UNIDIRECTIONAL FLUXES OF RHODAMINE-123 IN MULTIDRUG-RESISTANT CELLS -EVIDENCE AGAINST DIRECT DRUG EXTRUSION FROM THE PLASMA-MEMBRANE

P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multi drug-resistant tumor cells, is an ATPase thought to actively export cy totoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium (''vacuum cleaner'' hyp othesis). A possible mechanism for this model is that the Pgp is a fli ppase-i.e., it catalyzes the translocation of hydrophobic substrates f rom the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that th e apparent unidirectional influx of substrate should be less in Pgp-ex pressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblast s with different levels of Pgp expression to measure true unidirection al fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye t hat accumulates in mitochondria (hence, its cytosolic concentration re mains low at short times after external addition). The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors. The unidirectional influx of R123 was the same in sensitive and resistant cells-i.e., independent of the level o f Pgp expression and insensitive to inhibitors of R123 efflux. From th ese results, we rule out the vacuum cleaner and flippase hypotheses an d conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.