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Received for publication August 22, 2007.
Revised October 8, 2007.
Accepted for publication October 18, 2007.
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 apical proteins or predict biliary excretion in the intact organ is unclear. Following 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 1hr of isolation, and in cells cultured for 1hr. Oatp1a1 was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for co-localization of Oatp1a1 with P-gp or 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 ZO-1 staining. Functional analysis of rat hepatocytes cultured for 1hr demonstrated that the fluorescent anion and Mrp2 substrate, carboxydichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was MK571 (Mrp inhibitor)-sensitive and not observed in hepatocytes isolated from TR- rats (Mrp2-deficient). 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.
Key words:
ABC transporters, biliary excretion, drug efflux, drug transport, hepatobiliary transport, hepatocytes, membrane transport, organic anion transport, p-glycoprotein
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