1Departamento de Bioquímica, Facultad de Medicina, and 2Sección Biofísica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
Submitted 27 May 2004 ; accepted in final form 17 January 2005
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ABSTRACT |
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actin; epithelial sodium channel
Numerous factors appear to participate in the initiation and development of the healing process. Although the interruption of intercellular contacts may, in principle, play a role in triggering the repair response, a current view is that growth factor liberation by the injured cells specifically activates signaling pathways of the surviving neighboring cells (27, 51, 62). Also, an early Ca2+ wave of short duration, propagated from the leading edge toward the center of the monolayer, has repeatedly been detected immediately after experimental wounds (27, 51, 55). The activation of several signaling cascades has been reported, possibly triggered by the initiating signals represented by the growth factor liberation or the transient elevation of intracellular Ca2+. Thus, for instance, in the case of the purse-string mechanism, activation of members of the Rho family have been reported to be involved in the formation of the actin cable (9). This protein family also participates in the different stages of individual cell migration in general (for review, see Ref. 48) and particularly in the process of wound healing by lamellipodial crawling (1, 20, 31, 39). In addition, during the past few years, preeminent roles have been recognized for diverse ion transport systems in the establishment of the characteristic cell polarization and cytoskeletal rearrangements that take place during cell migration (18, 54). In the case of corneal epithelial wound healing, the development of induced K+ currents has been suggested to participate in the late stages of tissue restitution (58, 59). Finally, it also has been well established that transtissular electric fields spontaneously arise in wounded epithelia (2, 12), possibly generated by the electrogenic activity of the Na+ pump (40). The small direct currents provoked by these fields participate in the orientation and actin reorganization of migrating cells (34, 37, 41, 50) and may act via a complex interaction with enzymatic cascades (57).
In previous work (13), we showed that the nonspecific depolarization of the plasma membrane potential (PMP) provokes characteristic modifications in the cytoskeletal organization of bovine corneal endothelial (BCE) cells in culture. To determine possible physiological implications of these findings, we explored whether membrane depolarization participates in the cytoarchitectural modifications that take place in the course of epithelial wound healing. As mentioned above, the dramatic structural changes undergone by cells actively participating in the processes of wound healing require characteristic cytoskeletal reorganization. It has classically been accepted that corneal endothelial monolayers heal, both in vivo and in vitro, predominantly by cell migration (22, 49, 52). This has been described particularly for relatively large wounds (e.g., >3 mm in diameter). However, it has been suggested that narrower endothelial wounds may exhibit zones of actin cable formation at the leading edge (43). In this work, we report that linear wounds narrower than 150 µm produced on cultured BCE cell monolayers heal by a mechanism that simultaneously combines actin cable formation and cell crawling. The actin cable can be detected as early as 1 h after the injury and progressively extends to border almost the totality of the two edges of the wound. We specially report here the novel finding that, in this system, membrane potential depolarization occurs at the leading edge of the wounds and gradually extends inward toward the neighboring cells. We provide evidence that this spontaneous depolarization is involved in the development of the characteristic actin reorganization demonstrated by the healing cells. Thus the replacement of extracellular Na+ by choline (Ch) provokes a marked decrease in both the depolarization areas and actin cable formation. In addition, we obtained evidence suggesting that the membrane depolarization occurs mainly by an increase in epithelial Na+ channel (ENaC)-mediated Na+ permeability. Most remarkably, we found that the incorporation of ENaC inhibitors determines effects analogous to the ones produced by the replacement of the ambient Na+ by Ch and a marked decrease in the lamellar activity of the wound edges. Finally, the finding that Li+ can replace extracellular Na+ in the determination of the depolarization and cytoskeletal responses suggests that membrane depolarization of the leading cells, and not the increase in intracellular Na+, plays a role in the determination of the cytoskeletal reorganization involved in the healing process. The cellular electrical findings reported here might possibly be related to the generation of macroscopic transtissular electrical fields in the course of epithelial wound healing (see above), but the nature of this relationship is not analyzed in this work. The results reported here support the idea suggested previously by several authors (13, 23, 29, 42) that the PMP of nonexcitable cells may play a role as an intermediary of diverse cellular signaling processes.
