TRANSEPITHELIAL PARACELLULAR LEAKINESS INDUCED BY CHRONIC PHORBOL ESTER EXPOSURE CORRELATES WITH POLYP-LIKE FOCI AND REDISTRIBUTION OF PROTEIN-KINASE-C-ALPHA

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial el ectrical resistance (TER), continuation of exposure for 3 to 4 days re sults in a tachyphylactic response as TER begins to return to control levels, Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level, A reciprocal change in the transepithelial f lux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp -like foci (PLFs) across the otherwise one cell layer thick cell sheet s, The pattern of penetration of the electron dense dye, ruthenium red , from the apical surface, across the tight junction and into the late ral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA, However, wh en exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness, The decrease in TER following acute T PA exposure correlates with the translocation of protein kinase C-alph a (PKC alpha) into a membrane-associated compartment, with exposure of several days, only a trace of PKC alpha is visible by Western immunob lot, and this is in the membrane-associated compartment. Immunofluores cent microscopy indicates that the trace of PKC alpha seen in the West ern immunoblots is ascribable distinctly to cells of the PLFs, Monolay er areas between PLFs show no discernible immunofluorescent signal, Th e data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PL Fs. Possible implications of this model for in vivo epithelial tumor p romotion are discussed.