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 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. 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-alpha from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, 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-alpha 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-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha 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-alpha 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.
Clarke
H
H
Lankenau Medical Research Institute, 100 Lancaster Avenue, Wynnewood, Pennsylvania 19096, USA. Clarkeh@mlhs.org
Ginanni
N
N
Laughlin
K V
KV
Smith
J B
JB
Pettit
G R
GR
Mullin
J M
JM
D000818:Animals; D001692:Biological Transport; D054713:Bryostatins; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology; D018942:Macrolides; D008353:Mannitol / Q000493:pharmacokinetics; D008564:Membrane Potentials / Q000187:drug effects; D009097:Multienzyme Complexes / Q000378:metabolism; D011092:Polyethylene Glycols / Q000493:pharmacokinetics; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000502:physiology*
D054713:Bryostatins; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D007783:Lactones; D007976:Leupeptins; D018942:Macrolides; D009097:Multienzyme Complexes; C081021:Go 6976; C046785:bryostatin 1; D008353:Mannitol; D011092:Polyethylene Glycols; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde