ionic control of locomotion and shape of epithelial cells: ii. role of monovalent cations

Cell Motility and the Cytoskeleton 10528436 (1988)

Ionic Control of Locomotion and Shape of Epithelial Cells: II. Role of Monovalent Cations

Jurgen Bereiter-Hahn and Monika Voth

Cinematic Cell Research Group, Johann Wolfgang Goethe-Universitat, FrankfurVM., Federal Republic of Germany

The migration of keratocytes isolated from Xenopus tadpole epidermis has been investigated in vitro. In saline the cells move with a mean speed of 5-6 p d m i n . Migration is slowed down in saline with diminished sodium content and ceases in media containing not more than 4 mM sodium. Inhibition of the Nat/K+-2CI- cotransporter by piretanide reduces the speed of migrating cells to about one-third of the control level, the same accounts to inhibition of the Na+/H+ antiport with amiloride at pH 7.2. At pH 6.6, however, amiloride only slightly influences locomotion. Depolarization of the plasma membrane by increased extracellular K + concentration or by inhibition of the Na+/K+ pump by ouabain is only of minor influence during more than 1 h. Hyperpolarization of the cells using the sodium ionophore monensin impedes locomotion; this inhibition depends on an active Na+/K+ pump. Ionophore-mediated breakdown of the K + gradient strictly inhibits locomotion. The experiments have shown that a continuous flux of sodium ions is indispensable for the maintenance of cell locomotion. These ions may exert their action primarily by affecting cytosolic free calcium concentration and pH.

Key words: NdK-ATPase, keratocytes, Xenopus laevis

INTRODUCTION

As a cell migrates, a steady restructuring of the actomyosin network within its pseudopodia, filopodia, or lamellae must take place. This cytoskeletal turnover is the basis of motive force production [Bereiter-Hahn, 1987; Bereiter-Hahn and Strohmeier, 1987a,b; Oster, 19841 and is involved in the control of the direction of locomotion. A number of recent investigations revealed the significance of external free Ca2+ in determining cell shape and the direction of cell migration [Cooper and Schliwa, 1985; Gail et al., 1973; Koike, 1983; Mittal and Bereiter-Hahn, 1985; Strohmeier and Bereiter-Hahn, 19841.

Astonishingly few studies have been published on the control of cell locomotion by monovalent cations. External sodium and potassium do not seem to have any influence over a wide concentration range [Cooper and Schliwa, 19861. However, investigations on leukocyte motility and ruffling by Mukherjee and Lynn [1978] demonstrated the necessity of monovalent cation fluxes,

in particular of Nat . Chemotactic peptides seem to induce local ion influx and swelling at the leading edge of leukocytes. Other clues as to the importance of sodium fluxes in cytoplasmic motility come from the inhibition of fibroblast spreading by the sodium-specific ionophore monensin [El-Battari et al., 1986; Jones et al., 1985; Pizzey et al., 1983; Virtanen et al., 19821, the inhibition of spreading of megakaryocytes by amiloride and tetrodotoxin [Leven et al., 19831, and the dependence of neurite outgrowth on extracellular sodium [Koike, 19831. Therefore, we studied the influence of monovalent cation fluxes on the locomotion of Xenopus tadpole epidermis cells (keratocytes) in culture. This cell type shows a large advancing lamella that contains an actin network excluding larger organelles. The nucleus,

Received December 30, 1987; accepted March 24, 1988

Address reprint requests to Prof. Dr. J . Bereiter-Hahn, Cinematic Cell Research Group, Johann Wolfgang Goethe-Universitat, Senckenber- ganlage 27, D 6000 Frankfurt/M., F.R.G.

0 1988 Alan R. Liss, Inc.

Control of Cell Locomotion 529

mitochondria, dictyosomes, centrosomes, and interme- Experimental Procedure diate filaments are enclosed within the ovoid cell body located at the rear end of the lamella. This kind of cell organization seems to be typical for single epithelial cells of lower vertebrates [cf. Bereiter-Hahn, 1967; Bereiter- Hahn et al., 1981; Cooper and Schliwa, 1985; Radice, 1980; Kolega, 19861. The speed of locomotion of these cells is extremely high [Brown and Middleton, 19851, in the case of Xenopus epidermal cells up to 12 p d m i n , mainly around 5 p d m i n . The morphological and physiological properties make them a favorable object in which to study cell locomotion.

