Abstract
The treatment of endothelial cell monolayers with phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C (PKC) activator, leads to disruption of endothelial cell monolayer integrity and intercellular gap formation. Selective inhibition of PKC (with bisindolylmaleimide) and extracellular signal-regulated kinases (ERKs; with PD-98059, olomoucine, or ERK antisense oligonucleotides) significantly attenuated PMA-induced reductions in transmonolayer electrical resistance consistent with PKC- and ERK-mediated endothelial cell barrier regulation. An inhibitor of the dual-specificity ERK kinase (MEK), PD-98059, completely abolished PMA-induced ERK activation. PMA also produced significant time-dependent increases in the activity of Raf-1, a Ser/Thr kinase known to activate MEK ( approximately 6-fold increase over basal level). Similarly, PMA increased the activity of Ras, which binds and activates Raf-1 ( approximately 80% increase over basal level). The Ras inhibitor farnesyltransferase inhibitor III (100 microM for 3 h) completely abolished PMA-induced Raf-1 activation. Taken together, these data suggest that the sequential activation of Ras, Raf-1, and MEK are involved in PKC-dependent endothelial cell barrier regulation.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Blotting, Western
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Capillary Permeability* / drug effects
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Cattle
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Electric Impedance
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Endothelium, Vascular / cytology
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Endothelium, Vascular / drug effects
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Endothelium, Vascular / physiopathology*
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Enzyme Inhibitors / pharmacology
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MAP Kinase Kinase 1
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Mitogen-Activated Protein Kinase 1 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 1 / metabolism*
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Mitogen-Activated Protein Kinases / antagonists & inhibitors
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Mitogen-Activated Protein Kinases / metabolism*
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Myosin Light Chains / metabolism
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Phosphorylation / drug effects
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Precipitin Tests
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Protein Kinase C / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins c-raf / metabolism
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Tetradecanoylphorbol Acetate
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Vascular Diseases / chemically induced
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Vascular Diseases / metabolism
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ras Proteins / antagonists & inhibitors
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ras Proteins / metabolism*
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ras Proteins / pharmacology
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rho GTP-Binding Proteins / antagonists & inhibitors
Substances
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Enzyme Inhibitors
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Myosin Light Chains
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-raf
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Protein Kinase C
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 1
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Mitogen-Activated Protein Kinase Kinases
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ras Proteins
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rho GTP-Binding Proteins
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Tetradecanoylphorbol Acetate