulatory effect of NHE-mediated cell swelling on chemotaxis ( Rosengren et al. 1994). Both cell volume and pHi also regulate cytoskeletal architecture and cell migration ( Hallows et al. 1991; Van Duijn and Inouye 1991; Schwab et al. 1999).

Previously, we studied the role of a Ca2+-sensitive K+ channel for migration of transformed renal epithelial (Madin-Darby canine kidney, MDCK-F) cells (Schwab et al. 1994, Schwab et al. 1995). We suggested that the intermittent activity of this K+ channel supports the cytoskeletal mechanisms of migration by inducing a localized cell shrinkage at the trailing end of crawling MDCK-F cells (Schwab et al. 1999). However, these experiments left the following question open: by which mechanism is MDCK-F cell volume restored after K+ channel-mediated cell shrinkage? Previous studies on cell volume regulation (Lang et al. 1998) suggested NHE, anion exchanger, and Na+/K+/2Cl cotransporter as good candidates for this function. If volume uptake mediated by these transporters is required for migration, their inhibition should also impair locomotion. We showed earlier that this is indeed the case for Na +/K+/2Cl cotransport ( Schwab et al. 1994). In the present study, we set out to define the roles of the Na+/H+ and anion exchangers in cell migration. In particular, we wished to elucidate whether these transporters affect migration by modulation of pHi or by regulation of cell volume.

In addition, we studied the distribution of NHE and anion exchanger in migrating cells. Prompted by our own observation of polarized K+ channel activity (Schwab et al. 1995; Reinhardt et al. 1998) and by the finding that NHE is concentrated at the leading edge of fibroblasts ( Grinstein et al. 1993), we tested the hypothesis that NHE and anion exchanger support protrusion of the lamellipodium by mediating solute uptake–induced localized volume increase at this cell pole. Swelling due to this uptake of salt and water would then act in concert with the gel-osmotic swelling postulated to occur as a consequence of actin filament assembly at the cell's leading edge ( Oster and Perelson 1987).

METHODS

Cell Culture

Experiments were carried out on alkali-transformed MDCK-F cells ( Oberleithner et al. 1991) and with human melanoma cells transfected with actin-binding protein 280 (ABP280; Cunningham et al. 1992). Both cell types were kept at 37°C in humidified air containing 5% CO2. Cells were grown in bicarbonate-buffered MEM, pH 7.4, with Earle's salts (Biochrom) supplemented with 10% fetal calf serum (Biochrom). To maintain a constant selection pressure on ABP280-transfected melanoma cells, their culture medium contained geneticin (0.5 g/liter; Gibco Laboratories). For experiments, cells were plated on poly-l-lysine–coated coverslips (0.1g/liter; Serva Biochemicals). Experiments were performed between 1 and 2 d after seeding.

Migration Experiments

Migration of individual MDCK-F cells and human melanoma cells was monitored in paired experiments with video microscopy as described previously (Schwab et al. 1994). Migration was quantitated from calibrated videoprints as the distance lamellipodia advanced with time. During the entire course of the experiments, cells were superfused with prewarmed (37°C) Ringer solution that contained (mmol/liter): 122.5 NaCl, 5.4 KCl, 0.8 MgCl2, 1.2 CaCl2, 1.0 NaH 2PO4, 5.5 d-glucose, 10.0 HEPES, pH 7.4, titrated with 1 mol/liter NaOH. Indicated cells were alternatively superfused with CO2/HCO3-buffered Ringer solution (equilibrated with 5% CO2, pH 7.4) containing 24 mmol/liter NaHCO3 (isosmotically replacing NaCl). When applicable, 15 or 30 mmol/liter NaCl were replaced by Na-formate or Na-propionate. The protocol of all migration experiments was such that a 15-min control period was followed by a 10-min experimental period. The rate of migration during the experimental period is always normalized to that of the preceding control period.

Local Superfusion Experiments

Migration experiments with local superfusion of either cell body or lamellipodium were performed as described previously ( Schwab et al. 1997). A micropipette (2–3-μm tip diameter) was connected to a pressure-driven microinjection device (Eppendorf). The pipette was brought into close vicinity of either the cell body or lamellipodium of MDCK-F cells. Local superfusion was started by elevating the “injection” pressure. Then a well-defined stream of control Ringer's solution or Ringer's solution supplemented with ethylisopropylamiloride (EIPA), Hoe 694, or 4,4′-diisothiocyanate-stilbene-2,2′-disulfonic acid (DIDS) flowed over one pole of the cell under study. Simultaneously, cells were superfused “systemically.” The correct positioning of the local superfusate was verified after each experiment by superfusing the cell with a lissamine green–containing solution ( Schwab et al. 1997). Drug-induced changes in rate of cell migration were interpreted as functional evidence for altered activity of NHE or anion exchangers in the membrane of the cell pole under study.

