Ursodeoxycholate reduces ethinylestradiol glucuronidation in the rat: Role of prevention in estrogen-induced cholestasis

ABSTRACT Ethinylestradiol (EE) administration (5 mg/kg, s.c., daily for 5 days) to rats leads to cholestasis, and its derivative EE 17␤-glucuronide is a likely mediator of this effect. Coadministration of ursodeoxycholate (UDC) was shown to prevent ethinylestradiol-induced cholestasis. The aim of this study was to evaluate the inhibitory effect of UDC on EE glucuronidation in vivo and in vitro as a potential mechanism to explain UDC protection. UDC treatment (25 mg/kg, i.p., daily for 5 days) decreased the biliary excretion of EE 17␤-glucuronide in bile after administration of a trace dose of [ 3 H]EE and reduced microsomal EE 17␤-glucuronidation activity by 20% and expression of UGT2B1, one of the enzymes involved in EE conjugation, by 30%. Glucuronidation kinetic studies were performed in vitro using normal microsomes and isolated hepatocytes in the presence of tauroursodeoxycholate (TUDC), the major endogenous derivative of UDC in the rat. Kinetic enzymatic studies in microsomes showed a noncompetitive inhibition of EE 17␤-glucuronidation by TUDC, which was unique for this bile salt since other endogenous bile salts such as taurocholate, taurochenodeoxycholate, or taurodeoxycholate did not affect the enzyme activity. Studies in isolated hepatocytes confirmed the inhibitory effect of TUDC on EE glucuronidation and indicated that TUDC can reach the enzyme active site in intact cells. In conclusion, both in vivo and in vitro experiments indicate that UDC decreased the metabolic pathways involved in EE glucuronidation, hence decreasing the formation of the cholestatic derivative EE 17␤-glucuronide

Adaptive downregulation of Cl- /HCO3 - exchange activity in rat hepatocytes under experimental obstructive cholestasis

In obstructive cholestasis, there is an integral adaptive response aimed to diminish the bile flow and minimize the injury of bile ducts caused by increased intraluminal pressure and harmful levels of bile salts and bilirrubin. Canalicular bicarbonate secretion, driven by the anion exchanger 2 (AE2), is an influential determinant of the canalicular bile salt-independent bile flow. In this work, we ascertained whether AE2 expression and/or activity is reduced in hepatocytes from rats with common bile duct ligation (BDL), as part of the adaptive response to cholestasis. After 4 days of BDL, we found that neither AE2 mRNA expression (measured by quantitative real-time PCR) nor total levels of AE2 protein (assessed by western blot) were modified in freshly isolated hepatocytes. However, BDL led to a decrease in the expression of AE2 protein in plasma membrane fraction as compared with SHAM control. Additionally, AE2 activity (J(OH)-, mmol/L/min), measured in primary cultured hepatocytes from BDL and SHAM rats, was decreased in the BDL group versus the control group (1.9 +/- 0.3 vs. 3.1 +/- 0.2, p<0.005). cAMP-stimulated AE2 activity, however, was not different between SHAM and BDL groups (3.7 +/- 0.3 vs. 3.5 +/- 0.3), suggesting that cAMP stimulated insertion into the canalicular membrane of AE2-containing intracellular vesicles, that had remained abnormally internalized after BDL. In conclusion, our results point to the existence of a novel adaptive mechanism in cholestasis aimed to reduce biliary pressure, in which AE2 internalization in hepatocytes might result in decreased canalicular HCO3- output and decreased bile flow.This work was supported by grants from Spanish Carlos III Health Institute (ISCIII) [J. M. Banales (FIS PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129) cofinanced by "Fondo Europeo de Desarrollo Regional" (FEDER); "Instituto de Salud Carlos III" [CIBERehd: J. M. Banales], Spain; BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to J. M. Banales), Department of Health of the Basque Country (J. M. Banales: 2017111010) and Euskadi RIS3 (J. M. Banales: 2016222001, 2017222014, 2018222029). "Fundacion Cientifica de la Asociacion Espanola Contra el Cancer" (AECC Scientific Foundation, to J. M. Banales). F. A. Crocenzi was recipient of a Young Investigator Scholarship from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Dynamic localization of hepatocellular transporters in health and disease

