Alkaline phosphatase and gamma-glutamyl transpeptidase as polarization markers during the organization of LLC-PK1 cells into an epithelial membrane.

Confluent monolayers of LLC-PK1 cells, an epithelial cell derived from a normal pig kidney, contain high levels of alkaline phosphatase and gamma-glutamyl transpeptidase activities. Inhibition studies show a close similarity between alkaline phosphatase and the so-called liver-bone-kidney isoenzyme. Nearly complete recovery of both activities in the microsomal fraction demonstrates the membrane-bound characteristics of these enzymes. Histochemical localization of the enzymes activities on the apical membrane of LLC-PK1 cells confirms this observation. The activity of both enzymes decreases to very low levels when the cells are in exponential growth. Confluent monolayers of LLC-PK1 cells plated at saturation density with cells that were in active growth show a progressive increase in the activity of alkaline phosphatase and gamma-glutamyl transpeptidase. This increase is dependent on the synthesis de novo of RNA and protein and supports the conclusion that the activity of both enzymes is regulated at the transcriptional level. The development of these enzymes is delayed with respect to the development of the occluding junctions. This delay and the direct appearance of alkaline phosphatase activity in the apical membrane indicate that the different components of this membrane are inserted after the limits of the membrane have been established by the synthesis and assembly of the occluding junctions. Alkaline phosphatase activity of confluent monolayers can be further induced by reducing the concentration of phosphate in the medium. When the junctions are dissociated by incubating the monolayers in Ca2+-free medium, the alkaline phosphatase activity migrates freely beyond the limits of the apical membrane until it covers the entire cell surface. Both enzymes are synthesized even in the absence of contact between the cells, suggesting that the occluding junctions in LLC-PK1 monolayers are involved in the polarized distribution, but not in the modulation, of the synthesis of these enzymes. In addition, the progressive decrease in enzyme synthesis that is obtained by reducing the cell-substratum adhesion supports the idea that it is the cell-to-substrate and not the cell-to-cell interaction which is involved in the modulation of these enzymatic markers of the apical membrane.