Measurements of pHi

pHi of MDCK-F cells was measured by using video imaging techniques and the fluorescent pH indicator BCECF (Molecular Probes, Inc.). For dye-loading, MDCK-F cells were incubated in culture medium containing 2 μmol/liter BCECF-AM for 1–2 min. Coverslips were placed on the stage of an inverted microscope (Axiovert TV 100; Carl Zeiss, Inc.) and continuously superfused with prewarmed (37°C) Ringer's solution. Excitation wavelength alternated between 488 and 460 nm, respectively. The emitted fluorescence was monitored at 500 nm with an ICCD camera (Atto Instruments). Filter change and data acquisition were controlled by Attofluor software (Atto Instruments). Average fluorescence intensities (corrected for background fluorescence) were measured at 10-s intervals in several demarcated regions of interest placed over the projected cell surface.

At the end of each experiment, pHi measurements were calibrated by superfusing MDCK-F cells with a modified Ringer solution containing (mmol/liter): 125 KCl, 1 MgCl2, 1 CaCl2, 20 HEPES, and 10 μmol/liter nigericin using either two-point calibrations (pH 7.5 and 6.5) or four-point calibrations (pH 8.0, 7.5, 6.5, and 6.0).

The intracellular buffer capacity, β, was determined by a modification of the method described by Weintraub and Machen 1989. Cells were superfused with solutions in which Na+ was replaced by NMDG. After a 3-min pulse with 40 or 20 mmol/liter NH4Cl (isosmotically replacing NMDG-Cl), extracellular NH4Cl was reduced in a stepwise fashion while recording pHi. NH4+ transport was minimized by supplementing these solutions with 1 mmol/liter BaCl2 and 1 μmol/liter bumetanide. The ensuing changes of pHi and the calculated changes of the intracellular NH4 + concentration were used to determine the intracellular intrinsic (CO2/HCO3-independent) buffering capacity β.

Cell Volume Measurements

The effect of propionate on the volume of freshly trypsinized, suspended MDCK-F cells was determined electronically with a Coulter Counter. To obtain maximal propionate-induced swelling, we adopted a protocol described previously (Grinstein et al. 1984). Cells were immersed in a solution containing (mmol/liter): 140 Na-propionate, 1 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES, pH 6.7. When needed, this solution was substituted with 10 μmol/liter EIPA.

Immunofluorescence

MDCK-F cells growing on glass cover slips were rinsed twice with prewarmed PBS and fixed for 30 min with 3% paraformaldehyde in PBS. Fixed cells were washed with PBS, pH 7.4, PBS supplemented with 1% SDS ( Brown et al. 1996), and PBS (pH 8.0) containing 50 mmol/liter glycine and permeabilized with 0.1% Triton X-100 (4 min). Cells were incubated with primary antibody directed against the COOH-terminal peptide of the Cl/HCO3 exchanger AE2 (Stuart-Tilley et al. 1994) for 1 h at room temperature. Alternatively, cells were incubated with a mixture of antibody and blocking peptide (12 μg/ml) against which this antibody was raised or with an unrelated peptide (12 μg/ml). Peptide and antibody were preincubated overnight at 4°C. Before and after adding the Cy3-labeled secondary antibody (goat anti–rabbit IgG; Dianova), cells were washed with PBS, pH 7.4, supplemented with 2.7% NaCl. Cells were mounted in Vectashield (Vector Laboratories, Inc.) for conventional or confocal immunofluorescence microscopy. Specific staining of the anion exchanger AE2 is represented by the difference between immunofluorescence in the presence and absence of the blocking peptide.

Statistics

All data are presented as mean ± SEM. Paired or unpaired Student's t test were performed where applicable. Significance was assumed when P < 0.05.

RESULTS

NHE Modulates Migration and pHi

MDCK-F cells migrate at a rate of 0.92 ± 0.02 μm/min (n = 320) under control conditions. The specific NHE blocker EIPA (100 nmol/liter–25 μmol/liter) reduces the rate of migration to 52.0 ± 7.3% of control (Fig. 1 A) in a dose-dependent manner. 25 μmol/liter EIPA elicits indistinguishable inhibition of migration when MDCK-F cells are superfused with CO2 /HCO3-buffered Ringer solution (48.2 ± 5.8% of control; n = 13; data not shown). Another specific NHE blocker, Hoe 694, has a similar effect. 1 and 10 μmol/liter Hoe 694 reduce the rate of migration to 76.9 ± 7.7% (n = 20) and 63.2 ± 5.6% of control (n = 18), respectively. Together, these data suggest a requirement for NHE activity for normal migration of MDCK-F cells. The half maximal inhibition in the low micromolar range points to the involvement of NHE1 ( Noel and Pouyssegur 1995). In the presence of EIPA, NHE activity in MDCK-F cells may be partially replaced by K+/H + exchange. Simultaneous application of 10 μmol/liter EIPA and 10 μmol/liter omeprazole, a blocker of the H+/K+ -ATPase, causes a stronger inhibition of migration than EIPA alone (36.3 ± 8.1% of control; n = 12). Migration of ABP280-transfected human melanoma cells also depends on NHE activity. Under control conditions, melanoma cells migrate at a rate of 1.17 ± 0.18 μm/min. 25 μmol/liter EIPA reduces the rate of migration to 16.4 ± 5.9% of control (n = 15; Fig. 1 A).

Figure 1
(A) Migration of MDCK-F cells is inhibited by EIPA or Hoe 694 in a dose-dependent manner. Migration of human melanoma cells is almost completely inhibited by 25 μmol/liter EIPA. (B) EIPA and Hoe 694 also elicit a dose-dependent ...

As shown in Fig. 1 B, NHE blockade with EIPA or Hoe694 is followed by a dose-dependent reduction of pHi from the control value of 7.28 ± 0.01 (n = 91). There is no detectable standing gradient of pHi between cell body and lamellipodium. Both NHE inhibitors reduce pHi to similar extents. Half maximal intracellular acidification and half-maximal inhibition of migration exhibit similar IC50 values, consistent with the involvement of the same NHE isoform in both processes. However, careful inspection of Fig. 1A and Fig. B, reveals subtle differences between the inhibition of migration by EIPA and the decrease of pHi. Whereas 1 μmol/liter EIPA has no effect on migration, it reduces pH i by 0.1 pH units. This suggests that moderate intracellular acidification does not suffice to inhibit cell migration, and that the Na +/H+ exchanger may modulate migration by mechanisms other than its impact on pHi. Nonetheless, these experiments are consistent with inhibition of migration by EIPA or Hoe694, reflecting blockade of the Na+/H+ exchanger.

Migration of MDCK-F Cells Depends on NHE Activity Rather than on pH i

If inhibition of migration due to NHE blockade is due to the reduction of pHi below a threshold of 0.1 pH units acidification, this effect should be mimicked by acidifying MDCK-F cells with weak organic acids such as propionic and formic acid. Their protonated forms permeate the cell and rapidly dissociate (Boron 1983), in turn activating Na+/H+ exchange and leading to cellular accumulation of Na-propionate or Na-formate. Hence, application of weak organic acids should cause intracellular acidification and an increase of cell volume due to osmotically obliged water uptake ( Grinstein et al. 1984). As shown in Fig. 2 A, application of Na-propionate or Na-formate is indeed followed by rapid acidification of up to 0.5 pH units. The ensuing pHi recovery is abolished when Na-propionate is applied in the presence of 10 μmol/liter EIPA. Cell volume rises from 2,230 ± 80 fl to 2,420 ± 90 fl, or by 8.4 ± 1.0% within 5 min after the application of propionic acid (n = 10). When Na-propionate is applied in the presence of 10 μmol/liter EIPA, volume does not change (+1.7 ± 0.9%; n = 10).

Figure 2
Effect of weak organic acids on pHi and migration of MDCK-F cells and of human melanoma cells. (A) Formate and propionate induce a marked intracellular acidification of up to 0.5 pH units. Partial pH recovery from the acid load requires ...

