Effect of P-glycoprotein modulators on the human extraneuronal monoamine transporter

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Abstract

The aim of this work was to investigate the effect of P-glycoprotein modulators on human extraneuronal monoamine transporter (EMT)-mediated transport. The experiments were performed using a cell line from human embryonic kidney (HEK293 cells) stably transfected with pcDNA3hEMT (293hEMT), or with pcDNA3 alone (293control). Of the P-glycoprotein modulators tested, rhodamine123, verapamil and daunomycin concentration-dependently inhibited EMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+). The corresponding IC50's were found to be 3.6, 37 and 130 μM, respectively. By contrast, vinblastine, digitoxin and cyclosporine A were devoid of effect. The endogenous organic cation tyramine, but not choline, inhibited EMT-mediated transport (IC50 of 468 μM). Moreover, l-arginine and l-histidine (up to 1 mM) did not affect [3H]MPP+ uptake. Finally, MPP+ and tyramine trans-stimulated [3H]MPP+ uptake, but rhodamine123 had no effect, and verapamil and daunomycin trans-inhibited [3H]MPP+ uptake. In conclusion, this study shows that several cationic modulators of P-glycoprotein inhibit EMT-mediated transport. As a consequence, the interaction of P-glycoprotein modulators with EMT must be taken into account, and the consequences of this interaction must not be forgotten when using such drugs in vivo.

Introduction

The physiological actions of released catecholamines are terminated by transmembrane transport proteins. These transporters actively remove catecholamines from the extracellular space, thus decreasing their concentration at the receptor level. Several transport systems for these neurotransmitters have been described. The first to be recognised were uptake1, a Na+-driven, high-affinity, cocaine-sensitive neuronal transporter, and uptake2, a Na+-independent, low-affinity, high-capacity, corticosterone-sensitive extraneuronal transporter. Recently, other Na+-independent transmembrane transporters were also found to accept catecholamines as substrates. These include the organic cation transporter type 1 (OCT1; Breidert et al., 1998) and the organic cation transporter type 2 (OCT2; Gründemann et al., 1998a).

Uptake2 was first described more than 30 years ago (Iversen, 1965), but its primary structure was only recently elucidated Gründemann et al., 1998b, Kekuda et al., 1998. After the molecular cloning of uptake2, this transporter (now renamed extraneuronal monoamine transporter, EMT) was found to be a member of the amphiphilic solute facilitator (ASF) family. The ASF family of transporters constitutes a recently described protein family that includes transmembrane transporters of organic anions (OAT1) and organic cations (e.g. OCT1, OCT2) Gründemann et al., 1998b, Schömig et al., 1998.

EMT has a broad tissue distribution. It is found in sympathetically innervated tissues (e.g. myocardium, vascular and non-vascular smooth muscle and glandular cells; Trendelenburg, 1988), in central nervous system glia Staudt et al., 1993, Russ et al., 1996, Streich et al., 1996 and in the retina (Rajan et al., 2000). This transporter strongly contributes to the inactivation of circulating catecholamines Eisenhofer et al., 1996, Friedgen et al., 1996. EMT transports a wide array of organic compounds, all of which are organic cations (review by Trendelenburg, 1988). After analysis of the substrate specificity of EMT, this carrier was classified as a transporter for monoamine transmitters. Known endogenous substrates for EMT include catecholamines such as adrenaline and noradrenaline, histamine and serotonin Trendelenburg, 1988, Gründemann et al., 1998a, Gründemann et al., 1999.

P-glycoprotein, also known as the multidrug transporter, belongs to a large family of membrane transport proteins known as the ATP-binding cassete (ABC) superfamily of transporters. Considerable evidence has accumulated indicating that P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. P-glycoprotein confers resistance against a wide spectrum of compounds, but most of the compounds that are substrates for P-glycoprotein are hydrophobic organic cations of low molecular weight (reviews by Bellamy, 1996, Ambudkar et al., 1999).

The aim of this study was to investigate the possible effect of agents that can modulate P-glycoprotein-mediated transport on EMT-mediated transport. Two lines of evidence led us to this investigation. First, EMT is a transporter of organic cations, and substrates for P-glycoprotein are mostly hydrophobic organic cations (see above). Second, previous experiments from our group showed that some agents that can modulate P-glycoprotein-mediated transport interfere with the inward membrane transport of catecholamines and of the prototypical organic cation 1-methyl-4-phenylpyridinium (MPP+) in different cell types (rat hepatocytes, human intestinal epithelial (Caco-2) cells and rat brain microvessel endothelial (RBE4) cells) Martel et al., 1996, Martel et al., 1998a, Martel et al., 1998b, Martel et al., 1999, Martel et al., 2000, Martel et al., 2001a.

Section snippets

Cell culture

HEK 293 cells (ATCC CRL-1573, Rockville, MD, USA) stem from human embryonic kidney cells which have been transformed by human adenovirus type 5 (Graham et al., 1977). Cells stably transfected with pcDNA3hEMT (293hEMT) or with pcDNA3 only (293control) were a kind gift from E. Schömig. Cells were maintained in a humidified atmosphere of 5% CO2–95% air and were grown in Dulbecco's Modified Eagle's Medium (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum,

Time course of [3H]MPP+ uptake in 293hEMT and 293control cells

In previous experiments using the same cell lines, we verified that 293hEMT cells efficiently remove [3H]MPP+ from the medium, and that uptake of [3H]MPP+ by 293control cells is almost negligible (Martel et al., 2001b). Moreover, [3H]MPP+ uptake by 293hEMT cells was linear with time for the first 1–2 min of incubation. So, in this work, 293hEMT cells were incubated with [3H]MPP+ for 1 min, in order to measure initial rates of uptake.

Effect of agents that can modulate P-glycoprotein-mediated transport

The effect of several agents that can modulate

Discussion

The aim of this study was to investigate the possible effect of agents that can modulate P-glycoprotein-mediated transport on hEMT-mediated transport. Moreover, the effect of some endogenous organic cations and of cationic aminoacids was also studied. The experiments were performed using HEK293 cells stably transfected with hEMT, an hEMT-mediated transport model previously characterised Gründemann et al., 1998b, Gründemann et al., 1999, Martel et al., 2001b. Radiolabeled MPP+ was chosen as

Acknowledgements

The technical assistance of Ms. Luisa Vasques and Ms. Manuela Moura is gratefully acknowledged. The authors would like to thank Dr. Dirk Gründemann and Prof. Edgar Schömig (from the Department of Pharmacology, University of Heidelberg, Germany) for the generous supply of transfected HEK293 cells.

This work was supported by FCT and Programa Ciência, Tecnologia e Inovação do Quadro Comunitário de Apoio.

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