Monovalent cation fluxes were modified by using monensin, an ionophore that promotes Na+/H+ exchange, amiloride, which inhibits Na+/H+ exchange, the loop diureticum piretanide, which blocks the Nat/K+-2C1- cotransport, and by variation of the Na+ and K + concentration of the medium. Steady cation fluxes and low membrane potential are required for maintenance of the shape in moving cells and for locomotion.

MATERIALS AND METHODS Preparation of Cells

Tails from Xenopus laevis (Daudin) tadpoles, stages 49-52 [Nieuwkoop and Faber, 19561, were am- putated from anesthesized animals and minced in calcium-free, phosphate-buffered saline supplemented with 0.2% trypsin and 0.2% EDTA. The tissue homo- genate was suspended in 3 ml of this saline (for one tail) and heavily shaken for 5 min. The supernatant was diluted with an equal volume of amphibian culture medium (ACM; obtained from Gibco, Glasgow, U.K.) [Wolf and Quimby, 19621 and filled in Rose chambers [Rose et al., 19581. Trypsinization of the pellet was repeated three times. After 5 min for settling of the cells, the trypsin-ACM mixture was carefully removed and replaced by pure ACM. After 15 min epidermal cells have spread and started to migrate. Cultures were used for the experiments either immediately or they were stored at about 4°C for up to 5 h and warmed before starting the experiments. Cooling prevents the cultures from a loss of viability, which occurs if single cells are migrating and which results in slowing down of locomotion. Keratocytes organized in continuous cellular sheets survive for about 1 week [Bereiter-Hahn, 19671 and may undergo differentiation; in sparse cultures in which most of the cells are not grouped together viability is limited to a few hours. To improve adhesion and spreading of the epidermis cells, the supporting coverglasses were coated with fibronectin.

During the experiments ACM was replaced by Hanks’ solution (64 mM NaCl, 4.2 mM KC1, 1.4 mM CaC12, 0.65 mM MgS04, 20.9 mM NaHC03, and 0.7 mM NaH2P04) or by modified Ringer’s saline (90 mM NaCl, 18 mM KC1, 0.8 mM NaHC03, and 1.5 mM CaC12) to avoid inactivation of the inhibitors added by binding to serum proteins. Cell shape and motility were followed by reflection-interference contrast microscopy (RIC) on a Diavert (E. Leitz, Wetzlar, F.R.G.) inverted microscope using an HBO 50 lamp and a 570-nm long pass filter for protection of the cells against photody- namic damage [Beck and Bereiter-Hahn, 19841. The events were recorded on a time-lapse videorecorder (Panasonic Type NV805 1-E), using an image intensifier camera (Siemens K5). For evaluation of the speed of locomotion, the displacement of the middle of the rear boundary of the cell body during 5 rnin was determined. Because of the high evenness of locomotion of the epidermal cells, this procedure provides a reliable mea- sure of its speed.

Evaluation of the effect of the various inhibitors was performed by measuring the speed of locomotion of five cells prior to the addition of the drug and by measuring again the speed of five cells 30 min after its addition. It was not possible to retrieve the cells studied under control conditions after a time interval of 30 min or more. In all cases migrating cells were selected, thus the values (e.g., in Table I) do not represent the mean of speed changes of the whole cell population, only the mean of those cells that were still migrating in the presence of the drug.

Ouabain, amiloride, and piretanide were dissolved in the saline before use; monensin was dissolved in ethanol, and this 10-mM stock solution was diluted 1:1,000 with saline. Ouabain, amiloride, and monesin were obtained from Sigma (St. Louis, MO); piretanide was a generous gift of Prof. Geck (Frankfurt, F.R.G.).

