title: Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR(-)) rats.
abstract: Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km=3.2±0.8μM (SD), 9.0±3.1μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km=192.1±291.5μM (SD), 69.2±36.2μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h)=2.0±0.5) and vesicles (h=1.6±0.2) expressing Mrp2 and from SD (h=1.6±0.4) and Wistar (h=4.0±0.6) hepatocytes suggests transport involves more than one binding site. In TR(-) hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km=100.7±36.0μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.
authors.affiliation: Section of Translational Medicine, Division of Applied Medicine, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK. Electronic address: lzellis@dundee.ac.uk.
authors.forename: L C J
authors.initials: LC
authors.lastname: Ellis
authors.affiliation: Department of Biomedical Engineering, Bioengineering Unit, University of Strathclyde, Glasgow G4 0NW, UK.
authors.forename: M H
authors.initials: MH
authors.lastname: Grant
authors.affiliation: Section of Translational Medicine, Division of Applied Medicine, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK.
authors.forename: G M
authors.initials: GM
authors.lastname: Hawksworth
authors.affiliation: Biologie Servier, Drug Safety Research Centre, 905 Route de Saran, 45520 Gidy, France.
authors.forename: R J
authors.initials: RJ
authors.lastname: Weaver
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D005452:Fluoresceins / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D018345:Mice, Knockout; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
chemical_list: D018528:ATP-Binding Cassette Transporters; D005452:Fluoresceins; C053897:5(6)-carboxy-2',7'-dichlorofluorescein
keywords: Canalicular efflux; Drug transporters; Membranes and vesicles expressing rat Mrp2; Primary rat hepatocytes; Sinusoidal efflux