Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C.
Caco-2 cells are an enterocyte-like cell line derived from a human colonic adenocarcinoma. Paracellular permeability was assessed in monolayers of these cells by transmonolayer resistance and by the permeation of [3H]mannitol across the monolayer. Paracellular permeability was increased by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (50 nM), carbachol (500 microM), and the combination of carbachol (50 microM) and monolein (100 microM), an inhibitor of diacylglycerol kinase, as manifested by a decrease in transmonolayer resistance and an increase in mannitol permeation. The effects of all of these stimuli on transmonolayer resistance were inhibited by staurosporine (3 nM), an inhibitor of PKC. The effects of carbachol plus monolein were also inhibited by atropine (0.1 microM), a muscarinic antagonist. Treatment of the monolayers with each of the stimuli was associated with translocation of PKC activity from cytosol to a membrane-associated state. Stimulation of Caco-2 cell monolayers with phorbol myristate acetate or with the combination of carbachol and monolein was also associated with phosphorylation of the MARCKS protein, an endogenous substrate of PKC. These data support the hypothesis that intestinal paracellular permeability is regulated by the activity of enterocyte PKC and demonstrate that the increase in paracellular permeability induced by binding of carbachol to the muscarinic receptor is mediated by activation of PKC.
Stenson
W F
WF
Department of Medicine, Jewish Hospital of St. Louis, Missouri.
Easom
R A
RA
Riehl
T E
TE
Turk
J
J
D000230:Adenocarcinoma; D000470:Alkaloids / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D003110:Colonic Neoplasms; D004789:Enzyme Activation; D005989:Glycerides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D005989:Glycerides; D008353:Mannitol; D002217:Carbachol; C005953:monoolein; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate