Obstructive cholestasis leads to downregulated Cl-/HCO3- exchange activity in rat hepatocytes.

CROCENZI, FERNANDO A.; BAÑALES, JESÚS M.; ZUCCHETTI, ANDRÉS ERNESTO; BOAGLIO, ANDREA C; SÁEZ, ELENA; ROMA, MARCELO G; MEDINA, JUAN F.

Barcelona

Congreso; 47th Annual Meeting of the European Association for the Study of the Liver (EASL); 2012

EASL

Background and Aims: Cholestatic diseases, such as cholelithiasis, primary sclerosis cholangitis and primary biliary cirrhosis are associated with adaptive hepatocellular changes to reduce biliary pressure, including downregulation of Bsep and Mrp2. AE2 is a Cl−/HCO3− exchanger involved in both canalicular and biliary Cl−/HCO3− secretion that results in an osmotic driving force for bile formation. Our aim was to analyze the regulation of the hepatocellular AE2 activity under experimental cholestasis. Methods: Experimental cholestasis was performed by using the common bile-duct ligation (BDL) rat model. After 4 days of BDL, blood samples were taken for assessment of biochemical cholestatic markers, and the liver was perfused with collagenase for hepatocyte isolation. The Cl−/HCO3− 3 anion exchange (AE) activity was evaluated in cultured hepatocytes by microfluorimetric monitoring of pHi variations upon perfusion maneuvers using the pHsensitive probe 2’-7’-bis-(2-carboxyethyl)-5(6)-carboxyfluoresceinacetoxymethylester (BCECF-AM). AE activity was measured both baseline and in the presence of a cAMP-stimulation mixture (cAMP-SM) containing dibutyryl-cAMP, 3-isobutyl-1- methylxanthine (IBMX) and forskolin, and was expressed as transmembrane base fluxes (JOH−, mmol/L/min). Moreover, freshly isolated hepatocytes were used to assess both AE2 mRNA and protein expression by real-time PCR and Western-blot, respectively. Results: Total serum levels of bilirubin and alkaline phosphatase levels increased 8- and 5-times respectively (n = 3, p < 0.05 vs SHAM), demonstrating a fully installed cholestasis after 4 days of BDL. The AE activity in hepatocytes from BDL rats was decreased compared to hepatocytes from SHAM animals (JOH− = 2.43±0.14 vs 3.12±0.16 mmol/L/min, p < 0.005), and these differences were not associated with changes in the expression of AE2 mRNA or protein. On the other hand, stimulation with cAMP-SM was able to increase the AE activity in both BDL (JOH− = 3.54±0.22 mmol/L/min, p < 0.005) and SHAM (JOH− = 3.92±0.34 mmol/L/min, p < 0.05) hepatocytes. Increased AE activities in stimulated hepatocytes turned to be not significantly different between BDL and SHAM groups. Conclusions: Hepatocytes from BDL-rats show post-translational downregulation of basal Cl−/HCO3− exchange activity that can be reverted by increasing the cAMP levels. It is possible that the reverted effect be due to cAMP-induced exocytosis of intracellular vesicles containing AE2, which had been endocytosed by the cholestatic injury.