Multidrug resistance protein 1-mediated transport of saquinavir by microglia Shannon Dallas, Patrick T. Ronaldson, Moise Bendayan 1 and Reina Bendayan CA Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto,19 Russell Street, Toronto, Ontario M5S 2S2; 1 Department of Pathology and Cell Biology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada CA Corresponding Author: r.bendayan@utoronto.ca Received 5 February 2004; accepted 15 March 2004 DOI: 10.1097/01.wnr.0000126754.02091.c3 Regulation of CNS distribution of the human immunode¢ciency virus (HIV) protease inhibitor saquinavir may involve ATP-depen- dent membrane-bound e¥ux transport proteins that are ex- pressed in several brain cellular compartments. We recently characterized molecular and functional expression of one such transporter, multidrug resistance protein-1 (MRP1) in microglia, the primary brain cellular target of HIV. In the present study, we further examine subcellular localization of MRP1 in a microglia cell line (MLS-9) using immunogold cytochemistry and directly demon- strate MRP1-mediated export of saquinavir. MRP1 localized primar- ily to the plasma membrane of the MLS-9 cells. [ 14 C]Saquinavir e¥ux by MLS-9 monolayers was inhibited by well-established MRP1 inhibitors. These results indicate that MRP1 contributes, in part, to the overall low permeation of protease inhibitors in the brain. NeuroReport 15:1183^1186  c 2004 Lippincott Williams & Wilkins. Key words: Brain transport; Human immunode¢ciency virus; Microglia; Multidrug resistance protein 1; Saquinavir INTRODUCTION Human immunodeficiency virus (HIV)-associated dementia (HAD) is a debilitating clinical syndrome that affects B10% of HIV-infected adults [1]. Despite the availability of several antiretroviral medications, HAD continues to be refractory to standard pharmacotherapy. Saquinavir, the first protease inhibitor (PI) approved for use in the treatment of HIV infection, poorly penetrates into the brain [2]. One mechan- ism for this low permeability may be the presence of membrane-bound ATP-dependent efflux transporters at the blood–brain barrier, and within cellular brain targets of HIV, including microglia. Results from numerous in vitro, in vivo, and clinical studies, indicate that saquinavir interacts with several members of the ATP-binding cassette (ABC) super- family of efflux transporters, including P-glycoprotein (P- gp), multidrug resistance protein 1 (MRP1) and MRP2 [3–6]. In MRP1-overexpressing T-lymphoblastoid CEM cells, saquinavir reduced doxorubicin cytotoxicity by 10-fold [3]. Accumulation of the MRP1 substrate Calcein-AM was significantly increased by saquinavir in these same cells [3]. Using confocal microscopy, Miller and colleagues observed decreased accumulation of the fluorescent MRP1 probe sulforhodamine 101 by isolated brain capillaries, in the presence of 10–25 mM saquinavir [4]. While, these studies provide good evidence that saquinavir acts as an inhibitor of MRP1-mediated transport, they do not necessarily demon- strate that saquinavir itself is a MRP1 substrate [2]. Studies examining directly the transport properties of saquinavir by MRP1 are few [2,7], probably due to the unavailability of radiolabeled saquinavir from a commercial source. In addition, there are no reports characterizing the efflux properties of saquinavir by MRP1 in cellular targets of HIV in the brain. We have recently characterized the molecular and functional expression of MRP1 in a rat brain microglia cell line [8]. In the present study, we further investigate the cellular/subcellular location of rMRP1 in cultured microglia using immunogold cytochemistry and provide further direct evidence that MRP1 mediates saquinavir export in microglia. MATERIALS AND METHODS Chemicals: [ 14 C]Saquinavir (0.32 Ci/mmol) was a gener- ous gift from Roche Products Ltd. (Hertfordshire, UK). Genistein, sulfinpyrazone, and anti-rat IgG-colloidal gold (10 nm) were purchased from Sigma-Aldrich (Oakville, ON, Canada). MK571 was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The MRP1 mono- clonal antibody, MRPr1, was purchased from Kamiya Biomedical (Seattle, WA). Cell culture: Isolation of the microglia cell line (MLS-9) has been described in detail previously [8,9]. The MLS-9 cells show phenotypical attributes characteristic of microglia including pinocytosis of dyes (DiI-acetylated LDL, or Lucifer Yellow) and staining with microglia cell markers [9]. Monolayers of MLS-9 cells were cultured on 75 cm 2 polystyrene tissue culture flasks (Sarstedt, St.-Leonard, QC, Canada) in minimum essential medium (pH 7.2) containing 2 mM L-glutamine, 5.55 mM D-glucose, 5% (v/v) fetal bovine serum, 5% (v/v) horse serum and 0.5% (v/v) penicillin/streptomycin at 37 o C, in 5% CO 2 /95% air and 95% humidity. Subconfluent MLS-9 cells (B75% confluent) were subcultured using a 15 mM sodium citrate solution, NEUROPHARMACOLOGYAND NEUROTOXICOLOGY NEUROREPORT 0959-4965  c Lippincott Williams & Wilkins Vol15 No 7 19 May 2004 1183 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.