Abstract
Cumulative evidence suggests that several organic anions are actively effluxed from the brain to the blood across the blood-brain barrier (BBB). We examined the possibility of the presence of primary active transporters for organic anions (multidrug resistance associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT)) on the BBB by measuring the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C4(LTC4) into membrane vesicles prepared from a cell line derived from mouse brain capillary endothelial cells (MBEC4). The ATP-dependent uptake of DNP-SG into the membrane vesicles was osmotically sensitive and was also supported by GTP, but not by AMP or ADP. An ATPase inhibitor, vanadate, blocked the ATP-dependent uptake of DNP-SG. The ATP-dependent uptake process was saturable, withKm values of 0.56 and 0.22 μM, and Vmax values of 5.5 and 27.5 pmol/min/mg protein for DNP-SG and LTC4, respectively. Northern and Western blot analyses showed the expression of murine MRP but not cMOAT in MBEC4 cells. Western blot analysis of the rat cerebral endothelial cells indicated the expression of protein(s) that is detectable with MRPr1, an antibody against MRP. These results, together with previous findings that both DNP-SG and LTC4 are good ligands for MRP, suggest that MRP is responsible for the unidirectional, energy-dependent efflux of organic anions from the brain into the circulating blood across the BBB.
Footnotes
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Send reprint requests to: Dr. Yuichi Sugiyama, Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
- Abbreviations:
- BBB
- blood-brain barrier
- CNS
- central nervous system
- MRP
- multidrug resistance associated protein
- P-gp
- P-glycoprotein
- DNP-SG
- 2,4-dinitrophenyl-S-glutathione
- LTC4
- leukotriene C4
- hMRP
- human multidrug resistance associated protein
- cMOAT
- canalicular multispecific organic anion transporter
- ATP
- adenosine 5′-triphosphate
- AMP
- adenosine 5′-monophosphate
- GTP
- guanosine 5′-triphosphate
- SDS
- sodium dodecyl sulfate
- Received October 13, 1997.
- Accepted February 24, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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