Polarization of Na 1 / H 1 and Cl 2 / HCO 3 2 exchangers in Migrating Renal Epithelial Cells

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 1 /H 1 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 pH i . However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH i . Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na 1 . 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. key words: migration • Na 1 /H 1 exchanger • Cl 2 /HCO 3 2 exchanger • cell volume • pH I N T R O D U C T I O N 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 1 /H 1 exchanger (NHE) 1 (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 (pH i ) 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 pH i 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 Ca 2 1 -sensitive K 1 channel for migration of transformed renal epithelial (Madin-Darby canine kidney, MDCK-F) cells (Schwab et al., 1994, 1995). We suggested that the intermittent activity of this K 1 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 1 channel-mediated cell shrinkage? Previous studies on cell volume regulation (Lang et al., 1998) suggested NHE, anion exchanger, and Na 1 /K 1 /2Cl 2 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 1 /K 1 /2Cl 2 Drs. Klein and Seeger contributed equally to this work and should be considered co-first authors. Address correspondence to Albrecht Schwab, Physiologisches Institut, Röntgenring 9, D-97070 Würzburg, Germany. Fax: 49 931 312741; E-mail: albrecht.schwab@mail.uni-wuerzburg.de 1 Abbreviations used in this paper: DIDS, 4,4 9 -diisothiocyanate-stilbene-2,2 9 -disulfonic acid; EIPA, ethylisopropylamiloride; MDCK-F cells, Madin-Darby canine kidney cells; NHE, Na 1 /H 1 exchanger. on O cber 9, 2016 D ow nladed fom Published May 1, 2000