HEPATOLOGY 2010 Cytosolic

Ca2+ is a ubiquitous intracellular messenger.1 In the liver, Ca2+ signals have been linked to the regulation of such diverse functions PD0325901 cost as gene expression,2, 3 cell proliferation,4 apoptosis,5-7 paracellular permeability,8 glucose release,9, 10 and bile flow.11 Ca2+ signaling in hepatocytes is mainly controlled by the inositol 1,4,5-trisphosphate receptor (InsP3R), an InsP3-gated Ca2+ channel in the endoplasmic reticulum. InsP3Rs type I (InsP3R1) and type II (InsP3R2)12, 13 are the two InsP3R isoforms expressed in hepatocytes. InsP3R2 is the most highly expressed of these isoforms and is concentrated in the region of the endoplasmic reticulum near the canalicular membrane. Thus it is in close proximity to canalicular transporters,14 which selleck inhibitor are either inserted into the canalicular plasma membrane or localized to subplasmalemmal vesicles.15 Activation of InsP3Rs in the apical region of other epithelia generates a spatially restricted domain with Ca2+ concentrations as high as approximately 10 μM,16 which is sufficient to induce

vesicular exocytosis.17 Secretion of a wide variety of amphiphilic organic anions into bile, including bilirubin, glutathione S-conjugates, and oxidized glutathione, occurs through multidrug resistance-associated protein 2 (Mrp2), a member of the ATP binding cassette family of transporters.18 Mrp2 activity is partially regulated by its dynamic trafficking between the canalicular plasma membrane and a nearby endosomal compartment. Cholestatic agents such as estradiol,19 lipopolysaccharide,20 lithocholic acid,21 and phalloidin22 act in part by decreasing insertion of Mrp2 into the plasma membrane. Fusion of vesicles with the plasma membrane involves Ca2+-dependent proteins in nearly every cell type,23 and Ca2+ signals induced by tauroursodeoxycholic acid (TUDCA) have been linked to exocytosis in hepatocytes24, 25; yet the role of Ca2+ in regulating organic anion secretion is not known. Therefore, we investigated the role of InsP3R2-mediated Ca2+ release in the localization and activity of Mrp2. AM, fluo-4/acetoxymethyl ester; ANOVA,

analysis of variance; ATP, adenosine triphosphate; AVP, arg8-vasopressin; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; cAMP, cyclic adenosine monophosphate; CMFDA, 5-chloromethylfluorescein 上海皓元 diacetate; InsP3R, inositol 1,4,5-triphosphate receptor; GFP, green fluorescent protein; KO, knockout; Mrp2, multidrug resistance-associated protein 2; PKC, protein kinase C; TIRF, total internal reflection fluorescence; TUDCA, tauroursodeoxycholic acid; WT, wild-type; TLCA, taurolithocholic acid. Male Swiss black wild-type (WT) mice obtained from Taconic Farms Inc (Hudson, NY) and InsP3R2 knockout (KO) mice described previously26 were used for experiments. Animals were maintained on a standard diet and housed under a 12-hour light/dark cycle. All animal procedures were approved by the Yale University Institutional Animal Care and Use Committee.