RESULTS Influence of Na+ and K+ Content of the Saline

The normal concentration of the saline used is 86 mM for Nat and 4.2 mM for K’. Na’ was reduced to 21 mM by replacing NaCl with choliniumchloride. Further reductions were obtained by using Ringer’s saline with a low sodium bicarbonate content. In 21 mM Na+ the cells were still moving, but with a speed reduced to 65-82% (mean, 76 5 9%) of its initial level (Table I). Because of the high individual speed differences between the control group and the treated group of cells, this reduction was not statistically significant in a single experiment. Nevertheless, the mean of all the

530 Bereiter-Hahn and Voth

TABLE I. Cell Locomotion 1 speed

f i tn lmin Locomotor activity in percent of controls

Condition 30-60 min >60 min

21 mM Na 4 mM Na (15 mM KCI) No K Ouabain Monensin Monensin + ouabain Amiloride

pH 1.2 pH 6.6

pH 1.2 pH 6.6

Piretanide Piretanide + ouabain Piretanide + amiloride Piretanide + monensin Valinomvcin

Amiloride + ouabain

16 t 9 < 10

41 t 2.6 51 2 28 33 t 9 58 t 16

42 i 20 14 ? 21

18 t 12 38 t 2 30 t 11 21 t I 30 t 19 35 ? 12

0

N.D. N.D. N.D.

50 I 35 12 k 1.5 40 t 14

33 t 11 69 t 24

16 ? I N.D.

30 k I 12 t 5

N.D. 34 t 10

Locomotor activity in percent of control values (? standard devia- tions) before the respective treatment. Only those cells have been considered for measuring that still showed the approximately semicir- cular shape of migrating cells. N.D., Nondetermined (mostly because of detachment of the cells from the substratum). The concentrations were monensin, 10 pM; ouabain, 30 pM; amiloride, 0.2 mM; piretanide, 1 pM; valinomycin, 1 KM.

speeds measured in a series of three independent experiments following 15-20 cells each was lower in reduced Na+ without any exception. At 4 mM Nat (and 15 mM KCl), migration stopped or became reduced to less than 10% of its initial speed after 20-30 min (Fig. 1).

In saline with increased K+ content (64 mM) and 15 mM Na' even after 2 h the speed of locomotion was reduced to only 83% of the initial value. In control cultures kept for the same time and only being subjected to an exchange of saline, without changing its constitu- tion, cells maintained their initial speed. In K+-free medium (replaced by sodium) speed was reduced to about 41% after 30 min, and the cells were no longer able to move straightforward, but were rather migrating in a continuous curvature (Fig. 2). The morphological appearance of cells in modified Ringer's saline (4 mM Na' , 15 mM K + ) was characterized by a decrease of lamella area and an enlargement of the cell body projection area. These changes were similar to but more pronounced than in Kf-free saline (Fig. 2).

Equilibration of K+ Between Cytoplasm and Saline by Valinomycin

Valinomycin (0.1 or 1 pM) inhibits locomotion totally without any exception during 20-30 min after application at normal and at reversed Naf and K +

5 1

. . _ . . . 5 10 18 zz 27 32 37 42 47 sz 57 ez e i I? 77 mln

Fig. 1. Time course of speed reduction of a migrating keratocyte on exchange of normal Ringer's saline with a saline containing 4 mM Na+ (arrow). The sodium has been replaced by cholinium chloride; KCl concentration is 15 mM. Immediate reduction of speed is obvious; after the 47-min mark only shape changes occur. At the end of the measurements, the cell detached from the glass.

concentrations. Cytoplasmic motility, however, is differ- ent at normal and reversed Nat and K + concentrations: In normal K+ concentration of the external saline (4.2 mM) the lamella first becomes thin and clearly separated from the cell body (Fig. 3a,b); then its surface topography becomes irregular and nearly immobile, resembling a rigor state (Fig. 3c), and the cell body looses most of its adhesion area (Fig. 3c). Later the lamella is detached from the substratum. In saline with reversed Na/K concentrations agitation can be observed in the lamellar cytoplasm for up to 1 h; however, no locomotion takes place.

