Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes
Graphical abstract
Introduction
Hepatic drug transporters belonging to the solute carrier (SLC) or the ATP-binding cassette (ABC) transporter superfamilies are involved in hepato-biliary secretion of drugs and drug–drug interactions (Giacomini et al., 2010). They are located at the sinusoidal or canalicular pole of hepatocytes (Funk, 2008, Hirouchi et al., 2009) (See Fig. 1 for a schematic overview of drug transporter expression by mouse hepatocytes). Putative interactions of some hepatic transporters with new molecular entities developed by pharmaceutical companies have now to be characterized (Giacomini et al., 2010, Zhang et al., 2008). For this purpose, primary hepatocyte cultures have emerged as a valuable and useful tool (Ghibellini et al., 2006, Sahi et al., 2010, Soars et al., 2007).
Cultured hepatocytes can be used either in a monolayer standard configuration, i.e., hepatocytes plated on plastic dishes or collagen-coated dishes, or in a sandwich configuration, i.e., hepatocytes plated on collagen-coated dishes and overlaid with a second layer of collagen or matrigel (LeCluyse, 2001). These two configurations have been successfully retained to study drug transporter activity and regulation (Jigorel et al., 2006, Swift et al., 2010). Rat hepatocytes cultured in conventional monolayer conditions however fail to display bile canalicular structures and show a marked down-regulation of sinusoidal influx drug transporters, associated with a concomitant over-expression of the efflux transporter P-glycoprotein (Abcb1) (Fardel et al., 1993, Jigorel et al., 2005, Luttringer et al., 2002). By contrast, sandwich-cultured rat hepatocytes exhibit functional canalicular networks (LeCluyse et al., 1994, Liu et al., 1999b), even if they also display reduced expression of sinusoidal influx transporters with time in culture when compared to freshly isolated hepatocytes (Borlak and Klutcka, 2004, Kotani et al., 2011, Tchaparian et al., 2011). With regard to human hepatocytes cultured either in monolayer or sandwich conditions, expression of drug transporters appears to be much better preserved with time in culture when compared to rat counterparts (Hoffmaster et al., 2004, Jigorel et al., 2005, Kotani et al., 2011, Li et al., 2009, Schaefer et al., 2012, Takeba et al., 2011).
Unlike primary rat and human hepatocytes, cultured mouse hepatocytes remain poorly characterized with respect to drug transporter expression and activity, even if bile acid transport and multidrug resistance-associated protein (Mrp/Abcc) 4 expression have been recently investigated in sandwich-cultured mouse hepatocytes (Swift and Brouwer, 2010). Primary mouse hepatocytes likely represent an interesting in vitro model for studying liver drug transporters, because they can originate from various and already generated knockout mice in which specific liver transporter has been deleted, thus potentially allowing to address the function and the substrates of the disrupted transporter (Klaassen and Lu, 2008). Primary mouse hepatocytes may also served for identifying signaling ways governing transporter expression, through, for example, the use of hepatocytes from transgenic mice deficient in drug-sensing receptors such as pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) or constitutive androstane receptor (CAR), known to be involved in transporter regulation (Klaassen and Aleksunes, 2010). Before considering the use of primary hepatocytes from transgenic mice for transporter studies, accurate characterization of basal transporter expression in primary wild-type mouse hepatocytes is however required. The present study was therefore designed to carefully analyze expression, activity and regulation of drug transporters in mouse hepatocytes cultured in monolayer or sandwich configurations; the transporters studied in this work correspond to referent hepatic SLC and ABC transporters (Funk, 2008) and their cellular localization is indicated in Fig. 1.
Section snippets
Chemicals
Rhodamine 123, probenecid, verapamil, phenobarbital and dexamethasone were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France), whereas carboxy-2,7-dichlorofluoresceine (CF) diacetate and 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) were provided by Invitrogen/Life Technologies (Villebon sur Yvette, France) and Cambridge Isotope Laboratories (Andover, MA), respectively. [3H(G)] taurocholic acid (sp. act. 1.19 Ci/mmol), [6,7-3H(N)] estrone-3-sulfate (E3S) (sp. act. 57.3 Ci/mmol), [1-14C]
Morphology and canalicular network formation
Primary mouse hepatocytes maintained for 3 or 4 days after seeding either in monolayer on plastic or collagen or in sandwich configuration with collagen and matrigel formed confluent or nearly-confluent cultures (Fig. 2A). Hepatocytes cultured in monolayer exhibited a morphology rather flattener than that of sandwich-cultured counterparts, in agreement with previous data (Richert et al., 2002). Sandwich-cultured hepatocytes, but not monolayer-cultured counterparts, displayed extensive
Discussion
The present study demonstrated that expression of drug transporters in primary mouse hepatocytes is markedly influenced by time in culture, whatever the culture conditions used, i.e., monolayer/plastic, monolayer/collagen or sandwich/matrigel configurations. Indeed, mRNA expression of SLC transporters, especially those of Ntcp, Oatp1b2, Oct1, Oat2 and Mate1, rapidly and remarkably fall during primary culture. This is associated with a concomitant loss of Ntcp protein expression and a reduction
Acknowledgment
The authors thank the animal house platform of the SFR BIOSIT (University of Rennes 1, France) for mouse accommodation.
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