Second, InsP3R function was inhibited by EMD 1214063 nmr treating hepatocytes with the InsP3R inhibitor xestospongin C.32 This resulted in an 83% reduction in canalicular fluorescence

of CGamF relative to controls (Fig. 4A,B). Most InsP3Rs in hepatocytes are type II,21 so InsP3R2 expression was reduced by 70% (Fig. 5A) using an isoform-specific siRNA validated previously.22 This resulted in a 53% reduction in canalicular CGamF fluorescence relative to controls (Fig. 5C,D), similar to what was observed in matched preparations treated with BAPTA-AM (Fig. 5C,D). Interestingly, in InsP3R2-depleted cells there was a 40% decrease in Bsep expression (Fig. 5A). Finally, the importance of InsP3R2′s pericanalicular localization was examined. Cells were treated with mβCD to disrupt lipid rafts, which had no effect on the amount of InsP3R2 or Bsep that was expressed (Fig. 6A), but redistributed InsP3R2 and Bsep so that they were less concentrated in the canalicular region (Fig. 6B,C). This reduced canalicular CGamF fluorescence by 67% relative to controls, similar to what was observed in BAPTA-treated preparations (Fig. 6D,E). Together,

these findings provide evidence that Bsep activity is Ca2+-dependent, selleck chemicals and in particular depends on expression and pericanalicular localization of InsP3R2. In rats treated with either LPS or estrogen, InsP3R2 expression was significantly decreased (Fig. 7A,B). Moreover, InsP3R2 labeling in proximity to the canalicular membrane was decreased, and

InsP3R2 labeling was seen in a punctate pattern in the pericanalicular region (Fig. 7C). Quantification of InsP3R2 labeling confirmed that the receptor is distributed more diffusely Cyclin-dependent kinase 3 throughout the canalicular region in LPS- or estrogen-treated animals (Fig. 7D). Together, these findings raise the possibility that the mistargeting of canalicular transporters such as Bsep observed in canalicular cholestasis33, 34 may be related to decreased expression and/or mislocalization of InsP3R2. InsP3R2 is the major intracellular Ca2+ release channel in hepatocytes.16 Ca2+ release from pericanalicular InsP3R2 promotes the activity of Mrp2, in part by increasing Mrp2 insertion into the plasma membrane.22 The present study demonstrates that InsP3R2 also promotes the activity of Bsep. Pericanalicular Ca2+ signaling likely promotes Bsep activity by enhancing exocytic insertion, as with Mrp2. Vesicle fusion depends on a localized Ca2+ increase, which must be in the range of ∼10 μM for the form of exocytosis that governs transporter insertion.35 Apical clusters of InsP3R in other polarized cells are capable of producing such large amplitude, focal Ca2+ transients to regulate secretion.36 In Wif-B9 cells,37 Bsep constitutively recycles between a subapical endosomal pool and the canalicular membrane. Therefore, it would be predicted that Bsep would accumulate intracellularly and bile secretion would decrease without InsP3R2.