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

The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na⁺,K⁺-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na⁺,K⁺-ATPase polarity depends on interactions between the β subunits of Na⁺,K⁺-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these β subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.     

Changes in paracellular and cellular ionic permeabilities of monolayers of MDCK cells infected with influenza or vesicular stomatitis viruses

Summary MDCK cells (epithelioid line derived from the kidney of a normal dog) form monolayers which retain the properties of transporting epithelia. In these cells viruses bud asymmetrically: influenza from the apical, and vesicular stomatitis (VSV) from the basolateral membrane (E. Rodríguez-Boulán and D. D. Sabatini,Proc. Natl. Acad. Sci. USA 75: 5071–5075, 1978; E. Rodríguez-Boulán and M. Pendergast,Cell 20: 45–54, 1980). In the present study, we analyzed whether these viruses affect specific ion-translocating mechanisms located in the plasma membrane. We studied the effect of infection on membrane and transepithelial conductance, passive and active unidirectional fluxes of Na⁺ and K⁺, intracellular potentials, cellular content of Na⁺ and K⁺, and formation of blisters which, in these preparations, are due to the vectorial transport of fluid. Two main observations are derived from these studies. First, infection with VSV caused an increase in transepithelial electrical conductance, due to the opening of tight junctions, 5 to 6 hr after the start of infection, coincident with the accumulation of envelope protein in the cell surface and with the rise in the curve of virus budding. Infection with influenza, on the other hand, increased the transepithelial conductance only late in the infection (12 to 14 hr) when virus production has already stopped. Second, viruses did affect membrane permeability. Yet, the changes observed may not be ascribed to a perturbation of the specific translocating mechanisms for Na⁺ and K⁺ which operate in the same region of the plasma membrane that the viruses use to penetrate and leave MDCK cells. The methods used in the present study are not suitable to decide whether the nonspecific changes in permeability elicited by the viruses occur over the whole cell membrane or are restricted to a given region.     

A novel type of cell-cell cooperation between epithelial cells

Ma104 cells (renal, epithelial) have a peculiar way of resisting ouabain: their Na⁺,K⁺-pumps bind the drug with high affinity, cellular K⁺ is lost and cell division arrested, but cells do not detach as most cell types do. Then, if up to 4 days later the drug is removed, Ma104 cells recover K⁺ and resume proliferation (Contreras et al., 1994). In the present work, we investigate whether Ma104 cells are able to protect ouabain-sensitive MDCK cells in co-culture. The main finding is that they do, but in this case protection is not elicited by the usual mechanism of maintaining the K⁺ content of neighboring cells through cell-cell communications. Ma104 cells treated with ouabain simply remain attached to the substrate and to their MDCK neighbors, and both cells lose K⁺. This attachment includes tight junctions, because the transepithelial electrical resistance of the monolayers is not abolished by ouabain. Although the -subunit of the Na⁺,K⁺-ATPase is known to possess molecular characteristics of cell-cell attachment molecules, attachment between Ma104-MDCK cells does not seem to be mediated by this enzyme, as immunofluorescence analysis reveals that Na⁺,K⁺-ATPase is only inserted in the plasma membrane facing a neighboring cell of the same type.We wish to thank Dr. Enrique Rodríguez-Boulan (Cornell University Medical College) for the generous supply of Ma104 cells, as well as the generous economic support of COSBEL, SA de CV and the CONACYT of Mexico and the National Institutes of Health. Confocal experiments were performed in the Physiology Department's confocal microscopy unit, CINVESTAV.     

Amiloride and calcium effect on the outer barrier of the frog skin

Summary Amiloride (0.1mm) as well as Ca⁺⁺ (10mm) inhibit Na⁺ transport across frog skin by blocking Na⁺ entrance across the outer barrier of the epithelium. The inhibition produced by amiloride consists of an early and a late phase which together account for almost a total inhibition of the short-circuit current (SCC). The analysis of the time course indicates that the two phases are due to the inhibition of superficially and deeply located Na sites, respectively. Ca⁺⁺, instead, only blocks a fraction of the SCC, and this fraction seems to correspond to the inhibition of the same population of Na sites blocked by the late phase of amiloride effect. The location of the two populations of Na sites as well as the possible relationship between them are discussed in terms of maturation of the outermost cell layer.     

Studies on chloride permeability of the skin ofLeptodactylus ocellatus: I. Na+ and Cl− effect on passive movements of Cl−

Summary The outflux of chloride through the isolated skin (J ₃₁ ^Cl ) of the South American frogLeptodactylus ocellatus (L.) is carried by a mechanism that saturates at high concentration of chloride on the inside, and is stimulated by the presence of Cl^– in the outer solution (trans side). The presence of Na⁺ on the outside, by itself, does not increaseJ ₃₁ ^Cl . However, whenJ ₃₁ ^Cl is already increased by chloride on thetrans side, the addition of Na⁺ produces a significant further increase. At low concentration of Cl^– on the outsideJ ₃₁ ^Cl proceeds through a route which involves changes in electrical parameters. The results suggest that both mechanisms are located on the cell membranes and, therefore, that the fluxes would cross through the cytoplasm of the cells. Na⁺ stimulates the second mechanism only.     

Tight junctions and apical/basolateral polarity

Summary Physiological studies led to devise models of epithelial cells in which the membrane does not have its molecules distributed homogeneously, but polarized towards the apical or towards the basolateral regions. For a while, it was assumed that the TJ, acting as a fence between the two regions, would be responsible for this asymmetry. However, today the information available indicates not only that polarization may proceed independently of the TJ, but that this structure itself may attain its precise location due to a polarization process. Nevertheless, TJs may play a role in restricting to the apical or to the basolateral region those molecules that are free to diffuse in the plane of the cell membrane (e.g., lipids and protein that are not attached to cytoplasmic or extracellular structures).