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 normal cell-to-cell contacts. In a previous publication, we showed that VACs are rapidly exocytosed upon treatment with 8-Br-3',5'-cyclic adenosine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, and that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present work, we tested the hypothesis that cAMP may be a positive modulator of the 'constitutive' exocytic pathway. To mimic conditions in cells with 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 subtilin, thus allowing small molecules (including cAMP) to permeate and move out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellular 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 decreased rate of transport of an apical marker of the constitutive pathway (influenza virus hemagglutinin) from the trans-Golgi network to the apical plasma membrane. Likewise, the delivery of a number of cellular apical proteins to the plasma membrane was retarded at low cAMP concentrations. The inhibitors of adenylate cyclase failed to block basolateral delivery of vesicular stomatitis virus G protein. This differential modulatory effect may represent a differentiation-dependent control of the insertion of apical membrane in epithelial cells.

A specifically apical sub-membrane intermediate filament cytoskeleton in non-brush-border epithelial cells.

Although many pieces of evidence support the notion of a role for the cytoskeleton in epithelial polarization, no cytoskeletal component has been found to be specifically apical, except for some actin-binding proteins. Here we report the apical distribution of a 53 kDa cytokeratin. Furthermore, this cytokeratin co-purified with biotinylated apical plasma membrane proteins in high density complexes. Differential biotinylation of the basolateral domain showed that the 53 kDa protein is mainly attached to the apical membrane, although a companion 58 kDa protein attaches to both apical and basolateral membrane proteins. Immunoprecipitation experiments indicated that a number of apical components are directly or indirectly linked to the 53 kDa protein. These results indicate the existence of a terminal web-like structure in non-brush-border cells, which attaches to the apical domain and may play a role in apical polarization, especially during the acquisition of polarity from non-polarized cellular stages.

Exocytosis of vacuolar apical compartment (VAC) in Madin-Darby canine kidney epithelial cells: cAMP is involved as second messenger.

Vacuolar apical compartment (VAC) is a transient organelle originally observed in Madin-Darby canine kidney (MDCK) epithelial cells impaired from forming cell-cell contacts. VACs are large vacuoles which contain microvilli and apical plasma membrane markers (among others, a 184-kDa plasma membrane protein, AP2), but exclude basolateral membrane markers. Upon reestablishment of cell-cell contacts, VACs are rapidly (within 1 h) exocytosed toward intercellular spaces, after which the apical plasma membrane drifts toward its final destination (Vega-Salas, Salas, and Rodriguez-Boulan. 1988. J. Cell Biol. 107, 1717-1728). In this work, we studied the role of cAMP as a mediator for the exocytosis of VACs. We shifted confluent cells from low to normal calcium medium (thus reestablishing cell-cell contacts and causing VAC exocytosis), a shift which resulted in a significant rise of cellular levels of both total intracellular and protein-bound cAMP. The 8-Br analog of cAMP (8-Br-cAMP) (5-50 microM) caused externalization of the intracellular compartment of AP2 as measured by radioimmunoassay. A similar effect was observed with 3-isobutyl-1-methylxanthine. 8-Br-cAMP also caused the appearance of AP2-positive VAC images in nonpermeabilized cells, namely, VACs that become accessible to extracellular antibodies upon fusion with the plasma membrane. Lanthanum, which abolishes the peak of intracellular free calcium during a calcium switch, failed to block the exocytosis. On the other hand, 12-O-tetradecanoylphorbol-13-acetate induced only a modest exocytic response. Finally, 8-Br-cAMP induced VAC exocytosis in sparse MDCK cells grown in normal calcium medium. These data indicate that cAMP is a mediator between the extracellular signal provided by cell-cell contacts and VAC exocytosis.

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.