As shown in Fig. 2 B, the marked acidification of MDCK-F cells with Na-propionate or with Na-formate does not inhibit migration. To test whether propionate-induced activation of NHE is responsible for the pH insensitivity of MDCK-F cell migration, we applied 15 mmol/liter Na-propionate in the presence of 10 μmol/liter EIPA. Under these conditions, migration is inhibited to the same extent as with EIPA alone (51.7 ± 7.6% of control; Fig. 2 B).

Fig. 3 plots the migration of MDCK-F cells as a function of the decrease of pHi. It is apparent that migration of MDCK-F cells does not solely depend on pHi, but rather on NHE activity per se. We made a qualitatively similar observation in ABP280-transfected melanoma cells. Whereas NHE blockade with EIPA almost completely inhibits migration (16.4 ± 5.9% of control), melanoma cells are slowed down only to 54.8 ± 14.5% of control rates (n = 14) in the presence of 15 mmol/liter propionate ( Fig. 2 B). These observations raise the possibility that NHE activity may modulate migration due to its effect on cell volume. That increased NHE activity in the presence of propionate or formate is not followed by a stimulation of migration is probably due to the potentially inhibitory effect of lowered pHi on migration. However, this negative effect can be compensated for by increased NHE activity in MDCK-F cells.

Figure 3
Cell migration plotted as a function of pHi. When NHE is active, pHi can vary over a wide range (0.5 pH units) without inhibiting migration. Migration is only impaired when the decrease of pH i is due to blockade of NHE.

Anion Exchange Is Required for Migration

Removal of extracellular Cl in CO2/HCO3 -buffered Ringer's solution (isosmotically replaced by gluconate) demonstrates the existence of an anion exchanger in MDCK-F cells. Fig. 4 A reveals that pHi immediately rises at a rate of 0.11 ± 0.01 pH units/min upon removal of Cl (n = 36) and returns to baseline upon readdition of Cl to the bath. The pH recovery upon addition of Cl is sensitive to the anion exchange inhibitor, DIDS. Anion exchange activity is also evident in HEPES-buffered Ringer's solution. pHi rises at a rate of 0.06 + 0.01 pH units/min (n = 8) upon removal of extracellular Cl (data not shown). Taking the intracellular buffering power into account, which is lower in the absence of CO2/HCO3 , this corresponds to an approximately 10-fold lower transport rate of the anion exchanger than in the presence of exogenous CO 2/HCO3.

Figure 4
(A) Demonstration of Cl/HCO3 exchange in MDCK-F cells. Removal of extracellular Cl is followed by an intracellular alkalinization. pH recovery after readdition of Cl is inhibited by DIDS. (B) DIDS ...

As summarized in Fig. 4 B, DIDS slows migration of MDCK-F cells in a dose-dependent manner. 100 and 500 μmol/liter DIDS almost completely inhibit migration of MDCK-F cells (22.3 ± 7.7% and 12.5 ± 5.7% of control, respectively). The sensitivity to DIDS is enhanced in the absence of exogenous CO2 /HCO3. 25 μmol/liter DIDS elicit an almost complete inhibition of migration in HEPES-buffered Ringer's solution (20.7 ± 7.2% of control; Fig. 4 C). This is consistent with the lower anion exchange activity in the absence of exogenous CO2/HCO3 being a rate-limiting step for normal cell locomotion. To test for the contribution of endogenously produced HCO3, we also studied the effect of 100 μmol/liter acetazolamide, an inhibitor of carbonic anhydrase. Acetazolamide has no effect on migration by itself (104.7 ± 11.8% of control), nor does it accentuate the inhibition caused by 25 μmol DIDS in HEPES-buffered Ringer's solution (16.8 ± 5% of control; Fig. 4 C). DNDS (100 μmol/liter), another inhibitor of anion exchange, also slows MDCK-F cells to 68.3 ± 8.3% of control values (n = 11; data not shown). DIDS (100 μmol/liter) almost completely inhibits migration of human melanoma cells as well (24.8 ± 9.6%; n = 12; Fig. 4 B, inset). Thus, parallel operation of NHE and anion exchangers appears to be required for efficient locomotion of both cell types tested.

NHE Activity in MDCK-F Cells

We next determined the transport rate of the Na+/H+ exchanger in MDCK-F cells. Monitoring changes of pHi after stepwise reduction of extracellular NH4+ allowed calculation of the intrinsic buffer capacity β. In the physiological range of pHi, β is ~5 mmol−1 pH unit −1 (data not shown). NHE-mediated Na+ flux was calculated by multiplying β by the initial change of pH (dpH/ dt) after application of EIPA or after removal of extracellular Na+ (0.03 pH units/min). Under basal “resting” conditions (pHi between 7.2 and 7.3), with the assumption of 1:1 Na+/H+ transport stoichiometry, NHE mediates uptake of 1.5 mmol/liter Na+ per min, corresponding to ~1% of the total cellular cation content per min.

