NOX 2 plasma membrane expression is impaired by estradiol 17β-D-glucuronide in isolated and perfused rat livers.

LITTA, ALEN A.; SALAS, GIMENA; LAROCCA, M CECILIA; MARRONE, JULIETA; CROCENZI, FERNANDO A.

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias 2022; 2022

SAIC-SAI-SAFIS

All NADPH oxidases contain a catalytic transmembrane protein known as NOX, which requires its assembly with the membrane subunit p22phox, several cytosolic regulatory subunits and the small GTPase Rac, to get the active NOX oxidase complex. It was seen in different non-hepatic cells that NOX is mostly inactive in cholesterol rich lipid ̈rafts ̈, becoming active when present in ̈non- raft ̈ microdomains. Previously, we shown that estradiol 17β-D-glucuronide (E17G) impairs the canalicular transporter Mrp2 activity (which depends on raft localization) by inducing its migration to non-rafts and its clathrin-mediated endocytosis. Recently, we got evidence that E17G-induced alteration of Mrp2 activity is partially mediated by NOX2. Thus, we assessed the possible effects of E17G on NOX2 membrane localization and expression. We perfused liv- ers from male Wistar rats, and E17G (2 μmol/liver) or its vehicle (DMSO) were administered as a bolus injection. Twenty minutes lat- er, livers were snap-frozen and purified plasma membranes (PPM) were prepared by ultracentrifugation from liver homogenates; then, by treating PPM with Triton X-100 buffer we obtained purified detergent-soluble (non-raft) and detergent-insoluble (raft) fractions. Western blots from PPM, non-raft and raft samples were made to estimate the total expression of NOX2 in PPM as well as its distribution between microdomains. Caveolin and clathrin expression served as purity markers of raft and non-raft fractions, respectively; actin expression served as load control for PPM. E17G diminished the control PPM expression of NOX2 (1.86±0.08 vs 1.44±0.20; arbitrary values, mean±SEM, n=4; p