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.