NHE activity in MDCK-F cells was also measured as Na+ -dependent pHi recovery from imposed acid load. After acidifying cells in Na+-free Ringer's solution by removal of extracellular NH4+, readdition of extracellular Na+ initiated pHi recovery at a rate of 0.28 ± 0.03 pH units/min (n = 23; data not shown).

Local Superfusion Experiments

To contribute to cell migration, cell volume regulation should optimally be polarized within the cell. This hypothesis should be reflected by polarized distribution of volume regulatory ion transporters in the migrating cell. Therefore, we functionally tested the distribution of NHE and anion exchange activity in MDCK-F cells with a local superfusion technique (Schwab et al. 1997). When control Ringer's solution is directed to lamellipodium or to cell body, MDCK-F cells migrate at their normal rate (data not shown).

Fig. 5 summarizes the experiments with local superfusion of NHE inhibitors. When the lamellipodium is superfused with 25 μmol/liter EIPA, cells are slowed to the same extent as with superfusion of EIPA over the entire cell (50.5 ± 4.2% of control; n = 11). In contrast, when the cell body of migrating MDCK-F cells is exposed to EIPA, locomotion is not affected and cells move normally (103.4 ± 7.9% of control; n = 10; Fig. 5 A). Hoe 694 (50 μmol/liter) has the same differential effect as EIPA when consecutively superfused over both cell poles of one cell. As shown in Fig. 5 B, Hoe 694 slows migration only when applied to the lamellipodium of MDCK-F cells (41.2 ± 8.9% of control; n = 6), while it does not impair migration when directed to the cell body of the same cell (105.3 ± 4.2% of control; n = 6).

Figure 5
Local superfusion of NHE inhibitors. (A) EIPA (25 μmol/liter) inhibits migration only when applied to the lamellipodium of migrating MDCK-F cells. Exposing the cell body to EIPA does not impair migration. (B) Hoe 694 (50 μmol/liter) ...

We next investigated whether the anion exchanger is also unevenly distributed in migrating MDCK-F cells by local superfusion of DIDS (25 μmol/liter) in CO2/HCO3-buffered Ringer's solution (Fig. 6 ). When DIDS is applied to the lamellipodium of migrating MDCK-F cells, they are slowed to 48.6 ± 6.4% of control. This degree of inhibition is identical to that produced by application of DIDS to the entire cell (compare with Fig. 4 B). In contrast, when the cell body is exposed to DIDS, migration of MDCK-F cells is not impaired. MDCK-F cells move at the same rate (85.3 ± 5.8% of control; n = 9) of migration as when the cell body is superfused with control Ringer's solution. Taken together, these experiments suggest that functionally important NHE and anion exchangers are concentrated at the leading edge of migrating MDCK-F cells.

Figure 6
Local superfusion of DIDS, an inhibitor of the anion exchanger, in CO 2/HCO3 buffered Ringer's solution. Migration of MDCK-F cells is only inhibited when DIDS (25 μmol/liter) is directed towards the lamellipodium.

Immunocytochemical Localization of AE2 Anion Exchanger in Crawling Cells

As a correlate of the functional data on the localization of the anion exchanger, we visualized its distribution in MDCK-F cells with an anti–AE2 antibody (Stuart-Tilley et al. 1994). As evident from Fig. 7, functional and morphological localization of the anion exchanger match well in MDCK-F cells. In addition to staining within the cell body, there is a strong signal at the leading edge of migrating MDCK-F cells. Staining is specific since it can be almost completely blocked by incubating the antibody with the antigenic peptide (Fig. 7 C). Staining is not affected when the antibody is incubated with an unrelated peptide (not shown).

Figure 7
Visualization of the anion exchanger in MDCK-F cells. Both conventional (A) and confocal microscopy (B) show that in addition to intracellular staining, the specific antibody reveals concentration of the anion exchanger AE2 at the leading ...

