Cell adhesion is a crucial step in proliferation, differentiation, migration, apoptosis, and metastasis. In previous works we have shown that cell adhesion is modulated by ouabain, a highly specific inhibitor of Na ؉ ,K ؉ -ATPase, recently found to be a hormone. In the present work we pursue the investigation of the effect of ouabain on a special type of cell-cell interaction: the rescue of ouabainsensitive MDCK cells (W) by ouabain-resistant cells (R). In cultured monolayers of pure W cells, ouabain triggers the ''P3 A mechanism'' (from pump͞adhesion) consisting of a cascade of phosphorylations that retrieves adhesion-associated molecules occludin and ␤-catenin and results in detachment of the cell. When W cells are instead cocultured with R cells, the P3 A reaction is blocked, and W cells are rescued. Furthermore, in these R͞W cocultures ouabain promotes cell-cell communication by means of gap junctions by specifically enhancing the expression of connexin 32 and addressing this molecule to the plasma membrane. Ouabain also promotes the internalization of the ␤-subunit of the Na ؉ ,K ؉ -ATPase. These observations open the possibility that the crucial processes mentioned at the beginning would be under the control of the hormone ouabain.ardiotonic steroids started in the 18th century as unknown active principles when William Withering used foxglove tea (Digitalis purpurea) to treat dropsy (congestive heart failure). Once ouabain was isolated, the demonstration that it specifically inhibits the Na ϩ ,K ϩ pump (1) transformed ouabain into a crucial tool to identify the pump with the membrane enzyme Na ϩ ,K ϩ -ATPase (2) and unravel its intrinsic mechanisms. Ouabain was recently recognized to be a hormone (3) whose blood level increases in physiological (e.g., exercise) as well as pathological (e.g., arterial hypertension, chronic cardiac and renal failure, preeclampsia, etc.) conditions (4-7). Furthermore, we found that ouabain specifically decreases cell adhesion by triggering a P3A mechanism that involves the activation of protein tyrosine kinases and extracellular signal-regulated kinases, decreases the cell content of the small GTP-binding protein RhoA, and changes the degree of phosphorylation, internalization, and degradation of adhesion molecules such as occludin, E-cadherin, etc. (8, 9) (D. Flores-Benítez, personal communication). We have also shown that detachment may not be ascribed to the ensuing decrease of K content (8) and that a decrease of K content provoked by incubation of R or W cells in media with only 0.1 mM K ϩ (instead of the regular 4.0 mM) does not cause cell detachment (8). This finding is in keeping with the fact that changes in reduced͞oxidized glutathione modify the degree of ouabain resistance without reducing the cell K (10, 11).Because in the meanwhile ouabain was shown to be a hormone (3), in the present work we pursue the study of its effect on a special type of cell-cell interaction: metabolic cooperation (12). A typical example of cooperation is provided by R cells that have low...

Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (IK) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12-18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of IK also requires cell-cell contacts and the presence of 1.8 mM Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 microM TRH, 10 nM PMA or 200 micrograms/ml DiC8. drugs that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of IK triggered by Ca-dependent contacts can be blocked by 110 microM neomycin, 2.0 microM H7, and 250 nM staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca(2+)-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca(2+)-activated contracts. However, the effects of the chemicals tested on IK differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane.

The assembly and permanent sealing of tight junctions (TJs) depend crucially on cell-cell contacts containing E-cadherin. This poses a puzzling problem because, while TJs can be established between epithelial cells from different tissues and even different animal species ("heterotypic TJs"; Gonzalez-Mariscal et al. 1989, J Membr Biol 107:43), the cell-cell binding mediated by E-cadherin is a highly specific one (Takeichi 1995, Curr Opin Cell Biol 7:619). Yet the demonstration that TJs can be established at heterotypic borders is open to two distinct challenges. First, it is based on transepithelial electrical resistance (TER) and restriction to ruthenium red permeation only, which today are known to be just two of the many characteristics of TJs; and second some attributes of the TJs (e.g. the presence of specific molecules) have been found even in cells that do not establish these structures. This raised the question of whether heterotypic TJs were not true or full TJs. In the present work we demonstrate that heterotypic TJs in mixed monolayers of MDCK cells with a different cell type (LLC-PK1) are true TJs through several criteria, such as TER, the ability to stop the membrane diffusion of fluorescent sphingomyelin from the apical to the lateral domain, the presence of ZO-1, ZO-2, occludin, claudin-1 and claudin-2. We then turn to the presence of E-cadherin at heterotypic borders, and observe that it cannot be detected by the highly specific DECMA-1 antibody, in spite of the fact that this antibody does reveal the presence of E-cadherin at homotypic contacts of the same cell. Yet, ECCD-2, an antibody against another domain of E-cadherin, reveals that this molecule may be present at both types of borders. Thus, E-cadherin is present at heterotypic borders, yet it seems to be in a conformation unable to bind DECMA-1. Our results suggest: (1) that heterotypic borders can establish fully developed TJs; (2) that the sealing of these heterotypic TJs depends on E-cadherin; (3) but that this dependence is mediated through a cascade of chemical reactions involving two different G-proteins, PLC, PKC and calmodulin, which we have characterized elsewhere (Balda et al. 1991, J Membr Biol 122:193); and (4) hence molecules of E-cadherin that trigger junction formation can act from a distant homotypic contact.

The polarized distribution of K⁺ channels in MDCK cells is lost upon harvesting and restored upon re-seeding. Using semi-quantitative PCR, in the present work we find that (i) Cells do not “wait” for the normal recycling of membrane proteins to restore their lost channels, but trigger their replacement, suggesting that the membrane has a way of engaging the nucleus. (ii) Replacement channels do not come from an internal reservoir, as it is the case with Na⁺, K⁺-ATPase, but requires a de novo synthesis. (iii) Replacement is not an all-or-none response, since mRNA for MaxiK channels increases by 8-fold after re-seeding, but those for Kv1.6 and Kv1.7 are not affected by harvesting/re-seeding. (iv) TEA, charybdotoxin and iberiotoxin fail to trigger the replacement response in mature monolayers, suggesting that replacement is not due to suppression of channel function. (v) MDCK cells have a typical transporting epithelial phenotype (TEP) consisting of tight junctions (TJs) plus polarity. Although the polarized distribution of K-channels is a prominent attribute of TEP, blocking their function does not perturb the development of TEP, as gauged through the development of TJs, nor level of expression (Western blot) and distribution (confocal microscopy) of occludin, and claudins 1, 3 and 7.

No abstract