539 Inositol 1,4,5-Trisphosphate Receptor Type 2 Modulates Bsep Activity in Hepatocytes

Abstract

Hepatic bile salt secretion is mediated primarily by the bile salt export pump (BSEP), an ABC transporter on the canalicular membrane of the hepatocyte. Defects in bile salt secretion lead to the profound consequences of intrahepatic cholestasis, sometimes culminating in liver failure. Studies on the short term regulation of BSEP have implicated cAMP and other signaling pathways, but have yielded contradictory results regarding calcium. In order to clarify the role of calcium in BSEP regulation, we examined pericanalicular calcium signaling mediated by the inositol 1,4,5-trisphosphate receptor type 2 (InsP3R2), which is the principal calcium release channel in hepatocytes and is concentrated in the peri-canalicular domain. BSEP and InsP3R2 expression and function were examined in rat liver and in rat hepatocytes in collagen sandwich culture. Using confocal immunofluorescence microscopy, InsP3R2 and BSEP were both found in the region of the canalicular membrane. BSEP activity was examined bymonitoring the kinetics of secretion of CGamF, a fluorescent BSEP substrate, into the sealed canalicular vacuoles of hepatocytes in sandwich culture. CGamF secretion was significantly inhibited by either (1) treating hepatocytes with the calcium chelator BAPTA for 30 minutes; (2) inducing redistribution of InsP3R2 away from the canalicular region by depleting the plasma membrane of cholesterol with 5 mM methyl-beta-cyclodextrin for 30 minutes; (3) knocking down InsP3R2 expression using a specific siRNA; or (4) knocking down BSEP expression using BSEP-specific siRNA as a positive control for the secretion assay. Moreover, knockdown of InsP3R2 significantly decreased bile salt secretion without altering BSEP expression or localization, as confirmed by immunofluorescence microscopy. In separate studies, basal bile salt secretion was reduced in livers from InsP3R2 KO mice, despite maintenance of BSEP localization and expression, as assessed by immunofluorescence microscopy and western blot, respectively. These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion via post-translational regulation of BSEP.We postulate this regulationmay involvemodulating insertion of BSEP into the canalicular membrane .