1 Five 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), (e.g. atorvastatin,¯uvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P-glycoprotein (P-gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over-expresses the multi-drug resistance protein 1a/b (mdr1a/1b). 2 P-gp modulation was studied by a¯uorimetric assay and confocal microscopy by means of R123 eux and uptake experiments, respectively. 3 Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration-dependent manner. Lovastatin acid, simvastatin acid,¯uvastatin and pravastatin did not show a signi®cant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest anities for P-gp and results were comparable for both methods. 4 In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition tō uorimetric assay is a sensitive tool to study P-gp anity in drug screening that is especially useful for early phases of drug development.
Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acidactivated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5 flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at ؊13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/ retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias. (HEPATOLOGY 2007;46:1960-1970
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