Influence of Na+/K+-ATPase Inhibition The ouabain-sensitive Na+/K'-ATPase is the cen-

tral regulatory enzyme of cellular ion content because of its ability to maintain the gradient in Na+ and Kf concentrations between the cytoplasm and the extracellular space. Therefore, the influence of ouabain on the locomotion of keratocytes was studied. During the first 30 min the speed of locomotion was diminished to about one-half of its level prior to the addition of ouabain (Table I). No further decrease was observed during the following hour. After more than 3 h locomotion ceased in most cells. For about 2 h cells treated with ouabain (30 pM) looked more or less normal, with the exception of the frequent appearance of pinocytotic vesicles (Fig. 4b). Thus, cell morphology corresponded to the relatively small reduction of locomotion.


Fig. 2. Appearance and behavior of a keratocyte in K'-free saline (RIC-microscope). a: In normal saline. b-k In K+-free saline for 55 min (b), 68 min (c), 85 min (d), 98 min (e), and 113 min (f). In a the cell body is considerably smaller and the lamella larger than in all

other images. The trailing protoplasmic threads adhering to the glass mark the migratory path, which becomes circular after prolonged exposition to K+-free saline. Bar = 10 prn.

Fig. 3. RIC-microscope images of cellular reaction to valinomycin ( 1 pM) in normal saline. a: Control condition without valinomycin. b: 3 min and C: 8 min after addition ofthe ionophore. Note the steepening of the slope in the cell body/lamella transition region between a and b and the very irregular surface topography of the lamella in c. Bar =

10 wn.

Fig. 4. RIC-microscope images Of a keratocyte under control condition (a) and the Same Cell after 90 min in saline with 30 pM ouabain (b). No major change in cell morphology is apparent. Bar = 10 pm.

Influence of Monensin-Mediated Na+ Influx

Monensin ( 5 or 10 pM) in saline of normal Na+ content induced a decrease of locomotory s p e d to 33 9% of the initial value during 30 min. After 1 h the speed is further reduced to 12 * 1.5% of the control values. This inhibitory effect could be diminished if ouabain (30 pM) was added simultaneously. Then the speed of the

locomoting cells was reduced to only 58 2 16% after 30 min and to 40 * 14% after 1 h.

Monensin-treated cells differed morphologically from controls in the appearance of the lamella, which was more irregular in surface topography and the mar- ginal contour instead of being smooth (Fig. 5) . In many cases the cell body region was m ~ e spread than in


Fig. 5 . RIC-microscope image of a keratocyte exposed to the sodium ionophore monensin (10 pM) in saline with 86 mM NaCl. a: Control condition. b: 2 min 44 sec and c: 3 min 8 sec after monensin addition. Surface topography and contour of the lamella appear more irregular. The high motile activity becomes obvious by comparing correspond- ing lamella regions in b and in c, which are separated by only 22 sec. Bar = 10 pm.

Fig. 6. RIC-microscope image of a keratocyte in saline containing monensin (10 pM) and ouabain (30 pM). a: Control condition. b: 6 min and c: 8 rnin in presence of the drugs. Cell morphology is in the range of normal appearance. The trailing cytoplasmic threads indicate that the cell is still moving. Bar = 10 pm.

controls. Time lapse sequences of RIC microscopic images showed fast changes in surface topography from which an extraordinarily high cytoplasmic motility in the lamella was deduced. In the center or the distal part of the lamella large local thickenings appear comparable to the microcolliculi at the leading front of control cells [Bereiter-Hahn et al., 19811; however, contrary to controls, they do not arise at the edge. Rather they are closer to the cell body and become displaced along the length of the lamella (perpendicular to the direction of locomotion) (cf. Fig. 5b,c). The arrangement of F-actin does not deviate significantly from that in control cells. Some- times local thickenings in the lamella, which exhibit a strong fluorescence in RITC-phalloidin stainings, represent characteristic differences (cf. Fig. 7a,b), while no differences at all are found in the appearance of microtubules (cf. Fig. 7c,d).

