P-glycoprotein functions as an ATP-driven active efflux pump for many cytotoxic drugs. We now show that hydrophobic peptides and ionophores also interact with the multidrug transporter. Multidrug-resistant cells are cross-resistant to several hydrophobic peptides and ionophores, but not to some other membrane-active species. Linear peptides, cyclic peptides, and ionophores stimulated the ATPase activity of P-glycoprotein in plasma membrane vesicles by up to 2.5-fold. Drugs and chemosensitizers were able to block P-glycoprotein ATPase stimulation by verapamil, however, peptides and ionophores (with the exception of cyclosporine A) were unable to do so. Peptides and ionophores also effectively inhibited ATP-dependent drug transport by P-glycoprotein in plasma membrane vesicles. The median effect analysis was used to extract quantitative parameters from the drug transport inhibition data. Unlike drug substrates and cyclic peptides, linear peptides did not inhibit photoaffinity labeling of P-glycoprotein by [3H]azidopine. Taken together, these results indicate that certain hydrophobic peptides and ionophores are P-glycoprotein substrates, however, they affect the transporter in a different manner from drugs. Linear peptides interact with P-glycoprotein at a site distinct from those for verapamil and azidopine, whereas the interaction site for cyclic peptides and ionophores appears to be linked to these sites to varying degrees. Export of hydrophobic peptides may be an important physiological function of P-glycoprotein.

P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport.

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