UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.

ObjectiveThe endogenous, cholestatic metabolite estradiol 17ß-d-glucuronide (E217G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect.DesignERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E217G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis.ResultsE217G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E217G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E217G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2.ConclusionscPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E217G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.

Oxidative stress (OS) is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both OS from mitochondrial origin and cell death. Intracellular Ca²⁺ increase plays a permissive role in these events, but the underlying mechanisms are poorly known. We examined in primary cultured rat hepatocytes whether the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidant, model compound. tBOOH (500 μM, 15 min) induced MPTP formation, as assessed by measuring mitochondrial membrane depolarization as a surrogate marker, and increased lipid peroxidation in a cyclosporin A (CsA)-sensitive manner, revealing the involvement of MPTPs in tBOOH-induced radical oxygen species (ROS) formation. Intracellular Ca²⁺ sequestration with BAPTA/AM, CaM blockage with W7 or trifluoperazine, and CaMKII inhibition with KN-62 all fully prevented tBOOH-induced MPTP opening and reduced tBOOH-induced lipid peroxidation to a similar extent to CsA, suggesting that Ca²⁺/CaM/CaMKII signaling pathway fully mediates MPTP-mediated mitochondrial ROS generation. tBOOH-induced apoptosis, as shown by flow cytometry of annexin V/propidium iodide, mitochondrial release of cytochrome c, activation of caspase-3 and increase in the Bax-to-Bcl-xL ratio, and the Ca²⁺/CaM/CaMKII signaling antagonists fully prevented these effects. Intramitochondrial CaM and CaMKII were partially involved in tBOOH-induced MPTP formation, since W7 and KN-62 both attenuated the tBOOH-induced, MPTP-mediated swelling of isolated mitochondria. We concluded that Ca²⁺/CaM/CaMKII signaling pathway is a key mediator of OS-induced MPTP formation and the subsequent exacerbation of OS from mitochondrial origin and apoptotic cell death.