Cells treated simultaneously with monensin ( 10

Fig. 7. Fluorescence microscopic visualization of F-actin (a,b) and microtubules (c,d) in keratocytes. a,c: Control condition. b,d: After 30 min exposure to monensin in saline with 86 mM Na+, No differences can be found in the microtubule pattern, while the F-actin pattern is slightly modified by the ionophore treatment. The fibers in the lamella seem to be slightly thickened, and at the margin zones with bright fluorescence they are prominent. Bar = 10 pm.

FM) and ouabain (30 p.M) not only behave more normal than do those exposed to monensin alone (Table I), but they also look quite normal (Fig. 6). After a few minutes in both these inhibitors the lamella and the cell body/ lamella transition region decrease slightly in thickness.


the former lateral ends of the cells (Fig. 9) or the lamella may surround the whole cell body and it becomes a wavy contour (cf. Fig. 8b). During the first 10 min of action the lamella becomes thinner, and, by increasing the slope in the lamella/cell body transition region, these two cell regions are more clearly distinguished (cf. Fig. 9a,b). In its central part the lamella thickens and flattens in irregular cycles. The thickenings spread laterally and show no centripetal displacement as do the thickenings that arise at the front of the lamella in controls. After about 30 min the cell body is more spread than in controls.

An enlargement of the projection area of the cell body is also observed in amiloride-treated cells (Fig. 8b). In both cases k n o b i l e cell forms emerge. Lamella

Fig. 8. RIC-microscope image of a keratocyte a: in control condition and b: after being exposed to amiloride (0.2 mM at pH 7.2) for 22 rnin. The cell body region is enlarged, the contour of the lamella becomes wavy, and the width of the lamella is decreased. Bar = 10

thickness is slightly thinner than in controls, and motion in its central part is prominent.

After prolonged incubation the cell body becomes more spread, and the lamella area may be decreased. DISCUSSION

Influence of Na+ Influx Inhibition by Amiloride and Piretanide

These two inhibitors block the influx of sodium into the cytoplasm by Na+/H+ exchange and by the Na+/Kf-2C1- cotransport, respectively. Both these substances lead to a decrease of speed of cell locomotion to about one-third of its initial value (Table I).

The action of amiloride is sensitive to the pH in the bathing medium. Without amiloride, locomotion of keratocytes is not altered when pH is shifted from 7.2 to 6.6 (mean speed at pH 7.2 is 5.1 2 0.74 prdmin and at pH 6.6 is 5.5 ? 1.98 pm/min; N = 11 in both cases). Ouabain enhances the inhibition of locomotion exerted by amiloride, also depending on pH.

Morphologically, amiloride-treated cells differ from controls by an enlarged cell body and a smaller lamella often with an irregular margin (Fig. 8). In addition, an asymmetric position of the cell body in relation to the lamella is often observed. Cells treated with amiloride and ouabain simultaneously in most cases have a large lamella and a well-spread cell body. The lamella may surround the excentric cell body by more than 90". Although not migrating, the cells show cytoplasmic motility, as is indicated by fast changes in the surface topography of the lamella.

Piretanide-induced slowing down of speed is the same (approx. 2/3) from 1 pM to 2 mM. Its action is enhanced by the presence of ouabain, in particular during prolonged incubation, while simultaneous application of piretanide and amiloride does not reveal any additive effect. Time lapse videos of piretanide-treated cells reveal a high cytoplasmic motion in the lamella which may either become elongated with a bipartite lamella at

The results give a first overview on the involvement of monovalent cations in the control of cellular locomotion. It is certain that they represent one link only in a complex network of control reactions.

Our observations support the following statements: 1) Locomotion of keratocytes depends on the presence of a minimum concentration of external Na' , and external K + supports locomotion. 2) Hyperpolarization of the plasma membrane inhibits locomotion. 3 ) Depolarization of the plasma membrane by increased extracellular K+ concentration-or by inhibition of Na+/K+ -ATPase only-has a minor effect on cell locomotion during a time interval of at least 1 h. 4) Ionophore-mediated breakdown of the K+ gradient strictly inhibits locomotion. 5) Na+ influx in keratocytes can occur at least via the Na'/Kt-2C1- cotransport system and via an Na+H+ antiport.

