Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes
Introduction
Drugs given by the most convenient administration route, oral administration, are absorbed through the gastrointestinal tract. Thereafter, they reach the liver via the portal vein before entering the systemic circulation. For some drugs, transport proteins present in the gastrointestinal tract and in the liver play a significant role in drug absorption and drug bioavailability (Diaz, 2000). Multidrug resistance proteins (Mdr, also known as P-glycoprotein) and organic anion transporting polypeptides (Oatp) are major members in the group of transport proteins that regulate the disposition of orally administered drugs (Kim, 2002, Beringer and Slaughter, 2005). Oatp participates in the uptake of drugs from the blood into the hepatocytes where they may undergo significant phase I and/or phase II metabolism. Oatp has also been suggested to function as an excreting protein capable of transporting drug and drug metabolites back into the systemic circulation (Chandra and Brouwer, 2004, Beringer and Slaughter, 2005). The interplay between Oatp uptake of drugs into hepatocytes, their subsequent metabolism by hepatic enzymes and further excretion into bile through Mdr efflux system is a powerful detoxification pathway in the body. Besides their presence in the liver and the intestine, Oatp and Mdr are widely distributed in the human body where they play an important role in the disposition and distribution of certain drugs. In the kidneys, Oatp and Mdr1 are involved in renal secretion of xenobiotics; at the blood–brain barrier a range of drugs have limited brain penetration due to Mdr1-mediated efflux. Oatp is also expressed in the blood–brain barrier; this suggests its role in delivery of drugs into the brain and removal of metabolites from the brain (Ayrton and Morgan, 2001, Kim, 2002, Beringer and Slaughter, 2005).
Since the important role played by transport proteins in xenobiotic disposition and distribution is now understood, a great body of investigations of their effect on the disposition of new drugs are made by pharmaceutical industries as part of drug discovery screening. Investigations of Oatp activity have mostly been performed in vitro using freshly isolated hepatocytes. There are only very few reports of studies using cryopreserved hepatocytes for the assessment of Oatp transport activity. These studies show that some transport activity is retained in cryopreserved rat and human hepatocytes in suspension (Houle et al., 2003, Shitara et al., 2003). The use of cryopreserved rat hepatocytes for investigation of the functional activity of Mdr1 has not been reported. Moreover, sandwich culture is commonly used to assess the transport activity of Mdr1 and other canalicular transport proteins in freshly isolated rat and human hepatocytes by measurements of canalicular accumulation (Annaert et al., 2001, Hoffmaster et al., 2004, Annaert and Brouwer, 2005). The function of Mdr1 using freshly isolated rat hepatocytes has been demonstrated in conventional culture in a single report (Le Bot et al., 1994). However, this study assessed the transport activity as a decline in doxorubicin accumulation over time in culture as an indication of increased efflux of doxorubicin by Mdr1. The present study is the first, to our knowledge, to investigate the transport activity of Oatp and Mdr1 using cryopreserved rat hepatocytes in suspension, in conventional culture and in sandwich culture by the use of both substrates and inhibitors. Additionally, the protein expression and mRNA expression were investigated. A recent paper published by Bi et al. (2006) investigated MDR1 protein in cryopreserved human hepatocytes.
The presence of functional transport proteins in cryopreserved rat hepatocytes will make it possible to investigate these transport proteins for a number of scientists with limited access to freshly isolated hepatocytes. It also allows standardising these studies by using cells from same donors in several experiments and will reduce considerably the number of animals used in these studies.
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Materials
Dexamethasone, dimethyl sulfoxide (DMSO), ehthyleneglycol-bis(b-amino ethylether)-N,N,N′,N′-tetraacetic acid (EGTA), KCl, ketoconazole, Percoll, probenecid, rat tail collagen (type I), triton X-100, trypan blue and β-mercaptoethanol were obtained from Sigma–Aldrich (Bröndby, Denmark). Calcium and magnesium free Hanks’ balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), ITS (insulin (1.0 g/L), transferrin (0.55
Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression
The uptake of taurocholate using cryopreserved hepatocytes in suspension (day 0), in conventional culture (days 2–4) and in sandwich culture (days 2–4) is shown in Fig. 1. The results are presented as uptake of taurocholate after 15 min as it was shown that the uptake was not linear in the range of 0–15 min (data not shown). The highest uptake was seen in suspension treated few hours after thawing. Compared with the initial value in suspension (day 0), the uptake of taurocholate decreased, when
Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression
The present study documented that the uptake of taurocholate into cryopreserved rat hepatocytes in suspension was mediated by transport proteins; the transport activity declined when rat hepatocytes were cultured (Fig. 1). The results from Oatp mRNA investigation correlated with taurocholate update data. mRNA expressions for Oatp1a1 and Oatp1a4 were lower in cryopreserved rat hepatocytes in culture after 4 days as compared to data from cryopreserved rat hepatocytes in suspension (day 0).
It is
Conclusions
In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport. Cryopreserved rat hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to cryopreserved rat hepatocytes in culture. The presence of Mdr1 in cryopreserved rat hepatocytes cultured in both the conventional and the sandwich culture was confirmed at mRNA level, by protein expression
Acknowledgement
The study was supported by H. Lundbeck A/S, Valby, Denmark.
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