The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.

The tight junction (TJ) is not randomly located on the cell membrane, but occupies a precise position at the outermost edge of the intercellular space and, therefore, is itself considered a polarized structure. This article reviews the most common experimental approaches for studying this relationship. We then discuss three main topics. (a) The mechanisms of polarization that operate regardless of the presence of TJs: We explore a variety of polarization mechanisms that operate at stages of the cell cycle in which TJs may be already established. (b) TJs and polarity as partners in highly dynamic processes: Polarity and TJs are steady state situations that may be drastically changed by a variety of signaling events. (c) Polarized distribution of membrane molecules that depend on TJs: This refers to molecules (mainly lipids) whose polarized distribution, although not the direct result of TJs, depends on these structures to maintain such distribution.

The tight junction (TJ) was first noticed through its ability to control permeation across the paracellular route, but the homologies of its molecular components with peptides that participate in tumor suppression, nuclear addressing, and cell proliferation indicate that it may be involved in many other fundamental functions. TJs are formed by a dozen molecular species that assemble through PDZ and other protein-protein clustering promoting sequences, in response to the activation of E-cadherin. The TJ occupies a highly specific position between the apical and the basolateral domains. Its first molecular components seem to be delivered to such a position by addressing signals in their molecule and, once anchored, serve as a clustering nucleus for further TJ-associated molecules. Although in mature epithelial cells TJs and E-cadherin do not colocalize, a complex chain of reactions goes from one to the other that involves alpha-, beta-, and gamma-catenins, two different G proteins, phospholipase C, protein kinase C, calmodulin, mitogen-activated protein kinase, and molecules pertaining to the cytoskeleton, which keep the TJ sensitive to physiological requirements and local conditions (notably to Ca(2+)-dependent cell-cell contacts) throughout the life of the epithelium.

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 (J DEX ). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J DEX , 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.claudins | c-Src | ERK1/2 | Madin-Darby canine kidney | occludin S everal lines of evidence, including the high affinity and specificity of ouabain for Na + ,K + -ATPase, suggest that endogenous ouabain analogs might exist. In keeping with this possibility, Hamlyn et al.(1) demonstrated the presence of a substance in plasma that cannot be distinguished from ouabain even by specific antibodies and mass spectrometry (1-4). Endogenous ouabain levels are increased during exercise (5) and in pathological conditions such as arterial hypertension (6-9) and eclampsia (10), raising the possibility that ouabain functions as a hormone and prompting efforts to elucidate physiological role.We have previously demonstrated that ouabain acts on cell-substrate and cell-cell contacts in Madin-Darby canine kidney (MDCK) cells. At 1 μM, ouabain binding to the Na + ,K + -ATPase results in pump inhibition and disassembly of molecules from the tight, adherens, and focal junctions (i.e., a P→A mechanism from pump to adhesion) (11). Consistent with these results, Rajasekaran et al. (12) have observed that 0.5 μM ouabain acts on the tight junction (TJ), decreasing transepithelial electrical resistance (TER) and increasing mannitol and inulin permeability in cultures of human retinal pigment epithelial cells. Furthermore, we have shown that in cocultures of wild-type MDCK and ouabain-resistant MDCK cells, ouabain treatment increases the expression of connexin 32, but not 26 or 43, and increases cell-cell communication via gap junctions to rescue the wild-type cells (13). Although these effects indicate that toxic levels of ouabain affect the structure and function of cell-cell junctions, we do not know how lower levels of ouabain affect adhesive structures.In the present work, we focus on this question and demonstrate that 10 nM ouabain affects neither Na + ,K + -ATPase nor the K + balance of the cells and does not indu...

Transporting epithelia posed formidable conundrums right from the moment that Du Bois Raymond discovered their asymmetric behavior, a century and a half ago. It took a century and a half to start unraveling the mechanisms of occluding junctions and polarity, but we now face another puzzle: lest its cells died in minutes, the first high metazoa (i.e., higher than a sponge) needed a transporting epithelium, but a transporting epithelium is an incredibly improbable combination of occluding junctions and cell polarity. How could these coincide in the same individual organism and within minutes? We review occluding junctions (tight and septate) as well as the polarized distribution of Na+-K+-ATPase both at the molecular and the cell level. Junctions and polarity depend on hosts of molecular species and cellular processes, which are briefly reviewed whenever they are suspected to have played a role in the dawn of epithelia and metazoan. We come to the conclusion that most of the molecules needed were already present in early protozoan and discuss a few plausible alternatives to solve the riddle described above.