CYCLIC-AMP MODULATES THE RATE OF CONSTITUTIVE EXOCYTOSIS OF APICAL MEMBRANE-PROTEINS IN MADIN-DARBY CANINE KIDNEY-CELLS

Madin-Darby canine kidney and other epithelial cell lines (e.g. Caco-2 , MCF-10A and MCF-7) develop intracellular vacuoles composed of apical membrane displaying microvilli (VACs) when impaired from forming norm al cell-to-cell contacts. In a previous publication, we showed that VA Cs are rapidly exocytosed upon treatment with 8-Br-3',5'-cyclic adenos ine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, an d that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present w ork, we tested the hypothesis that cAMP may be a positive modulator of the 'constitutive' exocytic pathway. To mimic conditions in cells wit h incomplete intercellular contacts, the intracellular levels of cAMP were decreased by means of two independent approaches: (i) pores were induced in the plasma membrane with the polypeptidic antibiotic subtil in, thus allowing small molecules (including cAMP) to permeate and mov e out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellul ar levels of cAMP were measured and, in porated cells, equilibrated to simulate the corresponding physiological intracellular concentrations . The decrease in cAMP within the physiological range resulted in a de creased rate of transport of an apical marker of the constitutive path way (influenza virus hemagglutinin) from the trans-Golgi network to th e apical plasma membrane. Likewise, the delivery of a number of cellul ar apical proteins to the plasma membrane was retarded at low cAMP con centrations. The inhibitors of adenylate cyclase failed to block basol ateral delivery of vesicular stomatitis virus G protein. This differen tial modulatory effect may represent a differentiation-dependent contr ol of the insertion of apical membrane in epithelial cells.