Abstract
Freshly isolated hepatocytes are widely accepted as the “gold standard” for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and “legacy” network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.
Footnotes
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Research was supported by National Institutes of Health (NIH) Grant GM41935 and by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (NIEHS). All data in this project were collected with the assistance of the Fluorescence Microscopy and Imaging Center at the NIEHS.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.018200.
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ABBREVIATIONS: Mrp2, multidrug resistance-associated protein 2; BSA, bovine serum albumin; CDF, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein; CDF-DA, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate; Oatp1a1, organic anion transporting polypeptide 1a1; PBS, phosphate-buffered saline; MK571, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid; P-gp, P-glycoprotein; RT, room temperature; TR-, Mrp2-deficient Wistar rat; ZO-1, zonula occludens 1.
- Received August 22, 2007.
- Accepted October 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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