Transport of methotrexate in L1210 cells. Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide.
Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 microM CMPS or 175 microM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4 degrees C) after exposure to these inhibitors (at 37 degrees C). Methotrexate efflux was also inhibition by CMPS (50% at 4 microM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 microM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005033:Ethylmaleimide / Q000494:pharmacology*; D007939:Leukemia L1210 / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice; D010663:Phenylmercury Compounds / Q000494:pharmacology*
D002352:Carrier Proteins; D010663:Phenylmercury Compounds; D002731:4-Chloromercuribenzenesulfonate; D005033:Ethylmaleimide; D004229:Dithiothreitol; D008727:Methotrexate