Abstract
Brain expression of the multidrug resistance proteins (MRPs), a collection of membrane-associated ATP-dependent efflux transporters, is poorly understood. Although several studies have examined the expression of these proteins within the brain barriers (i.e., the blood-brain barrier and choroid plexus), little information is available with respect to brain parenchyma cells such as microglia and astrocytes. Because microglia are the primary brain cells infected by the human immunodeficiency virus type 1 (HIV-1), MRP1 expression within microglia may contribute to lower brain accumulation of anti-HIV drugs. To examine the expression pattern of MRP1 within microglia, we performed reverse transcriptase-polymerase chain reaction analysis and Western blotting on a rat brain microglia cell line MLS-9, and in primary cultures of rat microglia. Both rat MRP1 (rMPR1) mRNA and protein were expressed in the cell line, as well as the primary cultures. We then characterized rMRP1-mediated transport properties in MLS-9 cells using [3H]vincristine, a known MRP1 substrate. Vincristine accumulation by monolayers of MLS-9 cells increased significantly in the presence of several well established MRP1 inhibitors (MK571, genistein, sulfinpyrazone, probenecid, and indomethacin), protease inhibitors, or the ATPase inhibitor sodium azide. In addition, vincristine accumulation was significantly modulated by altering the intracellular concentration of the reduced form of glutathione, further suggesting the involvement of rMRP1-mediated transport. These results provide strong evidence that the MRP1 protein is both expressed and functional in microglia cells. They also suggest that brain parenchyma can act as a “second” barrier to drug permeability and regulate brain distribution/accumulation of various xenobiotics, including protease inhibitors.
Footnotes
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This work is supported by grants to R.B. from the Canadian Institutes for Health Research (HOP-56976) and the Ontario HIV Treatment Network, and to L.C.S. by Canadian Institutes for Health Research (MT-13657). S.D. has received a graduate studentship from the Ontario HIV Treatment Network. This work was presented in a preliminary format at The American Association of Pharmaceutical Scientists Annual Meeting, Denver, CO, October 2001, and the 10th Annual Canadian Association for HIV Research Conference, Toronto, ON, June 2001.
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ABBREVIATIONS. P-gp, P-glycoprotein; MRP, multidrug resistance protein; hMRP1, human multidrug resistant protein 1; rMRP1, rat multidrug resistance protein 1; HIV-1, human immunodeficiency virus type 1; BSO, l-buthionine-[S,R]-sulfoximine; RT-PCR, reverse transcriptase-polymerase chain reaction; TBS-T, Tris-buffered saline/Tween 20; EBSS, Earle's balanced salt solution; GSH, glutathione.
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DOI: 10.1124/jpet.103.054304.
- Received May 13, 2003.
- Accepted June 11, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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