Elsevier

Biochemical Pharmacology

Volume 70, Issue 4, 15 August 2005, Pages 500-510
Biochemical Pharmacology

Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4) that are present in human red cell membranes

https://doi.org/10.1016/j.bcp.2005.05.022Get rights and content

Abstract

Human erythrocyte membranes express the multidrug resistance-associated proteins, MRP1, MRP4 and 5, that collectively can efflux oxidised glutathione, glutathione conjugates and cyclic nucleotides. It is already known that the quinoline derivative, MK-571, is a potent inhibitor of MRP-mediated transport. We here examine whether the quinoline-based antimalarial drugs, amodiaquine, chloroquine, mefloquine, primaquine, quinidine and quinine, also interact with erythrocyte MRPs with consequences for their access to the intracellular parasites or for efflux of oxidised glutathione from infected cells. Using inside-out vesicles prepared from human erythrocytes we have shown that mefloquine and MK-571 inhibit transport of 3 μM [3H]DNP-SG known to be mediated by MRP1 (IC50 127 and 1.1 μM, respectively) and of 3.3 μM [3H]cGMP thought but not proven to be mediated primarily by MRP4 (IC50 21 and 0.41 μM). They also inhibited transport in membrane vesicles prepared from tumour cells expressing MRP1 or MRP4 and blocked calcein efflux from MRP1-overexpressing cells and BCECF efflux from MRP4-overexpressing cells. Both stimulated ATPase activity in membranes prepared from MRP1 and MRP4-overexpressing cells and inhibited activity stimulated by quercetin or PGE1, respectively. Neither inhibited [α-32P]8-azidoATP binding confirming that the interactions are not at the ATP binding site. These results demonstrate that mefloquine and MK-571 both inhibit transport of other substrates and stimulate ATPase activity and thus may themselves be substrates for transport. But at concentrations achieved clinically mefloquine is unlikely to affect the MRP1-mediated transport of GSSG across the erythrocyte membrane.

Introduction

Multidrug resistant-associated proteins (MRPs) mediate ATP-dependent transport of a wide range of compounds including glutathione conjugates and cyclic nucleotides. MRP1 (ABCC1) is a primary active transporter of organic anions including the oxidised form of glutathione, GSSG [1]. It is expressed in human erythrocytes where it accounts for the high affinity component of the transport of GSSG and dinitrophenyl-S-glutathione, DNP-SG [2], [3], [4]. In addition to MRP1 there are in red blood cells other efflux transporters, one or more of which account for the low affinity efflux of GSSG, DNP-SG and cGMP. The identities of these transporters have not been conclusively identified though MRP4 is thought to play a major role [4]. One of the most potent inhibitors of MRP-mediated transport is the LTD4 receptor antagonist MK-571, a quinoline derivative. Another quinoline-based compound, IAAQ, photolabels MRP1 and both chloroquine and MK-571 inhibit this labelling [5]. Thus it is probable that at least some quinolines in clinical use interact with MRPs. Quinoline-based antimalarials, especially chloroquine, have been used for decades to kill malaria parasites in the intraerythrocytic stage of their life cycle, If indeed these quinolines do interact with active transporters on erythrocytes, the possibility exists that they could affect access of drug to the parasite inside the cell. Interaction of mefloquine with P-glycoprotein, a mammalian ATP-binding cassette transporter has been documented [6], [7] but, while it has been sought, P-glycoprotein has not been detected in human erythrocyte membranes by immunoblotting [8] (Wu and Woodcock personal communication). Little or no information is available regarding the interaction of mefloquine with other ABC transporters such as the MRPs that are present in red blood cells.

The aim of the present study was to explore how some widely used quinoline-based antimalarial drugs affect activities of some MRPs known to be present in human erythrocytes. Using inside-out vesicles prepared from human red blood cells, we show that MK-571 and mefloquine directly inhibit MRP-mediated uptake of DNP-SG or cGMP. Confirmation of these interactions was obtained using inside-out vesicles prepared from MRP1-overexpressing tumour cells or transfected HEK 293 cells overexpressing MRP4. Further evidence of their interactions was obtained by observing their effects on accumulation and efflux of MRP substrates at the whole cell level. The effects of quinolines on ATPase activities and ATP binding to MRP1 and MRP4 protein provide further insight into the nature of the quinoline-MRP interactions and suggest that MK-571 and mefloquine appear to be substrates for transport.

Section snippets

Cell culture

Cells of the human large-cell lung tumour line L23/P and of its doxorubicin-selected MRP1-overexpressing multidrug-resistant variant L23/R [9], [10] were cultured as previously described [4]. Parental 293 human embryonic kidney cells (HEK 293 cells) and the MRP4-overexpressing HEK 293/4.63 cells [11] were generous gifts of Prof P. Borst (Division of Molecular Biology and Center for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands). HEK 293/4.63 cells were

Effects of quinoline derivatives on high affinity DNP-SG transport in human erythrocyte vesicles

ATP-dependent uptake of DNP-SG was measured over the course of 30 min in the absence or presence of quinolines. It has previously been shown [31] that DNP-SG uptake into human erythrocyte vesicles is linear for at least 60 min. The rate of uptake was significantly inhibited by MK-571 and by mefloquine with IC50 values of 1.1 ± 0.2 μM (n = 7, maximum percentage uptake 94 ± 4%) and 127 ± 17 μM (n = 4, maximum percentage uptake 98 ± 3%), respectively (see Fig. 1A). Other quinoline antimalarial drugs (amodiaquine,

Discussion

The purpose of this study was to investigate the interactions of mefloquine and other quinolines used in the treatment of malaria with MRPs present in red blood cell membranes. Of these, mefloquine was the most effective. It blocked the higher affinity DNP-SG transport observed in red blood cells and the MRP1-mediated DNP-SG transport in lung tumour cells as well as the principal cGMP transport in red blood cells and the cGMP transport measured in MRP4-transfected HEK cells with IC50 values of

Acknowledgements

We thank Dr. Gary Kruh (Fox chase Cancer Centre) for the gifts of plasmid PVL1393-MRP4 and anti-MRP4 and Dr. Piet Borst (The Netherlands Cancer Institute) for HEK 294/4.63 cells. CPW is supported by the Cambridge Commonwealth Trust Fund and by a visiting preCRTA award from the NCI, NIH.

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