Immunoblots of lysates from hepatocytes in culture revealed that, as in liver,16 InsP3R1 and InsP3R2 are expressed, whereas InsP3R-3 is absent (Fig. 1A). In addition, as in liver,16 InsP3R1 is expressed diffusely throughout the hepatocyte, whereas InsP3R2 is concentrated in the canalicular region (Fig. 1B). Both Bsep29 and InsP3R216 are expressed mainly in the canalicular region, so confocal immunofluorescence microscopy was used to compare their localization directly. Available Bsep29 and InsP3R230 antibodies are both generated in the rabbit, so each

protein was colocalized with multidrug resistance protein 2 (Mrp2), which resides in and immediately beneath the canalicular membrane.22 In rat liver Bsep and InsP3R2 were both in proximity to Mrp2 at the canalicular membrane (Fig. 2A), suggesting they are in proximity to each other as well. Similarly, PLX3397 manufacturer Bsep and InsP3R2 are expressed in close proximity in the canalicular region of cultured rat hepatocytes (Fig. 2B). Therefore, the expression and localization of InsP3R isoforms is preserved in rat hepatocytes in collagen sandwich click here culture, similar to what has been observed in mouse hepatocytes.22 These findings provide support for using this cell culture model to study the functional relationship between InsP3R2-mediated Ca2+ release and Bsep activity in rat liver. Despite the proximity of InsP3R2 and

Bsep, these proteins could not be coimmunoprecipitated in lysates from rat liver or rat hepatocytes in culture, even after treatment with crosslinking agents (not shown). This suggests that InsP3R2 and Bsep are in proximity but do not physically associate, consistent with

the observation that InsP3R2 is in a specialized region of ER beneath the canalicular membrane,23 whereas Bsep is located instead within subapical vesicles and the canalicular membrane itself.31 A confocal microscopy-based assay that monitors CGamF accumulation in the canalicular space was see more developed to detect the kinetics of bile salt secretion in hepatocytes in collagen sandwich culture. CGamF fluorescence was measured in untreated or scrambled siRNA-treated hepatocyte controls and in cells treated with siRNA to knockdown Bsep expression. Bsep siRNA reduced Bsep expression in hepatocytes by 75%, whereas scrambled siRNA had a negligible effect (Fig. 3A). Canalicular CGamF fluorescence was reduced by 80% in hepatocytes treated with Bsep siRNA, relative to hepatocytes treated with scrambled siRNA or untreated hepatocytes (Fig. 3B,C). These results demonstrate the specificity of canalicular CGamF secretion for Bsep activity. Several experiments were performed to determine the role of InsP3R2 in modulating Bsep activity. First, canalicular fluorescence was measured after treatment with the cytosolic Ca2+ buffer BAPTA-AM (Fig. 4A,B).