title: Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures.
abstract: P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany. khirsche@med.uni-goettingen.de
authors.forename: K I
authors.initials: KI
authors.lastname: Hirsch-Ernst
authors.forename: C
authors.initials: C
authors.lastname: Ziemann
authors.forename: I
authors.initials: I
authors.lastname: Rustenbeck
authors.forename: G F
authors.initials: GF
authors.lastname: Kahl
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005079:Excipients / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism / Q000493:pharmacokinetics*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D017830:Octoxynol / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D020112:Rhodamine 123 / Q000378:metabolism / Q000493:pharmacokinetics*; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D005079:Excipients; D005456:Fluorescent Dyes; D020112:Rhodamine 123; D014638:Vanadates; D016572:Cyclosporine; D017830:Octoxynol; D014700:Verapamil