Point 1

Lowering external Na+ concentration by replacing Na+ either by K+ or by cholinium chloride results in the enhanced appearance of immobile cell forms and in a profound decrease of locomotory speed in those cells still migrating. In both experimental conditions the ionic strength of the medium remained constant; therefore, the effects are specifically related to the lack of Na' . The demand for K+ in the extracellular fluid can be ascribed to a block of the Na+/K+ pump [Kim et al., 19871; however, the locomotory behavior of the cells treated with ouabain differs from that in K+-free medium, because the loss of directionality of locomotion (Fig. 2) is not observed in ouabain. The high amount of cytoplasmic threads remaining attached to the glass delineat-


Fig. 9. RIC-microscope image of a keratocyte a: in control condition and b: after being exposed to piretanide ( 1 pM) for 7 min or c: for 30 min. The main difference during the beginning of piretanide action is a decrease in cell thickness, as indicated by the disappearance of most

of the interference lines in the lamellaicell body transition region. In c the formation of a bipolar shape by local retraction of the lamella is shown. Bar = 10 pm.

ing the migratory tracks of the keratocytes points to an increased adhesion to the substrate in K+-free medium. The reasons for the differences are unknown.

does 100 pM ouabain [Kim et al., 19871, which coin- cides with a similar amount of inhibition of cell locomotion in tadpole keratocytes by ouabain and in K+-free

Point 2

Hyperpolarization can be expected to result from monensin treatment, which mediates Na'/H+ exchange. Thus it causes alkalinization and primarily increases cytoplasmic Na+ concentration. This in turn activates the Na+/K+ pump, transporting 3-K+ into the cell for 2-Nat leaving the cell, resulting in an increase of membrane potential and intracellular ion concentration. The latter shifts the osmotic balance, causing swelling of the cells. These three effects (hyperpolarization, swelling, and alkalinization) do not appear if the Na+/K+- ATPase is inhibited by ouabain. Then only a short increase of Na+ influx is to be expected until an equilibrium is reached between the cytoplasm and the medium. The small change in pH may be counteracted by the cellular regulatory mechanisms. The cells should appear and behave similar to controls, which is the case indeed.

Point 3

Ouabain reduces the speed of migrating keratocytes by about SO% during a time interval up to 2 h. The total inhibition after prolonged exposure could well be a nonspecific toxic effect, e.g., by the lack of an Na+ gradient needed for the uptake of amino acids and glucose. According to experiments with cardiomyocytes, ouabain toxicity is due to increasing free cytoplasmic Ca2+ concentration [Kim et al., 19871. Lack of extracellular K + has the same effect on cardiomyocytes as

saline. In keratocytes, as in many other cells, an increase

of Ca2* has a dual effect, causing solation of myosin- free cytoplasm (in the lamella) and inducing myosin- based contractions [Cooper and Schliwa, 1986; Taylor and Fechheimer, 1982; Mittal and Bereiter-Hahn, 1985; Strohmeier and Bereiter-Hahn, 19841. Contractions occur at much higher concentrations than solation of actin gels cross-linked with Ca2+ -sensitive proteins. The swelling of cells that are still moving in the presence of ouabain could be explained by a slight increase in Ca2+, which is sufficient to induce solation of the lamella cytoplasm, resulting in swelling caused by the hydrostatic pressure present in these cells [Bereiter-Hahn and Strohmeier, 1987a,b], while the Ca2+ level controlling contraction still remains regulated.

