ESTRADIOL 17β-D-GLUCURONIDE-INDUCED NADPH OXIDASE IMPAIRS Mrp2 ACTIVITY BY CONTRIBUTING TO ITS CELLULAR INTERNALIZATION IN SANDWICH CULTURED RAT HEPATOCYTES

LITTA, AA; ANDERMATTEN, RB; SALAS, G; MEDEOT, AC; SCHUK, VS; CROCENZI FA; BASIGLIO CL

Mar del Plata

Congreso; Reunión Conjunta Sociedades de Biociencias; 2022

SAFIS

Estradiol 17β-D-glucuronide (E17G) alters canalicular secretion viaseveral kinase-mediated signaling pathways which induce endocytosisand intracellular retention of canalicular transporters, the MEKERK1/2 pathway contributing to the second process. Previously,we found that NADPH oxidase (NOX)-generated reactive oxygenspecies (ROS) are partly responsible for the impairment of canalicularMrp2 transport activity induced by E17G, and that NOX sharesthe MEK-ERK1/2 signaling pathway, downstream these kinases.We aimed to evidence that E17G-induced NOX-mediated functionalimpairment of Mrp2 transport is dependent on its internalization.Intracellular distribution of Mrp2 was assessed by immunofluorescenceand confocal microscopy analysis in sandwich cultured rathepatocytes (SCRH). First, SCRH were treated with vehicle (DMSO,control), E17G (200 μM, 20 min) or pre-treated with apocynin (Apo,NOX inhibitor, 100 μM, 30 min) prior to E17G. Then, SCRH werefixed, permeabilized, and incubated with a specific antibody to Mrp2,and then with a secondary antibody and Alexa Fluo 568-conjugatedphalloidin for F-actin staining, which is mainly pericanalicular(demarcating the canaliculi) and barely evident in the basolateralmembrane. Mrp2 internalization was evaluated in confocal images(n=12 canaliculi, from 3 independent cultures) by calculating thepercentage of Mrp2-associated staining within the canaliculi to totalcellular staining. Mrp2, mainly confined to the canaliculi in controls,was internalized to intracellular vesicles by E17G. Apo preventedthis alteration, showing a control-like distribution pattern. Apo, whichinhibits migration of the cytosolic p47phox subunit of NOX, impedingthe formation of the active NOX complex in the plasma membrane,prevents internalization of Mrp2 by E17G. This finding supports thelocation of NOX in the MEK-ERK1/2 signaling pathway, which is involvedin E17G-induced internalization of canalicular transporterssuch as Mrp2.