Estradiol 17beta;-D-glucuronide-induced NADPH oxidase impairs Mrp2 activity by contributing to its cellular internalization in sandwich cultured rat hepatocytes.

SALAS, GIMENA; LITTA, ALEN A.; SCHUCK, VIRGINIA SOLEDAD; MEDEOT, ANABELA CAROLINA; ANDERMATTEN, ROMINA B.; BASIGLIO, CECILIA L; CROCENZI, FERNANDO A.

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias 2022; 2022

SAIC-SAI-SAFIS

Estradiol 17β-D-glucuronide (E17G) alters canalicular secretion via several kinase-mediated signaling pathways which induce endocytosis and intracellular retention of canalicular transporters, the MEK- ERK1/2 pathway contributing to the second process. Previously, we found that NADPH oxidase (NOX)-generated reactive oxygen species (ROS) are partly responsible for the impairment of canalicular Mrp2 transport activity induced by E17G, and that NOX shares the MEK-ERK1/2 signaling pathway, downstream these kinases. We aimed to evidence that E17G-induced NOX-mediated function- al impairment of Mrp2 transport is dependent on its internalization. Intracellular distribution of Mrp2 was assessed by immunofluorescence and confocal microscopy analysis in sandwich cultured rat hepatocytes (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 were fixed, permeabilized, and incubated with a specific antibody to Mrp2, and then with a secondary antibody and Alexa Fluo 568-conjugated phalloidin for F-actin staining, which is mainly pericanalicular (demarcating the canaliculi) and barely evident in the basolateral membrane. Mrp2 internalization was evaluated in confocal images (n=12 canaliculi, from 3 independent cultures) by calculating the percentage of Mrp2-associated staining within the canaliculi to total cellular staining. Mrp2, mainly confined to the canaliculi in controls, was internalized to intracellular vesicles by E17G. Apo prevented this alteration, showing a control-like distribution pattern. Apo, which inhibits migration of the cytosolic p47phox subunit of NOX, impeding the formation of the active NOX complex in the plasma membrane, prevents internalization of Mrp2 by E17G. This finding supports the location of NOX in the MEK-ERK1/2 signaling pathway, which is in- volved in E17G-induced internalization of canalicular transporters such as Mrp2.