Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: Evidence against direct drug extrusion from the plasma membrane

Guillermo A. Altenberg, Carlos G. Vanoye, Julie K. Horton, L. Reuss*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

109 Scopus citations

Abstract

P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ('vacuum cleaner' hypothesis). A possible mechanism for this model is that the Pgp is a flippase-i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains 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 of Pgp expression and insensitive to inhibitors of R123 efflux. From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.

Original languageEnglish (US)
Pages (from-to)4654-4657
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number11
DOIs
StatePublished - May 24 1994

Keywords

  • P-glycoprotein
  • doxorubicin
  • multidrug resistance
  • vinblastine

ASJC Scopus subject areas

  • General

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