Multidrug resistance protein (MRP) 4- and MRP 5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine by microglia.
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 microM [(3)H]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.
Dallas
Shannon
S
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, ON M5S 2S2, Canada.
Schlichter
Lyanne
L
Bendayan
Reina
R
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000502:physiology*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology; D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics*; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D006801:Humans; D017628:Microglia / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000502:physiology; D063065:Organophosphonates; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014316:Tritium
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000225:Adenine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D063065:Organophosphonates; D012333:RNA, Messenger; D014316:Tritium; C073492:ABCC4 protein, human; C120809:ABCC5 protein, human; C489542:Abcc4 protein, rat; C492375:Abcc5 protein, rat; C089823:bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine; C053001:adefovir