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Original Article |
albrecht.schwab{at}mail.uni-wuerzburg.de
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ABSTRACT |
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 Top ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
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Key Words: migration • Na+/H+ exchanger • Cl–/HCO3– exchanger • cell volume • pH
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INTRODUCTION |
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TopABSTRACTINTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
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; 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).
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METHODS |
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) 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.
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RESULTS |
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). 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).
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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).
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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.
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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).
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). 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).
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DISCUSSION |
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TopABSTRACTINTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
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; 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 1
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.
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.
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ACKNOWLEDGMENTS |
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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.
Submitted: 29 December 1999
Revised: 13 March 2000
Accepted: 20 March 2000
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TopABSTRACTINTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
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