Functional Expression of Sinusoidal Drug Transporters in Primary Human and Rat Hepatocytes

Abstract

ABSTRACT:Primary hepatocyte cultures are considered as a useful in vitro system for pharmacological/toxicological studies. Although expression of drug-metabolizing enzymes and canalicular drug transporters has been well documented in this cellular model, less information is available about sinusoidal drug transporter activities. This has led us to investigate functional expression of the major sinusoidal transporters in primary human and rat hepatocytes. Membrane transport proteins belonging to the solute carrier (SLC) superfamily of transporters play a major role in the initial sinusoidal influx of drugs to hepatocytes (van Montfoort et al., 2003). Organic cation transporters (OCT1/SLC22A1 and Oct1/Slc22a1) thus mediate the transport of small organic cations such as tetraethylammonium (TEA) (Jonker and Schinkel, 2004). Organic anion-transporting polypeptides (OATP-A/SLCO1A2, OATP-B/SLCO2B1, OATP-C/ SLCO1B1, and OATP8/SLCO1B3 in human hepatocytes and Oatp1/ Slco1a1, Oatp2/Slco1a4 and Oatp4/Slco1b2 in rat hepatocytes) are implicated in sodium-independent uptake of organic anions such as estrone-3-sulfate (ES), biliary acids, the antihistamine fexofenadine, and the cholesterol-lowering pravastatin (Kim, 2003); some of these OATPs also transport amphipathic compounds like digoxin, handled by OATP8 and Oatp4. Organic anion transporters (OAT2/SLC22A7 and Oat2/Slc22a7) mediate the influx of organic anions like paraaminohippurate (PAH) and salicylates (Sekine et al., 1998), whereas the Na ϩ -taurocholic acid-cotransporting polypeptides (NTCP/ SLC10A1 and Ntcp/Slc10a1) are involved in Na ϩ -dependent uptake of biliary acids such as taurocholate (Trauner and Boyer, 2003).Primary hepatocytes represent a major in vitro model for studying the activity and regulation of liver-detoxifying pathways and can therefore contribute to prediction of hepatic elimination of xenobiotics (Modriansky et al., 2000;Gebhardt et al., 2003). Such a liver cell culture system has been extensively characterized with respect to expression and activity of drug-metabolizing enzymes and canalicular transporters Payen et al., 2000;Gomez-Lechon et al., 2003;Annaert and Brouwer, 2005). By contrast, activity of sinusoidal membrane transport proteins has, as yet, been less studied, to our knowledge, in cultured hepatocytes, especially in primary human hepatocytes, although these influx transporters play a crucial role in xenobiotic pharmacokinetic and drug-drug interactions (Chandra and Brouwer, 2004;Shitara et al., 2005). The present study was therefore designed to investigate functional expression of the major sinusoidal transporters in primary hepatocyte cultures. Owing to known interspecies differences in liver-detoxifying pathways, this work was conducted with both human and rat hepatocytes. Materials and Methods Chemicals.[ 3 H(G)]Taurocholic acid (specific activity 1.19 Ci/mmol), [6, Cell Isolation and Culture. Hepatocytes from adult male Sprague-Dawley rats weighing 150 to 200 g were isolated by a perfusion of the liver as previously described ,...

Regulation of Drug Transporter Expression in Human Hepatocytes Exposed to the Proinflammatory Cytokines Tumor Necrosis Factor-α or Interleukin-6

“…Human hepatocytes were obtained from adult donors undergoing hepatic resection for primary and secondary tumors via the Biological Resource Center (Rennes, France). Cells were prepared by perfusion of histologically normal liver fragments using a collagenase solution (Jigorel et al, 2005). They were primary cultured on plastic dishes in Williams' E medium as already reported (Chouteau et al, 2001;Jigorel et al, 2005;Le Vee et al, 2008).…”

“…Cells were prepared by perfusion of histologically normal liver fragments using a collagenase solution (Jigorel et al, 2005). They were primary cultured on plastic dishes in Williams' E medium as already reported (Chouteau et al, 2001;Jigorel et al, 2005;Le Vee et al, 2008). All of the experimental procedures complied with French laws and regulations and were approved by the National Ethics Committee.…”

Characterization of a Rat Multi-Cell Type 3D-Liver Microtissue System

“…Hepatocytes cultured as monolayers rapidly lose the expression and functional activities of key cytochrome P450 enzymes, multi-drug resistance proteins, proteins required for albumin and transferring, and glucocorticoid regeneration within the first 24-48 h [43][44][45]. The prediction of drug-induced hepatotoxicity needs to consider bioactivation as well as detoxification and clearance of reactive metabolites.…”

Differential regulation of drug transporter expression by all-trans retinoic acid in hepatoma HepaRG cells and human hepatocytes

“…Immunofluorescence analyses were performed as previously described substrates, using a defined sodium-and calcium-containing transport assay medium (Jigorel et al, 2005). Incubation times were 3 min (for taurocholate and E3S) or 5 min (for TEA); preliminary experiments indicated that uptakes of substrates were linear over these periods (data not shown).…”