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MATERIALS AND METHODS |
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Cell cultures and general experimental procedures. BCE cells were obtained and cultured as described previously (13). The biological materials employed to obtain the cellular cultures utilized in this work come from animals butchered at a slaughterhouse, as mentioned in MATERIALS AND METHODS and ACKNOWLEDGMENTS. We did not handle any live animals whatsoever for this study. Briefly, fresh bovine eyes obtained from the slaughterhouse were processed within 4 h of enucleation. The cornea was dissected and treated with trypsin (0.25%) and EDTA (0.02%) in Ca2+- and Mg2+-free PBS for 2030 min in the tissue culture incubator. The endothelial cells of each cornea were carefully scraped with a blunt spatula and placed in a 35-mm tissue culture plate containing minimum essential medium (MEM) supplemented with 10% serum, 50 µg/ml gentamicin, 0.25 µg/ml amphotericin B, and 50 µg of total protein/ml of retinal extract. For this study, we used cells from passages 15, which were grown on glass coverslips and had achieved visual confluence at least 5 days before the experiments.
Linear incision wounds were created manually on the monolayers using a fresh 21-gauge syringe at a speed of 5 mm/s. After the occurrence of dead cell detachment (see below), the measured width of the wounds was 150 µm. The injured cells were then kept in medium or in any of the experimental solutions described above for the corresponding time period at 37°C. The fact that the wound-healing processes exhibited similar electrical and cytoskeletal modifications (see below), both in medium and in saline CS lacking HCO3, permitted us to exclude possible effects caused by the lack of this anion involved in several transport processes in BCE cells (7). Figure 1A shows a typical phase-contrast image of a wound 2 h after injury. At this time, it is possible to observe that a wide area of extracellular matrix, which was left denuded as a consequence of the loss of the overlapping dead cells, has not yet been covered by the restituting tissue. Also, some cells at the wound borders exhibit lamellar activity.
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The velocity of wound healing was determined on images taken after phalloidin staining. The resulting images were processed with Adobe PhotoShop software as follows: 1) The denuded area was selected with the magic wand tool, and the total number of pixels contained in this area was determined using the histogram command; 2) the quotient between this value and the wound length (expressed in pixels), obtained using the rule tool, was determined and considered to represent the average width of the wound; and 3) the micrometric scale of the corresponding image was used to convert the width value in pixels to micrometers.
Determination of PMP and intracellular Na+ changes. The modifications in the PMP were detected using fluorescence microscopy and the anionic dye Oxonol V [bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethine oxonol; Molecular Probes, Eugene, OR]. Oxonol V, kept as a 0.7 mM stock solution in ethanol at 4°C, was freshly diluted to a final concentration of 3 µM in the corresponding solution before each experiment. For the experiments, the coverslips containing wounded monolayers were incubated for 30 min at room temperature in the appropriate solution containing 3 µM Oxonol V. In the studies conducted immediately after wounding, the cells were incubated in the oxonol-containing solutions and then injured. In every case, the coverslips were then mounted in a custom-made chamber (13) containing the same solution and placed under a fluorescence microscope. To detect dead cells, all of these solutions also contained 1 µg/ml propidium iodide (PI). The fluorescent images were obtained using a rhodamine filter set and recorded as described in the previous section. Figure 1B shows a typical image of an oxonol-loaded monolayer in which the overall distribution of the dye between the cells is approximately homogeneous, although some isolated cells may exhibit a larger fluorescent signal (Fig. 1B, arrow). As described by Dall'Asta et al. (16), the dye accumulates in the cellular vesicles, thus determining a somewhat heterogeneous intracellular distribution of the fluorescent signal. As also shown in Fig. 1, the cell nuclei appear as negative areas. In our cells, a juxtanuclear area of low vesicle density was frequently observed (Fig. 1B, arrowheads); this was confirmed by staining the intracellular vesicles with acridine orange (results not shown). The higher oxonol intensity displayed by some isolated cells may thus reveal a more depolarized membrane potential, a greater vesicle density, or both. For all of these reasons, the PMP measurements needed to be performed by averaging image regions containing several cells (see below). To enhance the PMP modifications, pseudocolor images were developed from the original ones by transforming the spectrum of intensity of the monochromatic signals into a color spectrum using Scion Image Beta 4.0.2 software.