DISCUSSION

We combined migration experiments with measurements of pHi and indirect immunofluorescence to study the functional roles of NHE and anion exchanger in cell migration. Our experiments clearly show that both transport activities are required for efficient locomotion of transformed renal epithelial cells and of human melanoma cells. NHE dependence of locomotion is also known for other cell types such as granulocytes ( Simchowitz and Cragoe 1986; Rosengren et al. 1994; Ritter et al. 1998) and keratinocytes (Bereiter-Hahn and Voth 1988). Thus, it appears that NHE activity is a general requirement for locomotion. To our knowledge, the role of the anion exchanger in cell migration has not yet been studied in detail.

NHE and anion exchanger are both involved in regulation of intracellular pH and cell volume homeostasis, both of which have been shown independently and in different cell types to be important for cell migration (Simchowitz and Cragoe 1986; Rosengren et al. 1994; Van Duijn and Inouye 1991; Ritter et al. 1998; Schwab et al. 1999). Similarly, pHi and cell volume modulate the organization of the cytoskeleton, which provides the directed force for locomotion ( Tonetti et al. 1990; Hallows et al. 1991; Edmonds et al. 1995; Schwab et al. 1999). In particular, NHE activity has been linked to RhoA-induced stress fiber formation (Vexler et al. 1996). The intimate relation between pH and volume regulation complicates efforts to determine which, if either, might be responsible for inhibition of migration after NHE blockade. Our experiments provide circumstantial evidence favoring cell volume as being of greater importance in mediating the effect of NHE on migration. 1 μmol/liter EIPA reduces pHi by 0.1 pH units, but migration is not inhibited. Even when cells are acidified by 0.5 pH units with 30 mmol/liter formate, they crawl at their normal rate. Conversely, the degree of inhibition of migration by 10 μmol/liter EIPA is not further increased by simultaneous application of EIPA and propionate, although pHi is 0.1 pH units more acidic in the latter condition. Finally, parallel operation of NHE and anion exchangers in the lamellipodium suggests that they might modulate migration via their influence on cell volume. Thus, our experiments indicate that increased NHE activity uncouples cell migration from pHi. This observation may be of pathophysiological relevance for leukocyte or tumor cell migration, since these cells often migrate into or out of acidotic tissue.

How can changing cell volume modulate migration? In our view, the distribution of NHE and anion exchanger points to a possible explanation. They are both localized at the leading edge of the lamellipodium. One of the mechanisms involved in the protrusion of the lamellipodium is gel-osmotic water flow into migrating cells at their leading edge ( Oster and Perelson 1987). Due to their localization in the same region of the cell, NHE and anion exchanger can support the protrusion of the lamellipodium by causing osmotic water uptake (a form of isosmotic regulatory volume increase) at the leading edge. NHE mediates the uptake of ~1% of the cellular cation content per minute. Taking the osmotically obliged water uptake into account, the transporters in tandem would account for the uptake of 1% of total cell volume per minute at the leading edge of the lamellipodium. If, for the sake of argument, the resulting swelling exclusively affects the protruding leading edge [a cross-linked actin filament network resists osmotic water flow to some extent (Ito et al. 1992)], one can calculate that the lamellipodium will advance at a rate of ~1 μm/min, exactly the normal rate of migration. Although an oversimplification, this example demonstrates that salt and water uptake at the leading edge of migrating cells can be of a magnitude sufficient to contribute to lamellipodial protrusion.

By measuring cell volume of MDCK-F cells with the atomic force microscope, we provided indirect support for the idea of localized osmotic swelling at the leading edge of migrating cells. The volume of lamellipodium and cell body can change independently from each other since activating or inhibiting a Ca2+-sensitive K+ channel exclusively affects the volume of the cell body ( Schneider et al. 2000). Physiologically, the Ca2+ -sensitive K+ channel is responsible for intermittent shrinkage of the cell body (Schwab et al. 1995; Schneider et al. 2000). Thus, with respect to salt and water transport, migration can be modeled as regulatory volume increase at the leading edge of the cell coordinated with regulatory volume decrease at the cell body (Lang et al. 1998).

