Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line.
The characteristics of the transport of the dipeptide glycylsarcosine were studied in the human colon carcinoma cell line Caco-2 grown as a monolayer on impermeable plastic support. Transport of glycylsarcosine in these cells was found to be Na(+)-independent, but was stimulated by an inwardly directed H+ gradient. This H(+)-dependent transport of glycylsarcosine was inhibited by di- and tri-peptides and also by the beta-lactam antibiotic cephalexin, but was unaffected by the amino acids glycine and leucine. The transport system exhibited a Michaelis-Menten constant (Kt) of 1.1 +/- 0.1 mM for glycylsarcosine. The specific activity of the transport system in this cell line was found to be maximal when the cultures were confluent. Treatment of the cells with phorbol esters which activate protein kinase C resulted in a significant inhibition of the transport system. This inhibition was specific and could be blocked if treatment was done in the presence of staurosporine, an inhibitor of protein kinase C. Kinetic analysis revealed that the inhibition was associated with a decrease in the maximal velocity, the Kt remaining unaffected. The phorbol-ester-induced inhibition of the peptide-transport system was not prevented by co-treatment with cycloheximide, an inhibitor of cellular protein synthesis. In addition, there was no change in the intracellular pH following treatment with the phorbol ester, suggesting that the effect was not due to alterations in the transmembrane pH gradient. It is concluded that the peptide/H+ co-transport system, which is known to exist in the normal intestine, is expressed in Caco-2 cells and that the function of the transport system is under the regulatory control of protein kinase C.
Brandsch
M
M
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Miyamoto
Y
Y
Ganapathy
V
V
Leibach
F H
FH
D000470:Alkaloids / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division; D003110:Colonic Neoplasms; D004151:Dipeptides / Q000378:metabolism*; D004224:Diterpenes; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D019311:Staurosporine; D013729:Terpenes / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D002352:Carrier Proteins; D004151:Dipeptides; D004224:Diterpenes; D013729:Terpenes; C004194:glycylsarcosine; C011309:mezerein; D006859:Hydrogen; D012964:Sodium; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate