Multidrug Resistance Protein (MRP) 4- and MRP 5-Mediated Efflux of 9-(2-Phosphonylmethoxyethyl)adenine by Microglia

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Abstract
The pathogenesis of human immunodeficiency virus (HIV)-associated dementia has been linked to microglial responses after infection. We have recently confirmed expression of several ATP-dependent efflux transporters in microglia, namely, multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-gp). In the present study, we investigated whether cultured rat microglia express two additional MRP family members, rMRP4 and rMRP5. Using reverse transcriptase-polymerase chain reaction, rMRP4 and rMRP5 mRNA was detected in primary cultures of microglia and in a rat microglia cell line, MLS-9. Western blot analysis further confirmed protein expression of the two MRP isoforms in MLS-9 cells. Bis(pivaloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine [bis(POM)PMEA], a lipophilic ester prodrug of the well characterized MRP4 and 5 substrate 9-(2-phosphonylmethoxyethyl)adenine (PMEA), was chosen to examine transport characteristics in MLS-9. Using thin layer chromatography, we verified that more than 90% of radioactivity recovered in MLS-9 loaded with 1 μM [3H]bis(POM)PMEA for 1 h under ATP-depleting conditions was converted to PMEA. Efflux of PMEA by MLS-9 cell monolayers was ATP-dependent, glutathione-independent, and significantly inhibited by several MRP inhibitors (i.e., sulfinpyrazone, genistein, indomethacin, and probenecid) as well as the antiretroviral drug azidothymidine-monophosphate. Similar results were not observed in MRP1- or P-gp-overexpressing cell lines, suggesting that PMEA is not a substrate for either P-gp or MRP1. These studies provide further evidence that microglia express multiple subfamilies of ATP-binding cassette transporters (i.e., P-gp, MRP1, MRP4, and MRP5) that could restrict permeation of several different classes of antiretroviral drugs in a brain cellular target of HIV-1 infection.