It is notable that NHE blockade inhibits migration to the same degree whether in HEPES buffer or in CO2/HCO3-buffered Ringer's solution. The absence of exogenous HCO3 might be expected to inhibit anion (Cl /HCO3) exchange in a HEPES-buffered solution and diminish the impact of NHE activity on cell volume. Intracellular HCO3 production appears not to drive Cl/HCO3 exchange activity since blockade of carbonic anhydrase with 100 μmol/liter acetazolamide has no effect on cell migration in nominally CO2/HCO3-free solutions and since it does not pronounce the inhibitory effect of DIDS in HEPES-buffered Ringer's solution. However, we showed that the anion exchanger in MDCK-F cells also accepts OH ions as counterions, although at an ~10-fold lower transport rate than its Cl/HCO3 exchange rate. A similar observation was made for recombinant AE2 Cl/HCO3 exchanger, which can also function at ambient CO 2 conditions (Jiang et al. 1994). This lower transport rate of the anion exchanger is of a magnitude similar to that of NHE and thereby suffices for normal locomotion in HEPES-buffered solution. Moreover, AE2-mediated Cl/base exchange in Xenopus oocytes exhibits nearly equivalent rates at room air and in 5% CO 2 when assessed during the hypertonic phase of secondary regulatory volume increase (RVI; Jiang et al. 1997). It is possible that the lamellipodium of a crawling MDCK-F cell, undergoing a form of localized RVI, presents a similar local environment, either accelerating Cl/OH exchange or enhancing its affinity for intracellular HCO3.

While we think that local regulation of cell volume is of major importance for the effect of NHE and anion exchanger on migration, we do not know the exact molecular mechanism by which these transporters elicit their action. Presently, we cannot rule out the possible involvement of changes in pHi. In amoeba, bundling and cross linking of actin filaments by elongation factor 1α is increased when pHi falls (Edmonds et al. 1995). Thus, pHi can affect the structural plasticity of the cytoskeleton, which is important for its remodeling during migration. The relative role of pHi for migration probably varies between different cell types. Melanoma cells appear to be more sensitive than MDCK-F cells to propionate-induced intracellular acidification since propionate inhibits migration of human melanoma cells, but not of MDCK-F cells.

NHE1 is concentrated at sites of focal contact ( Grinstein et al. 1993; Plopper et al. 1995). This localization appears to be important for RhoA-dependent formation of stress fibers (Vexler et al. 1996) and for integrin-mediated signaling events during cell adhesion (Schwartz et al. 1991). Recently, NHE was shown to interact via a regulatory cofactor, NHE-RF, with merlin. Both NHE-RF and merlin colocalize to membrane ruffles, filopodia, and microvilli; i.e., to similar structures, as shown for NHE1 in fibroblasts (Grinstein et al. 1993; Murthy et al. 1998). Merlin and related ERM proteins have actin binding sites (Martin et al. 1997), and they have been implicated in Rho-dependent cytoskeletal reorganization ( Mackay et al. 1997). Thus, it appears feasible that NHE exerts its effect on the actin-based cytoskeleton through a physical interaction with NHE-RF and MERM proteins. In gastric parietal cells, the anion exchanger AE2 colocalizes at the basolateral membrane with a form of ankyrin (Jöns and Drenckhahn 1998) to which it may ( Jöns and Drenckhahn 1998) or may not ( Morgans and Kopito 1993) bind. A similar scaffolding or binding protein may serve to concentrate both ion exchangers at the front of MDCK-F cells. However, despite the many open questions that remain, our findings of the polarization of NHE and anion exchanger in MDCK-F cells emphasize that distinct membrane domains are present not only in sessile neuronal or epithelial cells, but also in migrating cells.

Acknowledgments

We thank Dr. J. Reinhardt for help with confocal microscopy, and Drs. M. Gekle, H. Oberleithner, and S. Silbernagl for fruitful discussions during the course of the experiments. We thank Dr. H.-J. Lang (Hoechst AG) for providing us with EIPA, Hoe 694, and bumetanide.

This work was supported by Deutsche Forschungsgemeinschaft (SFB 176 A6) and by the National Institute of Diabetes and Digestive and Kidney Diseases (43495) and the Harvard Digestive Disease Center. S.L. Alper is an Established Investigator of the American Heart Association. Portions of this study were done by Magnus Klein and Ponke Seeger in partial fulfillment of the requirements for their Medical Degree.

Footnotes

Drs. Klein and Seeger contributed equally to this work and should be considered co-first authors.

Abbreviations used in this paper: DIDS, 4,4′-diisothiocyanate-stilbene-2,2′-disulfonic acid; EIPA, ethylisopropylamiloride; MDCK-F cells, Madin-Darby canine kidney cells; NHE, Na+/H+ exchanger.

References

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