Boldine enhances bile production in rats via osmotic and Farnesoid X receptor dependent mechanisms Jolana Cermanova a,1 , Zuzana Kadova a,d,1 , Marie Zagorova a , Milos Hroch a,b , Pavel Tomsik b , Petr Nachtigal c , Zdenka Kudlackova c , Petr Pavek d , Michaela Dubecka d , Martina Ceckova d , Frantisek Staud d , Tomas Laho a , Stanislav Micuda a, ⁎ a Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic b Department of Medical Biochemistry, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic c Department of Biological and Medical Sciences, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Czech Republic d Deparment of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Czech Republic abstract article info Article history: Received 29 October 2014 Revised 26 February 2015 Accepted 3 March 2015 Available online 12 March 2015 Keywords: Boldine Bile production Bile salt export pump Farnesoid X receptor Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 μM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine. © 2015 Elsevier Inc. All rights reserved. Introduction Bile formation (BF) is an essential function of the liver providing an important route for the excretion of many endo- and xenobiotics and depends on the transporter-mediated secretion of osmotically active compounds by hepatocytes and cholangiocytes. These compounds attract water to form bile in relation to their actual secretion. Two princi- pal pathways are involved: bile acid-dependent bile flow (BADF), and bile acid-independent bile blow (BAIF) (Esteller, 2008). Regarding BADF, bile production is achieved by bile acids, the major endogenous os- motic bile constituents. Moreover, bile acids (BA) demonstrate strong in- direct mechanisms, such as activation of the FXR (Farnesoid X receptor) nuclear receptor or post-transcriptional modulation with consequent change in expression, localization and finally function of bile acid trans- porters and enzymes responsible for bile acid synthesis and metabolism (Dombrowski et al., 2006; Boyer, 2013). Key to BAIF is the function of Mrp2 transporter (multidrug resistance-associated protein 2) which me- diates biliary secretion of osmotically active glutathione (GSH). This tripeptide is the main mechanism of antioxidant defense protecting the hepatocytes against injury imposed by reactive oxygen species and elec- trophiles (Ribas et al., 2014). However, biliary secretion of GSH together with its oxidized form (GSSG) is also the major determinant of BAIF (Ballatori and Truong, 1992). As a consequence, modulation of mecha- nisms responsible for BADF or BAIF may in turn change the rate of elim- ination of their endogenous as well as exogenous substrates. Therefore characterization of influence of the agents administered to organism as drugs or food ingredients on these mechanisms may prevent serious interactions. Choleretics are agents that stimulate bile production, and work through activation of BADF and/or BAIF, or may also possess direct Toxicology and Applied Pharmacology 285 (2015) 12–22 ⁎ Corresponding author at: Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Simkova 870, 500 38 Hradec Kralove, Czech Republic. E-mail address: micuda@lfhk.cuni.cz (S. Micuda). 1 Authors that contributed equally. http://dx.doi.org/10.1016/j.taap.2015.03.004 0041-008X/© 2015 Elsevier Inc. 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