Vesicle-based trafficking of hepatocellular transporters involves delivery of the newly-synthesized carriers from the rough endoplasmic reticulum to either the plasma membrane domain or to an endosomal, submembrane compartment, followed by exocytic targeting to the plasma membrane. Once delivered to the plasma membrane, the transporters usually undergo recycling between the plasma membrane and the endosomal compartment, which usually serves as a reservoir of pre-existing transporters available on demand. The balance between exocytic targeting and endocytic internalization from/to this recycling compartment is therefore a chief determinant of the overall capability of the liver epithelium to secrete bile and to detoxify endo and xenobiotics. Hence, it is a highly regulated process. Impaired regulation of this balance may lead to abnormal localization of these transporters, which results in bile secretory failure due to endocytic internalization of key transporters involved in bile formation. This occurs in several experimental models of hepatocellular cholestasis, and in most human cholestatic liver diseases. This review describes the molecular bases involved in the biology of the dynamic localization of hepatocellular transporters and its regulation, with a focus on the involvement of signaling pathways in this process. Their alterations in different experimental models of cholestasis and in human cholestatic liver disease are reviewed. In addition, the causes explaining the pathological condition (e.g. disorganization of actin or actin-transporter linkers) and the mediators involved (e.g. activation of cholestatic signaling transduction pathways) are also discussed. Finally, several experimental therapeutic approaches based upon the administration of compounds known to stimulate exocytic insertion of canalicular transporters (e.g. cAMP, tauroursodeoxycholate) are described

G-protein-coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis

Estradiol-17ß-D-glucuronide (E17G) activates different signaling pathways (e.g., Ca21- dependent protein kinase C, phosphoinositide 3-kinase/protein kinase B, mitogenactivated protein kinases [MAPKs] p38 and extracellular signal-related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistanceassociated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G-protein-coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G-induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G-induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30-AC-PKA. PKA inhibition prevented E17G-induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30-AC-PKA pathway totally prevented E17G-induced alteration in Abcb11 and Abcc2 function and localization. Conclusion: In conclusion, activation of GPR30-AC-PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation.Fil: Zucchetti, Andrés E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Barosso, Ismael R. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Boaglio, Andrea C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Miszczuk, Gisel Sabrina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Larocca, María Cecilia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); ArgentinaFil: Ruiz, M. Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Davio, Carlos A. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Laboratorio de Farmacología de Receptores; Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Crocenzi, Fernando A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sánchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

Chemoprophylactic activity of metformin during <i>E</i>. <i>granulosus</i> cyst development.

<p>(A) Box plot showing the comparative distribution of the weight (g) of cysts recovered from untreated (C) and Met-treated (Met, 50 mg/kg/d) mice. A significant cyst weight reduction (**<i>p</i> < 0.01) was achieved in treated animals. (B) Representative SEM (a, b, e, f) and TEM (c, d, g, h) images of hydatid cysts recovered from untreated control mice (a-d) compared with Met-treated mice (e-h). ll, laminated layer; mt, microtriches; dc, distal cytoplasm; gl, germinal layer; nu, nucleus; ly, lysosomes; gly, glycogen storage; a, autophagosomes; al, autophagolysosome; cc, calcareous corpuscles. Bars indicate: 50 μm in (a, e), 10 μm in (b, f) and 1 μm in (c, d, g, h). (C) Number of cysts obtained from untreated and Met-treated mice as indicated in (A).</p

Therapeutic efficacy study in <i>E</i>. <i>granulosus</i> infected mice.

<p>(A) Box plots showing the comparative distribution of the weight (g) of cysts recovered from untreated mice (C) and treated with albendazole (ABZ, 5 mg/kg/d), metformin (Met, 50 mg/kg/d) and the combination of both drugs (ABZ+Met) for 60 days. The weight of cysts was significantly decreased upon all treatments compared with the control (***<i>p</i> < 0.01), but the decrease was more prominent in the group receiving the combined treatment than in those with either drug alone (**<i>p</i> < 0.05); in turn, weight reduction was greater with Met than with ABZ (*<i>p</i> < 0.2). (B) Representative SEM (a, d, g, j) and TEM (b, c, e, f, h, i, k, l) images of hydatid cysts recovered from untreated mice (a, b, c) or treated with Met (d, e, f), ABZ (g, h, i) and ABZ+Met (j, k, l). ll, laminated layer; mt, microtriches; dc, distal cytoplasm; gl, germinal layer; nu, nucleus; ly, lysosomes (arrowheads); ve: vesicles (double-headed arrow); gly, glycogen storage; a, autophagosomes. Bars indicate: 20 μm in (a, d, g, j), 1 μm in (b, e, f, h, I, k, l), and 0.2 μm in (c). (C) Intracystic concentrations of Met from cysts recovered from Met- or ABZ+Met-treated mice in the experiment indicated in (A). (D) Number of cysts obtained from untreated and pharmacologically treated mice as indicated in (A). *Statistically significant difference (<i>p</i> < 0.05) in (C) and (D).</p

Prevention of estradiol 17β-d-glucuronide–induced canalicular transporter internalization by hormonal modulation of cAMP in rat hepatocytes

Glucagon- and salbutamol-derived cAMP prevents estrogen-induced alteration of canalicular transporter localization and function via different pathways. Glucagon-derived protection depends on PKA activation, whereas salbutamol protection is exerted through a pathway that depends on Epac/MEK and microtubules