▪ Abstract 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 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 β-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 β₁-subunit from the dog kidney (CHO-β). 4) This may be attributed to the adhesive property of the β₁-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-β cells shows that the expression of dog β₁-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of β₁-subunit forces CHO-β cells to coexpress endogenous α-subunit. Together, our results indicate that MDCK cells express Na⁺,K⁺-ATPase at a given border provided the contacting cell expresses the dog β₁-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 sodium pump, or Na⁺/K⁺-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na⁺) and potassium (K⁺) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA’s role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell–cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA β-subunits as cell adhesion molecules in glia and epithelial cells.

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.