PHYSIOLOGY OF EPITHELIAL MONOLAYERS GROWN IN VITRO

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When MDCK cells (an epithelioid line derived from the kidney of a normal dog) is obtained by trypsination of previous monolayers and plated at high densities on a permeable and transluscent support, they develop an uninterrupted monolayer which has many of the structural and functional characteristics of transporting epithelia. The main two features of these cells are: 1) they contact each other and form occluding junctions which confer to the monolayers the ability to act as effective permeability barriers; and 2) the apical and the basolateral aspects are structural and funcionally different, indicating that, as in natural transporting epithelia, cells are polarized. These characteristics are exhibited 15 hours after plating. Our overall aim is to explore how these characteristics are installed and, the specific aim of this particular project is: 1) To study the establishment of occluding junctions in newly plated cells, and compare it with the establishment in confluent monolayers that have been kept without Ca for 20 h, situation where we know that junctions have never been sealed; 2) To study with immunofluorescent antibodies against ATPase whether polarization into apical/basolateral elements depends on the formation of occluding junctions; 3) To characterize the ion translocating mechanisms by using microelectrodes and measuring tracer fluxes. This will be done in steady state monolayers (plated for more than 20 h.), and in those just plated, in which trypsin might have cleaved the proteins involved, and give us a chance to follow how are the mechanisms resinthesized and reinstalled; 4) We have a mutant of MDCK cell which is resistant to high dosis of ouabain. We want to study ion translocating mechanisms, 3H-ouabain binding, etc. to learn why they resist. 5) When the common, wild MDCK cell, is co-cultured in mixed monolayer with the mutant, it is protected from ouabain. We plan to elucidate the mechanisms of this protection.