Abstract
Dexamethasone (DEX) is a well established inducer of CYP3A. These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes. Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 μM, 0–48 h and 0.1–100 μM, 48–96 h). The expression of sinusoidal [(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na+-dependent taurocholate cotransporting polypeptide (Ntcp)] and canalicular [bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2] transport proteins was determined by Western blot analysis. The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 μM, 10 min), rhodamine 123 (Rh123; 10 μM, 30 min), and carboxy-2′,7′-dichlorofluorescein (CDF; 10 μM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes. DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep. The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 μM). The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged. Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins. Modest alterations in hepatic transport protein function were consistent with changes in protein expression.
Footnotes
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This research was supported by Pfizer Global Research and Development and National Institutes of Health Grant RO1 GM41935. Data were presented at the North American Meeting of the International Society for the Study of Xenobiotics in Providence, RI, October 2003 and are included in the doctoral dissertation of Ryan Z. Turncliff.
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ABBREVIATIONS: SC, sandwich-cultured; DEX, dexamethasone; Bsep, bile salt export pump; Mrp, multidrug resistance-associated protein; Rh123, rhodamine 123; Mdr, multidrug resistance protein; GR, glucocorticoid receptor; PXR, pregnane X receptor; Ntcp, Na+-dependent taurocholate cotransporting polypeptide; Oatp, organic anion transporting polypeptide; FXR, farnesoid X receptor; DMEM, Dulbecco's modified Eagle's medium; ITS, insulin-transferrin-sodium selenite; FBS, fetal bovine serum; TC, taurocholate; CDF-DA, carboxydichlorofluorescein diacetate; DMSO, dimethyl sulfoxide; BEI, biliary excretion index; HBSS, Hanks' balanced salt solution; BCA, bicinchoninic acid; BC, bile canaliculi.
- Received December 19, 2003.
- Accepted May 18, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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