Glutathione-conjugate transport by human colon adenocarcinoma cells (Caco-2 cells).
The secretion of a glutathione-S-conjugate, dinitrophenyl-glutathione (GS-DNP) was studied in the Caco-2 cells, a cultured human colonic adenocarcinoma cell line with many of the characteristics of enterocytes. The labelled glutathione conjugate was generated within the cell by incubation with 14C-labelled 1-chloro-2,4-dinitrobenzene (CDNB). This compound is hydrophobic and enters the cell by simple diffusion. Cells incubated with CDNB at 10 degrees C form only one metabolite, GS-DNP. After secretion into the medium GS-DNP is partly converted into one or two slightly more hydrophobic products. This must represent hydrolysis of the glutathione moiety by the action of gamma-glutamyltransferase (EC 2.3.2.2.; gamma-GT) because the reaction was completely inhibited by acivicin, an inhibitor of gamma-GT. Secretion of GS-DNP was a temperature-sensitive, saturable process with an apparent Km of 1.03 +/- 0.26 nmol/mg of protein and a Vmax of 111 +/- 17 pmol/min per mg of protein. The secretion was not sensitive to trans-stimulation by extracellular concentrations of GS-DNP up to 2.5 mM. Furthermore the initial GS-DNP secretion rate was sensitive to dissipation of the membrane potential and correlated closely with the cellular ATP content. Caco-2 cells cultured on nitrocellulose filters secreted GS-DNP significantly faster over the basolateral membrane than over the apical membrane (146 +/- 25 versus 90 +/- 18 pmol/min per mg respectively). Secretion over both membrane domains of the cell was sensitive to ATP depletion. In conclusion, Caco-2 cells contain an active-transport system that is primarily involved in the secretion of glutathione conjugates and that is present in both plasma membrane domains of the cell.
Oude Elferink
R P
RP
Department of Gastrointestinal Diseases, Academic Medical Center, Amsterdam, The Netherlands.
Bakker
C T
CT
Jansen
P L
PL
D000230:Adenocarcinoma / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000378:metabolism*; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004140:Dinitrophenols / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004137:Dinitrochlorobenzene; D004140:Dinitrophenols; D007555:Isoxazoles; D000255:Adenosine Triphosphate; D005723:gamma-Glutamyltransferase; D005982:Glutathione Transferase; D005978:Glutathione; C006303:acivicin