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

Mitogen-activated protein kinases are involved in hepatocanalicular dysfunction and cholestasis induced by oxidative stress

In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca2+-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 μM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.Fil: Toledo, Flavia D. 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: 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: Zucchetti, Andrés E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sanchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

Bilirubin is an endogenous antioxidant with cytoprotective properties, and several studies highlight its potential in the treatment of pro-oxidant diseases. We demonstrated that oxidative stress (OS), a key feature in most hepatopathies, induces cholestasis by actin cytoskeleton disarrangement and further endocytic internalization of key canalicular transporters, such as the bile salt export pump (Bsep) and the multidrug resistance-associated protein 2 (Mrp2). He re, we evaluated the capability of physiological concentrations of unconjugated bilirubin (UB) to limit OS and the impairment in biliary secretory function induced by the model pro-oxidant agent, tert-butylhydroperoxide (tBuOOH). UB fully prevented the formation of reactive oxygen species (ROS) and membrane lipid peroxidation induced by tBuOOH in isolated rat hepatocytes. In the isolated rat hepatocyte couplet model, UB (17.1 µM) prevented the endocytic internalization of Bsep and Mrp2 and the impairment in their secretory function induced by tBuOOH. UB also prevented actin disarrangement, as evaluated by both plasma membrane bleb formation and actin fluorescent staining. Finally, UB prevented tBuOOH-induced cPKC activation. Experiments in isolated perfused rat livers showed that UB prevents the increase in oxidized glutathione biliary excretion and the drop in bile flow and the biliary excretion of specific Bsep and Mrp2 substrates. We conclude that physiological concentrations of UB are sufficient to prevent the biliary secretory failure induced by OS, by counteracting actin disarrangement and the consequent internalization of canalicular transporters relevant to normal bile formation. This reveals an important role for UB in preserving biliary secretory function under OS conditions.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Toledo, Flavia D. 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: Arriaga, Sandra Mónica María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Bioquímica Clínica; Argentina.Fil: Ochoa, Justina E. 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.Fil: Mottino, Aldo D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

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

Effect of the inhibition of p38, ERK1/2 and JNK1/2, or the coinhibition of cPKC-ERK1/2, PI3K-p38, or p38-ERK1/2, on E₂17G-induced impairment of the canalicular accumulation of the Bsep and Mrp2 fluorescent substrates in IRHCs.

<p>IRHCs were incubated with E₂17G (200 µM, 20 min) (or DMSO in controls), with or without pretreatment for 15 min with the JNK1/2 inhibitor SP600125 (1 µM), the ERK1/2 inhibitor PD98059 (PD; 5 µM), and/or the p38 inhibitor SB203580 (SB; 1 µM), together or not with the cPKC inhibitor Gö6976 (Gö; 1 µM) or PI3K inhibitor wortmanin (WM; 100 nM). Canalicular accumulation CGamF (Bsep substrate, panel A) and GS-MF (Mrp2 substrate, panel B) was determined as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation. The results are expressed as percentages of the control group and are shown as mean ± SEM (<i>n</i> = 3–4). *P<0.05 <i>vs.</i> E₂17G, and ^#P<0.05 <i>vs.</i> E₂17G-WM, E₂17G-Gö, E₂17G-PD or E₂17G-SB.</p

Schematic representation of the signalling events involved in E₂17G-induced cholestasis by endocytic internalization and further retention of canalicular transporters relevant to bile formation (Bsep, Mrp2).

<p>p38, acting downstream of cPKC, triggers endocytic internalization of the apical carriers presumably towards apical early endosomes (AEE), the first intracellular endosomal compartment receiving internalized proteins from the apical membrane, in a microtubule-independent manner (solid arrow). These transporters traffic to, and accumulate into, apical recycling endosomes (ARE), from where they can be retargeted to the apical membrane during the recovery of the cholestatic process, in a microtubule-dependent manner (dashed arrows). Activation of the PI3K/Akt/ERK1/2 signalling pathway halts this latter process, thus explaining the increased colocalization of Bsep/Mrp2 with Rab11a, an ARE marker. This prolongs the cholestatic effect of E₂17G by impeding the fast, spontaneous retargeting of intracellular transporters that would lead to a rapid recovery from the cholestatic injury.</p

Effect of inhibition of p38 or ERK1/2, and coinhibition of cPKC-ERK1/2, PI3K-p38, or p38-ERK1/2 on E₂17G-induced retrieval of Bsep and Mrp2 in IRHCs.

<p>The upper panels show representative confocal immunofluorescence images of the localization of Bsep and Mrp2 in DMSO-treated (control) or E₂17G (200 µM)-treated IRHCs, with or without the p38 inhibitor SB203580 (SB; 1 µM) or the ERK1/2 inhibitor PD98059 (PD; 5 µM), in combination or not with the cPKC inhibitor Gö6976 (Gö; 1 µM) or PI3K inhibitor wortmanin (WM; 100 nM). The lower panels show the densitometric analysis of the fluorescence intensity along a line (8 µm) perpendicular to the center of the canalicular vacuole (from +4 to −4 µm). The statistical analysis of the profiles of fluorescence showed a significant change in the E₂17G-treated group (P<0.05; number of analyzed canalicular vacuoles >10), but this reverted to normal in the E₂17G-SB, E₂17G-PD, E₂17G-PD-SB, E₂17G-Gö-PD and E₂17G-WM-SB groups for Bsep and Mrp2.</p

E₂17G activates the MAPK signalling pathway.

<p><i>(A)</i> left panel: representative Western blottings of phospho (p)-p38, p-ERK1/2, p-JNK1/2 and total forms of all these MAPK types were obtained from whole cellular lysates of primary-cultured rat hepatocytes incubated with E₂17G (200 µM) for 10 to 60 min, or with E₂17G (200 µM) for 20 min in cells pretreated with the PI3K inhibitor wortmanin (WM, 100 nM) or with the cPKC inhibitor Gö6976 (Gö, 1 µM) for 15 min. <i>A</i> (right panel), and <i>B</i> and <i>C panels</i> show phosphorylation status of all MAPK types evaluated (calculated as the p-MAPK to total MAPK ratio for each experimental condition). An arbitrary value of 100 was assigned to the band of highest densitometric intensity in every Western blot before the ratio was calculated. The results are shown as mean ± SEM (<i>n</i> = 5). *P<0.05 <i>vs.</i> control (cells treated only with DMSO), and ^#P<0.05 <i>vs.</i> E₂17G (20 min).</p

Effect of E₂17G on colocalization of Rab11a with Mrp2 or Bsep in IRHCs.

<p>Immunofluorescence confocal images showing staining of Mrp2 or Bsep (green) and Rab11a (red) in IRHCs treated with DMSO (control), the ERK1/2 inhibitor PD98059 (PD; 5 µM), the p38 inhibitor SB203580 (SB; 1 µM), or both inhibitors together. Colocalization of Rab11a with Mrp2 or Bsep in the E₂17G-treated group is indicated by orange-yellow fluorescence in merged images. Insets depict F-actin staining, which was used to demarcate the limits of the canalicular vacuoles.</p

Sequential Activation of Classic PKC and Estrogen Receptor α Is Involved in Estradiol 17ß-D-Glucuronide-Induced Cholestasis

<div><p>Estradiol 17ß-d-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways <em>via</em> estrogen receptor alpha (ERα), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ERα. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ERα inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ERα in canalicular transporter internalization induced by E17G was confirmed in ERα-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ERα shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ERα and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ERα, indicating that cPKC activation precedes that of ERα. Conclusion: ERα is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC.</p> </div