Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures.
We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT-PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P-glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P-glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin-1 polyclonal H-97 antibody demonstrated that P-glycoprotein is physically associated with caveolin-1 in both cell culture systems. The accumulation of [(3)H]digoxin (an established P-glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P-glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P-glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.
Ronaldson
Patrick T
PT
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
Bendayan
Moise
M
Gingras
Diane
D
Piquette-Miller
Micheline
M
Bendayan
Reina
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D051242:Caveolin 1; D022461:Caveolins / Q000096:biosynthesis; D002478:Cells, Cultured; D004077:Digoxin / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D004077:Digoxin