Isabel Larre

Ouabain modulates epithelial cell tight junction

Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell–cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K+ pumping nor disturb the K+ balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell–cell contacts.

Control of tight junctional sealing: roles of epidermal growth factor and prostaglandin E2.

Epithelia can adjust the permeability of the paracellular permeation route by regulating the degree of sealing of the tight junction. This is reflected by a transepithelial electrical resistance (TER) ranging from a few tenths to several thousand ohms times square centimeters, depending on the difference in composition between the fluid in the lumen and the interstitial fluid. Although teleologically sound, such correlation requires a physiological explanation. We have previously shown that urine extracts from different animal species increase the TER of Madin-Darby canine kidney (MDCK) monolayers and that these effects are mediated by epidermal growth factor (EGF) contained in the flowing intratubular fluid that eventually reaches the urine. This increase in TER is accompanied by an enhanced expression of claudin-4 (cln-4) and a decrement of cln-2. These changes are transient, peaking at approximately 16 h and returning to control values in approximately 24 h. In the present work we investigated how EGF provokes this transient response, and we found that the activation of extracellular-regulated kinases 1/2 (ERK1/2) by EGF is essential to increase TER and cln-4 content, but it does not appear to participate in cln-2 downregulation. On the other hand, prostaglandin synthesis, stimulated by EGF, functions as a negative feedback, turning off the signal initiated by EGF. Thus, PGE(2) blocks ERK1/2 by a mechanism that involves the G alpha(s) protein, adenylyl cyclase as well as protein kinase A in MDCK cells. In summary, the permeability of a given segment of the nephron depends on the expression of different claudin types, which may be modulated by EGF and prostaglandins.

The Polarized Distribution of Na+,K+-ATPase: Role of the Interaction between β Subunits

Na+,K+-ATPase polarity depends on the interaction between the β subunits of Na+,K+-ATPases located on neighboring cells. In the present work, we use energy transfer methods (FRET), in vivo to demonstrate that these β subunits interact directly at the intercellular space of epithelial cells.

Ouabain modulates ciliogenesis in epithelial cells

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

Na+,K+-ATPase is the membrane receptor of hormone ouabain

At 10 nM, ouabain elicits changes in cell contacts, which are independent and usually in opposite direction to effects occurring at μM levels, suggesting that these depend on entirely different mechanisms.1 However, this does not discard the possibility that in both instances ouabain would act on the same receptor. We demonstrate that such is the case by comparing the response of wild and ouabain-resistant MDCK cells on a very special type of cell contact, the tight junction (TJ).

Ouabain Modulates Cell Contacts as well as Functions that Depend on Cell Adhesion

Ouabain, a toxic of vegetal origin used for centuries to treat heart failure, has recently been demonstrated to have an endogenous counterpart, most probably ouabain itself, which behaves as a hormone. Therefore, the challenge now is to discover the physiological role of hormone ouabain. We have recently shown that it modulates cell contacts such as gap junctions, which communicate neighboring cells, as well as tight junctions (TJs), which are one of the two differentiated features of epithelial cells, the other being apical/basolateral polarity. The importance of cell contacts can be hardly overestimated, since the most complex object in the universe, the brain, assembles itself depending on what cells contacts what other(s) how, when, and how is the molecular composition and special arrangement of the contacts involved. In the present chapter, we detail the protocols used to demonstrate the effect of ouabain on the molecular structure and functional properties of one of those cell-cell contacts: the TJ.

Ouabain Increases Gap Junctional Communication in Epithelial Cells

Background/Aims: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). Methods: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. Results: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. Conclusion: Ouabain 10 nM increases GJC in MDCK cells.

Na + /K + -ATPase Drives Most Asymmetric Transports and Modulates the Phenotype of Epithelial Cells

Usually the history of an enzyme is the narrative of the works to isolate and purify it, measure its molecular weight, determine its crystal configuration, measure its activity, and so on, along, say, 2 or 3 years. On the contrary, the history of Na+/K+-ATPase is a tortuous road full of pitfalls, skirmishes with physical chemistry, thermodynamics, and even philosophy. Fortunately it had a happy end, because it was the first known molecule to produce vectorial movement of ions using chemical energy; to cyclically modify its selectivity; to participate in the action potential; to act as a self-adhesion molecule at cell-cell contacts; to act as receptor of the hormone ouabain, whose main physiological role is to modulate cell contacts; and to generate a Na+ gradient that enables co- and counter-transporters to transport net amount of ions, sugars, and amino acids, i.e., to act as secondary pumps. It is sufficient to say that one of its crucial properties, i.e., to be expressed polarizedly at the intercellular membrane of transporting epithelial cells, involves the β-subunit of the pump that happens to be an adhesion molecule which plays a crucial role in that polarization mechanism.

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells.

Background/Aims: The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. Methods: We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. Results: Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. Conclusion: Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.