Calibration curves for the membrane potential were performed using the standard procedure of modifying the external K+ concentration in the presence of the selective ionophore valinomycin (24). For this procedure, NaCl in CS was replaced by potassium gluconate and ChCl such that the corresponding external concentrations satisfied the equation [potassium gluconate] + [ChCl] = 137 mM. In every case, valinomycin was added to a final concentration of 2.3 µM. A linear relationship was found between the logarithm of the oxonol fluorescence intensity of the original images and the membrane potential, calculated assuming a Nernst equilibrium for K+ in the interval between 5 and 130 mM of external K+ concentration as predicted by previous studies (16, 24). For the determinations, the intracellular K+ concentration was assumed to be 130 mM. Because of the heterogeneity of the intracellular distribution of the oxonol signal (see above; see also Ref. 16), the quantitative analyses were performed by determining average intensities of the complete original images or of groups of cells within these images. Also for this reason, the pseudocolor images shown have a qualitative character. From the calibration curves, the PMP of the BCE cells in noninjured monolayers kept in CS was determined to be 48.3 mV (SD 8.7; n = 5), a value in fair agreement with previous determinations (63).
The changes in intracellular Na+ concentration ([Na+]i) were determined using the fluorescent intracellular Na+ indicator sodium green tetraacetate (Sodium Green; Molecular Probes). After the experiments, the cells were incubated for 30 min at room temperature in the corresponding solution containing 5 µM Sodium Green. The coverslips were washed three times and mounted in the same solution plus 1 µM PI in a chamber similar to the one used for the determination of PMP changes. The fluorescent signals were analyzed analogously to the oxonol studies (see above), but using a fluorescein filter set.
As suggested by the manufacturers, and as commonly described in the literature (32, 33, 61), the [Na+]i was calculated from the determined value of fluorescence intensity (F) using the following equation
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RESULTS |
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In the next portions of our present study, we sought to determine the mechanisms involved in the spontaneous depolarization of the PMP demonstrated by the cells at the wound border and to establish whether this depolarization plays a role in the cytoskeletal rearrangements that take place in the course of wound healing.
Dependence of actin reorganization and membrane depolarization on extracellular Na+. To assess whether membrane depolarization at the border cells occurs via an increase in Na+ conductivity, we performed double-staining experiments to study the oxonol signal and [Na+]i (Fig. 4, AD). The pseudocolor images shown in Fig. 4, AD, reveal that, in effect, concomitant with the plasma membrane depolarization, there is an increase in [Na+]i of the cells at the wound edges. Quantitative analysis of the original images (see MATERIALS AND METHODS) yielded an average maximum value for [Na+]i of the cells at the wound border of 81.3 mM (SD 9.1; n = 3). As also shown in Fig. 4, AD, the increase in the Na+ signal propagated toward the rest of the epithelium according to the same pattern observed for membrane depolarization.
