The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-alpha

The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and br yostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryos tatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promot er. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial ele ctrical resistance (R-t) across LLC-PK1 epithelial cell sheets, while 10(-7 ) M bryostatin 1 caused only a 30% decrease in R-t, which spontaneously rev ersed after 5 h, Simultaneous exposure of cell sheets to 10-7 M TPA and 10- 7 M bryostatin 1 blunted the increase in epithelial permeability observed w ith 10(-7) M TPA alone, Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R-t at the 6- h time point and inhibited the recovery in R-t seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-a from the cyt osol to the membrane of the cell where it remained elevated. Bryostatin 1 t reatment resulted in a slower translocation of PKC-alpha from the cytosol t o the membrane and a much more rapid downregulation of PKC-alpha, with disa ppearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R-t seen with TPA, Treatment of cells wit h TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregula tion profile which more closely resembled that seen with bryostatin 1 alone . Go-incubation of cells with MG-132 and bryostatin 1 caused a slower downr egulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment o f cells expressing a dominant/negative form of PKC-alpha resulted in a slow er and less extensive decrease in R-t compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membra ne-associated fraction of the treated cells correlated closely with increas ed transepithelial permeability. Due to its transient effect on tight junct ion permeability, bryostatin 1 offers a novel pharmacological tool to inves tigate junctional physiology. (C) 2000 Academic Press.