The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy . As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca" concentration and fails if this ion is replaced by Mg`or Ba ++ . The removal of Ca" and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance . Resealing was achieved within 10-15 min by restoring Ca" . Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca", was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components . Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction . The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca" on the extracellular side and low Ca'c oncentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7 .5°C) . Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components . The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels ; the Na'/Cl -and the cation/cation selectivity were recovered with the same time-course as the electrical resistance . The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article (Meza et al ., 1980, f. Cell Biol ., 87 : 746-754.) . intermixed with "leaky" ones (7).As in the case of leaky epithelia, the monolayer has the ability to discriminate Na' from Cl -with a 9:1 permeability ratio, and the monovalent cations of the series IA follow Eisenman's 6th pattern (corresponding to negative sites with a medium force field) .The occluding junctions of MDCK monolayers can be opened by removing Ca" and adding EGTA to the bathing medium . Restoration of Ca" reseals the junction (5). functional resealing, which is completed in 2-3 h, offers the opportunity to study the factors that participate in the reassembly process of the cellular contacts by monitoring changes in transmural electrical resistance and by modifications of the freeze-fracture electron microscopy pattern of the junctions.

Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.

When twitch muscle fibres of the frog were equilibrated in chloride free saline with 2.5–20 mM tetraethylammonium sulphate [(TEA) 2 SO 4 ], the action potential was followed by a long depolarizing response. This response was greatly reduced by adding chloride ions (10 mM). In 20 mM (TEA) 2 SO 4 the response consisted of an initial depolarization of – 23 mV lasting several seconds, followed by a slow delayed spike reaching + 23 mV. After the delayed spike the cell remained continuously depolarized creeping to a steady depolarization of + 25 mV (mean values). The response can be attributed to an increase in membrane conductance to calcium since the phenomenon was abolished by removing external calcium or by adding cobalt or D-600, and was not greatly affected by reducing or removing external sodium or by adding tetrodotoxin.

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.

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