3). The two kinetic parameters used to characterize these differences were the amplitude of the Ca2+ signal and the rise time, which is the time required EPZ-6438 cell line for the signal to increase from 25% to 75% of the maximum response (Fig. 3A). The amplitude of the Ca2+ signal was significantly reduced in InsP3R2 KO cells stimulated with either AVP (Fig. 3B) or ATP (Fig. 3C). The rise time for both agonists was significantly prolonged in InsP3R2 KO hepatocytes compared with WT cells (Fig. 3D, E). These differences demonstrate impaired InsP3-mediated Ca2+ signaling in InsP3R2

KO hepatocytes, similar to what has been reported in rat hepatocytes in which InsP3R2 expression was reduced by antisense.12 To further characterize the effects of InsP3R2 expression on liver function, bile secretion and serum bilirubin concentration were examined in WT and InsP3R2 KO mice. Age-matched and weight-matched animals were used to measured basal bile flow

in both groups of mice. The average liver weight in WT mice was greater than in InsP3R2 KO mice (1.483 g ± 0.0502, n = 9, and 1.234 g ± 0.0437, n = 6, respectively; P < 0.005). However, there was no significant difference in bile flow between AG14699 the two groups (0.9486 μL/g liver/minute ± 0.0618, n = 9; and 1.116 μL/g liver/minute ± 0.030, n= 6, respectively). There also was no significant difference in the total bilirubin concentration in the serum of WT and InsP3R2 KO animals (0.2170 mg/dL ± 0.01813, n = 10; and 0.2096 mg/dL ± 0.02917, n = 9, respectively). Because translocation of Mrp2 to the MCE公司 plasma membrane is stimulated by protein kinase C alpha (PKCα) and this kinase is Ca2+-activated, we hypothesized that organic anion secretion might be impaired in InsP3R2 KO animals. To test this, we first measured the secretion of cell tracker green CMFDA, a fluorescent Mrp2 substrate,36 by hepatocytes in collagen sandwich cultures using time-lapse confocal microscopy. This cell system was used because it preserves structural and functional polarity of hepatocytes.37, 38 CMFDA

accumulated in the canalicular lumen of hepatocytes (Fig. 4A), and the kinetics could be quantified as a measure of Mrp2 function (Fig. 4B). Canalicular accumulation of CMFDA fluorescence was observed in both WT and InsP3R2 KO hepatocytes, but luminal fluorescence in InsP3R2 KO hepatocytes reached only 25.8% of what was observed in WT cells (Fig. 4C). Luminal fluorescence was reduced by a similar extent in WT cells treated with the cytosolic Ca2+ chelator BAPTA/AM (50 μM; Fig. 4C). To understand the significance of this finding in vivo, bile flow was measured in WT and InsP3R2 KO livers infused with the cholestatic bile acid TLCA (5 uM) with or without co-infusion of taurolithocholic acid (TUDCA) (25 μM). This model was chosen because TUDCA stimulates biliary exocytosis in a Ca2+-dependent fashion24, 25 and reverses the cholestatic effect of TLCA.39 As shown in Fig.