Point 4

On destruction of the K + gradient by application of the ionophore valinomycin in a concentration range of 0.1 to 1 pM, locomotion ceases at normal and at high extracellular K + concentration; however, the loss of K+ in saline of the normal low K t concentration is much more injurious to the cells than is valinomycin at high external K f , as shown by the detachment from the substrate during a few minutes in medium of low K+ content. The inhibitory effect in medium with a high K f content might well be a consequence of a lack of energy supply by uncoupling of mitochondria, which occurs at valinomycin concentrations far below those needed for


are consistent with the Na+ requirements for ADP- induced spreading of megakaryocytes , which is associ- ated with a permanent membrane depolarization by Na+ influx [Leven et al., 19831. Ruffling and motility of polymorphonuclear lymphocytes also depend on the presence of Na+ influx at the leading edge, thus inducing local swelling of cytoplasm. The resulting promotion of motility, however, was observed at low Na+ concentrations only (0.1 mM). At 1.0 mM, motility was inhibited [Mukherjee and Lynn, 19781. The demand for Na+ can reside either in regulation of the cytoplasmic Ca2+ content via the Na+/Ca2+ exchange or in its role in membrane depolarization [see, e.g., Lee and Clusin, 19871.

In summary, the observations show that a continuous influx of sodium is indispensable for maintenance of cell locomotion. The inhibitory effect of monensin may be related to cytoplasmic alkalinization abolishing a promoting effect of sodium influx. The monovalent cations are thought to exert their action primarily by affecting cytosolic free calcium concentration and pH. The latter by itself is involved in regulation of calcium fluxes. Therefore, at the moment, any discussion must be very speculative and has to be tested by experiments monitoring the changes induced in cytosolic free Ca2+ and pH.

permeabilizing plasma membranes [Kleuser et al., 19851. At low external K+ concentrations the inhibition may be a consequence of acidification of the cytoplasm by valinomycin-mediated K +/H + exchange rather than a breakdown of the K + gradient. Impedance of cytoplasmic motility by loss of K + can also be deduced from the reduction of coated pits in human fibroblasts and hepatocytes, as observed by Larkin et al. [1985].

Point 5 The fact that both piretanide and amiloride inhibit

cell locomotion is a strong argument for both routes of cation influx being active in toad tadpole keratocytes. This could be a particular property of anuran epidermal cells, which are well known to be active in transepithelial ion transport [for review, see Katz, 19861; however, the widespread occurrence of furosemide- (an analog of piretanide) and amiloride-sensitive cation uptake points to a general physiological significance of these ways in many cells [e.g., Geck et al., 1981; Hoffmann, 1982; Saier and Boyden, 1984; Sachs et al., 19861.

Locomotion is significantly reduced by amiloride only at slightly alkaline pH (7.2), not at a slightly acid pH (6.6). This indicates a pH dependence of the activity of the Na+/Ht antiport. The action of both the antiport and the piretanide-sensitive cotransporter is considerably enhanced by ouabain. When applied separately, many cells are found still migrating, although at reduced speed. However, if applied together with ouabain, amiloride (still depending on the pH of the medium) and piretanide block locomotion nearly completely, and no further inhibition is achieved by adding all three inhibitors together (results not shown). It has to be clarified whether this behavior results from an ouabain-induced increase of amiloride sensitivity of the Na+ influx, as observed by Kim et al. [1987] in cardiomyocytes. The fact that simultaneous application of piretanide and amiloride is not more inhibitory to cell locomotion than if applied separately could indicate either the existence of an additional route for Naf influx or the consequence of a down regulation of the Na+/K+ pump in the presence of one of the inhibitors, thus pointing to a block of Na+ current being the main factor of importance for the maintenance of locomotor activity. However, restoring Na+ influx in piretanide-treated cells with monensin does not restore motility, probably because of a prevalent alkalinization of the cytoplasm.

Role of Monovalent Cations in the Control of Cell Locomotion

For maintenance of locomotion a minimum concentration of cytoplasmic Naf is required, as shown by the inhibitory effect of low concentrations of external Na+ and of amiloride and piretanide. These observations

ACKNOWLEDGMENTS

We thank Prof. Dr. Faulstich (Heidelberg) for his generosity in giving us TRITC-phalloidin. Support for this study from the Gesellschaft der Freunde und For- derer der Johann Wolfgang Goethe-Universitat and from the Deutsche Forschungsgemeinschaft (Be 423/ 12) is gratefully acknowledged.

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