Repolarization of Na+-K+ pumps during establishment of epithelial monolayers

Abstract

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.

Cell Adhesion, Polarity, and Epithelia in the Dawn of Metazoans

“…This approach gave us an opportunity to study the development of the transporting epithelia phenotype. Thus we used it to study the synthesis, assembly, and sealing of tight junctions (TJs) (69,74,75,78,145,183,297), as well as the polarity of Na ϩ -K ϩ -ATPase (61,62,96,102); Enrique Rodriguez-Boulan introduced the use of viruses that bud either apically (e.g., Flu) or basolateraly (e.g., stomatitis) to track the fate of specific proteins during apical or basolateral polarity (283, 284, 379 -382), and Carlos A. Rabito analyzed the onset of polarized cotransporters (368,370,371). M. Taub perfected the approach by developing totally defined culture media (440 -442), and complementary procedures were refined to open and reseal the TJs by removing and restoring Ca 2ϩ (69,97,101,184,186), follow the cascades of phosphorylation involved (20,21,85), the role of the cytoskeleton (311, 312), the participation of protein synthesis and sorting (196,376,379,381), the polarized distribution of ion channels (317,359,360,361,421,439), the involvement of E-cadherin (199 -201), the effect of agents that induce differentiation (280), and the vectorial movement of receptors (302).…”

“…When cells are plated in the presence of Ca 2ϩ but this ion is removed after allowing 20 -40 min for attachment to the substrate, the relatively small fraction of Na ϩ -K ϩ -ATPase and ion channels expressed at the plasma membrane cells is not polarized, and these molecules, as well as TJ-associated molecules, remain trapped in intracellular membrane vesicles (96,286,361,395). Ca 2ϩ added at this time triggers polarization and junction formation in a few hours, a maneuver called "Ca 2ϩ switch" (184).…”

“…The assembly and sealing of TJs occurs so quickly that a fraction of the Na ϩ -K ϩ -ATPase is trapped on the apical side but is thereafter removed from this location (96). The polarized insertion of Na ϩ -K ϩ -ATPase triggered by Ca 2ϩ can be blocked with inhibitors of protein synthesis, yet the proteins whose synthesis is required are neither the ␣-nor the ␤-subunits of Na…”

“…1). Yet in model systems of MDCK, either as monolayer (61,62,96,98,102) or as cysts (465), Na ϩ -K ϩ -ATPase is not expressed on the basal side, but restricted to the lateral plasma membrane facing the intercellular spaces. For the macroscopic parameters mentioned above, it is irrelevant whether the pump is located at the basal or at the lateral side, but as we shall discuss below, the expression on the lateral side may be crucial for the polarized distribution of the pump, which is the basis of the whole asymmetric behavior of epithelial cells.…”

“…Clear-cut demonstrations that TJs are not responsible for the polarized distribution of membrane proteins stem from the study of receptors for immunoglobins, ferritin, and cholesterol-binding-protein and from the observations already mentioned above that upon switching confluent MDCK cells to Ca 2ϩ , TJs form so rapidly that a fraction of Na ϩ -K ϩ -ATPase is trapped in the apical (wrong) side, but is thereafter removed and the cell attains the normal polarization of this enzyme in the next couple of hours (96). However, as discussed below, TJs may be in fact responsible for the polarized transport of fluid.…”