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The mechanisms involved in ethinyl estradiol-induced cholestasis are controversial. Basal bile flow was reduced by ethinyl estradiol administration, with a half time (t1/2) of 12.5 +/- 0.6 h. In contrast, initial taurocholate uptake was not significantly reduced until 3 days to 59% of control and to 13 and 10% of control at 5 and 7 days, respectively. The t1/2 was 4.3 +/- 0.1 days. These physiological changes were correlated with measurement of protein mass and steady-state mRNA for Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase), Na(+)-dependent taurocholate transporter, organic anion transporters, and membrane lipid fluidity. Ethinyl estradiol significantly decreased Na(+)-K(+)-ATPase activity and membrane fluidity. However, neither Na(+)-K(+)-ATPase alpha-subunit nor beta-subunit mass was altered by ethinyl estradiol administration. In contrast, protein content of the Na(+)-dependent taurocholate transporter was significantly reduced to 21% of control (P < 0.001) at 5 days. The Na(+)-dependent taurocholate transporter was identified in sinusoidal membrane fractions as a doublet with a molecular size estimated to be 51 and 56 kDa. Although both bands were reduced with ethinyl estradiol treatment, the 56-kDa band was decreased more rapidly and to a greater extent than the 51-kDa band. The estimated t1/2 of 4.8 +/- 0.6 days for the doublet was similar to that for Na(+)-dependent taurocholate uptake. The organic anion transporter protein mass was similarly reduced with time of ethinyl estradiol administration to 21% of control (P < 0.01) at 5 days. Ethinyl estradiol also rapidly decreased the steady-state mRNA levels of Na(+)-dependent and organic anion transporters to approximately 50% and 15% of control at 5 days, respectively. These studies indicate early generalized abnormalities of the sinusoidal membrane lipid fluidity, Na(+)-K(+)-ATPase activity, and bile acid transport protein content.

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Proinflammatory cytokines such as TNF-alpha and IL-1beta lead to downregulation of hepatic organic anion transporters in cholestasis. This adapted response is transcriptionally mediated by nuclear hormone receptors and liver-specific transcription factors. Because little is known in vivo about cytokine-dependent regulatory events, mice were treated with either TNF-alpha or IL-1beta for up to 16 h. Transporter mRNA expression was determined by Northern blot analysis, nuclear activity, and protein-expression of transactivators by EMSA and Western blotting. TNF-alpha induces a sustained decrease in Ntcp, Oatp1/Oatp1a1, and Bsep mRNA expression but exerts only transient [multidrug resistance-associated protein 2 (Mrp2)] or no effects (Mrp3) on Mrps. In addition to Ntcp and Oatp1/Oatp1a1, IL-1beta also downregulates Bsep, Mrp2, and Mrp3 mRNAs to some extent. To study transcriptional regulation, Ntcp and Bsep promoters were first cloned from mice revealing a new distal Ntcp hepatocyte nuclear factor 1 (HNF-1) element but otherwise show a conserved localization to known rat regulatory elements. Changes in transporter-expression are preceeded by a reduction in binding activities at IR-1, ER-8, DR-5, and HNF-1alpha sites after 4 h by either cytokine, which remained more sustained by TNF-alpha in the case of nuclear receptors. Nuclear protein levels of retinoid X receptor (RXR)-alpha are significantly decreased by TNF-alpha but only transiently affected by IL-1beta. Minor reductions of retinoic acid receptor, farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor nuclear proteins are restricted to 4 h after cytokine application and paralleled by a decrease in mRNA levels. Basolateral and canalicular transporter systems are downregulated by both cytokines, TNF-alpha and IL-1beta. Activity of HNF-1alpha as regulator of mNtcp is suppressed by both cytokines. Decreased binding activities of nuclear receptor heterodimers may be explained by a reduction of the ubiquitous heterodimerization partner RXR-alpha.

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