Role of OAT2 as Endoplasmic Reticulum Membrane Transporters in Bumetanide Glucuronidation

Abstract

The glucuronidation reaction, which is catalyzed by the UDP‐glucuronosyltransferase (UGT) superfamily of enzymes, plays an important role in metabolism of numerous endogenous and exogenous compounds. Since the active site of UGT resides in the lumen of the endoplasmic reticulum (ER), permeation of substrates across ER membranes is necessary for its glucuronidation. The aim of the present study is to clarify the involvement of ER transporters in drug transport across the ER membranes and investigate the role in drug glucuronidation. Time‐dependent increase of bumetanide glucuronide formed was observed up to 30 min in mouse primary cultured hepatocytes. Glucuronidation of bumetanide was inhibited by anionic inhibitor nateglinide with IC50 value of 45.4 μM, while its glucuronidation in mouse liver microsomes was not inhibited. Moreover, uptake of bumetanide was inhibited by nateglinide with IC50 value of 365 μM. These results suggest active ER transport of bumetanide. Moreover, knockdown of OAT2 in HepaRG cells reduced bumetanide glucuronide without inhibition of bumetanide uptake into the cells. Immunohistochemical study revealed that mOat2 was localized in intracellular compartment in mouse liver, and that myc‐tagged OAT2 was localized in the ER in HEK293 cells. In the western blotting, a signal for mOat2 was abundantly detected in mouse liver microsome fraction. In conclusion, transporters inhibited by nateglinide are likely to participate in bumetanide glucuronidation after uptake process of bumetanide across plasma membranes in mouse primary cultured hepatocytes and HepaRG cells. OAT2 localized in ER may be involved in drug transport across the ER membranes and glucuronidation of various UGT substrates.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.