Replacement of extracellular Na+ by Ch, a nonpermeant cation, determined a decrease in the depolarization areas (Fig. 4, E and F) and conspicuous modifications in the processes of actin remodeling that takes place during the course of the healing of narrow wounds (Fig. 4, GJ). The oxonol images (Figs. 4, E and F) also reveal that, in general, the fluorescence intensity was lower in the Ch-treated layers (Fig. 4F) than in the ones that healed in the presence of normal extracellular Na+ concentrations (Fig. 4E). Concomitant with the modifications in oxonol fluorescence, the replacement of Na+ by Ch provoked an almost complete interruption of the actin changes observed during the healing process (Fig. 4, GJ). While the control monolayers developed an actin cable at the wound border and exhibited characteristic rearrangements of the actin organization that extended to several rows of cells in the form of relocalization of the peripheral actin toward the cytoplasm (Fig. 4, G and I, see also Fig. 2C), the cells that healed in the presence of Ch did not develop a continuous actin cable and conserved their typical circumferential localization of actin (Fig. 4, H and J). Also, lamellar protrusions were markedly diminished (Fig. 4, I and J). In fact, in the Ch experiments, the border cells exhibited an organizational pattern of actin similar to that observed immediately after wounding (data not shown), although some individual cells may form thin, cablelike structures. The possibility that the observed Ch effects could have been provoked by the stimulation of putative acetylcholine receptors, found to be present in several epithelia (60), was discarded by performing experiments similar to the ones described in this section in the presence of 1 µM atropine or 0.1 mM D-tubocurarine (results not shown).
ENaC participation in membrane depolarization and actin reorganization during wound healing.
Taken together, the results described in the previous sections are highly suggestive that plasma membrane depolarization occurs spontaneously in the cells at the border of narrow wounds produced on cultured BCE monolayers and that this depolarization is involved in the development of the characteristic remodeling of actin. In addition, the evidence also suggests that membrane depolarization occurs mainly as a consequence of an increase in Na+ permeability. To obtain some evidence regarding the possible mechanism underlying the depolarizing response, we explored whether the augmented Na+ permeability could be determined by an increase in ENaC activity of the border cells, because this channel has been detected and demonstrated to play a role in fluid transport in situ and in cultured corneal endothelial cells (28, 38). Figure 5, AD, shows characteristic images of the effects of phenamil, a specific ENaC inhibitor (21), on the PMP and Na+ concentration of the BCE cells 1 h after wounding. The presence of phenamil in the incubation medium strongly inhibits the [Na+]i increase of the leading cells compared with the control cells (Fig. 5, A and C). The depolarization areas are correspondingly decreased (Fig. 5, B and D), suggesting that the ENaC-mediated increase in Na+ conductance represents the main mechanism of plasma membrane depolarization in these cells. After 1 h of healing in the presence of phenamil, the effects of the drug on the cytoskeletal modifications of the border cells (data not shown) are similar to those induced by the replacement of ambient Na+ by Ch (Fig. 4, GJ). In analogous experiments (i.e., maintaining the monolayers in MEM not supplemented with serum) performed for longer periods of time, phenamil started to produce morphological alterations of the cells and eventually cell death. For this reason, to study the effect of phenamil on the velocity of healing, the wounded monolayers were left in MEM supplemented with 5% serum, which permitted us to prolong the experiments for 6 h without noticeable effects on cellular morphology and viability (Fig. 5, E and F). As shown, under these conditions, the phenamil-treated monolayers exhibited significantly less actin cable and a smaller degree of wound closure. The average velocities determined for the control and phenamil-treated monolayers (see MATERIALS AND METHODS) were 8.7 µm/h (SD 2.3) and 3.2 µm/h (SD 0.9) (data from 3 independent experiments in which a total of 6 fields/experiment were processed), respectively, for each border.
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The augmented presence of ENaC at the wound borders was demonstrated using indirect immunofluorescence with an anti--ENaC antibody (Fig. 6, AD). Immediately after the wound was created, the ENaC fluorescence intensity was extremely low throughout the cell monolayer (Fig. 6B). At that point, the corresponding actin image (Fig. 6A) exhibited the typical aspect of the initial stages. From then on, the ENaC fluorescent signal increased following a time course and distribution similar to the membrane depolarization and to the increase in [Na+]i. In Fig. 6D, we show a typical image obtained 2 h after injury. As shown, the area exhibiting an increase in the ENaC fluorescence corresponds to the area of actin reorganization and cable formation (Fig. 6C).
