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

The role of PKC-α in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10^-7 M induced a sustained 95% decrease in transepithelial electrical 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 reversed 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 with 10^-7 M TPA alone. Coincubation 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-α from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-α from the cytosol to the membrane and a much more rapid downregulation of PKC-α, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R(t) seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-α translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-α from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-α resulted in a slower and less extensive decrease in R(t) compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-α in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology. (C) 2000 Academic Press.