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DISCUSSION |
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The concurrent findings of this work that phenamil is a potent inhibitor of the depolarization response and of the actin rearrangement, that Li+ can replace Na+ in the generation of the response, and that the ENaC immunofluorescent signal increases in the course of the healing process support the idea that the augmented activity of this channel may be responsible for the increase in [Na+]i and the concomitant plasma membrane depolarization exhibited by the border cells. In this respect, it has been demonstrated that modifications in the number and open probability of ENaC mediates PMP depolarization responses in cultured alveolar epithelium (30). On the basis of our findings, a plausible hypothesis of the sequence of events leading to membrane depolarization could consider the transcriptional activation of genes involved in ENaC expression and its membrane insertion. This activation could be promoted, for instance, by the liberation of growth factors and/or by the intracellular Ca2+ increase that take place immediately after the injury (see above). The wound-induced increase in intracellular Ca2+ has indeed been reported to activate genes involved in the regulation of cellular motility (55). On the basis of our present results, the appearance of the ENaC immunofluorescent signal seems to follow a time course similar to that of the depolarization area. This supports the idea that, for both the border and neighboring cells, membrane depolarization is achieved via an increase in ENaC activity and not by the intercellular propagation of electrical or chemical signals. This notion is further supported by our finding that heptanol and octanol, commonly used blockers of gap junctions (11), did not affect the propagation of the depolarization areas (results not shown). Also in this respect, it is interesting to note that the early Ca2+ wave, which could promote the initiation of the healing response, does not seem to be propagated via gap junctions (27).
The participation of diverse ion channels and transporters in cell motility and migration is well documented (for review, see Ref. 54). Although several possible mechanisms have been suggested to account for this participation, there is a considerable body of evidence relating ion transport systems and the cytoskeleton (18, 19, 46, 54). Thus, several reports point to the crucial role of the ion channels and transporters in the anchoring of the cortical cytoskeleton (4, 19). In turn, the cytoskeleton exerts control over ion transport activity by modulating ionic currents (5, 10, 18). This interrelationship may underlie the participation of some ion transport systems in cell migration. For instance, in the case of the Na+/H+ exchanger, both the cytoskeletal anchoring and the ion translocation are necessary for cell migration and wound healing of PS-120 fibroblasts (18). Similarly, ENaC may participate in actin cable-mediated wound healing via an analogous interaction with some cytoskeletal components. In this respect, it has been reported that the -subunit of ENaC associates with spectrin, a member of the cortical cytoskeletal network (64). Also, direct interaction of ENaC with actin results in modifications of the channel conductance (6, 15).
Finally, we remark that, under the conditions used in this study, linear narrow wounds produced in corneal endothelial monolayers predominantly heal by a purse-string mechanism. In this mechanism, the adherens junctions play a key role by providing the sites of anchorage of the actin cable, which allows the constitution of a continuous functional string all along the wound border (17). As emphasized above, one of the central findings of this study is that the development of the actin string is dependent on plasma membrane depolarization of the border cells. This finding is consistent with recent results showing that the actin reorganization induced by the nonspecific depolarization of the PMP of undamaged cultured epithelium occurs during the initial stages concomitant with conservation of the cadherin disposition (14).
In summary, the results described herein permit us to conclude that the actin cable formation and reorganization observed at the edges of narrow wounds produced on BCE monolayers are determined by the spontaneous depolarization of the PMP of the cells present at or near the wound borders. The depolarization seems to be generated by a rise in the ENaC activity of these cells as suggested by the fact that inhibitors of this channel greatly suppress the electrical and cytoskeletal responses, as well as by the increase in the immunofluorescent signal of the channel observed after the production of the wound. Also, the fact that Li+ ions can replace Na+ ions in the determination of the response supports the concept that the membrane depolarization, not the increase in the [Na+]i, is mainly responsible for it. This is consistent with our previous observations that the nonspecific membrane depolarization of confluent BCE cells and other epithelia in culture elicits a characteristic reorganization of the actin cytoskeleton (13). Hence, the findings of the present study further support the idea, suggested by several authors (13, 23, 29, 42), that the PMP of nonexcitable cells may play a role in the regulation of diverse cellular processes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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