However, we also observed a small but significant decrease in Bsep expression in the absence of InsP3R2. This may reflect that the half-life of Bsep in the intracellular compartment is shorter than

in the membrane. Evidence Dabrafenib for this comes from studies of rodent liver treated with estrogen, which induces rapid internalization of Bsep.33 In this model, total protein content of the transporter decreases without a change in mRNA levels, suggesting a posttranslational mechanism of Bsep down-regulation such as increased protein turnover.38 Our findings suggest not only that InsP3R2 promotes bile secretion but that canalicular cholestasis (in both estrogen and LPS models) is associated with loss of InsP3R2 expression. Bsep and Mrp2 are mistargeted in both of these models and overall expression of these proteins is reduced secondary to posttranslational mechanisms.33, 34, 38-41 Moreover, in the case of Bsep this defect is reversed by UDCA,40 which increases Ca2+,42 and stimulates exocytosis.19 Considering our current and previous Selleckchem FK228 findings,22 it may be that decreased expression and localization of canalicular transporters seen in canalicular cholestasis is secondary to loss of pericanalicular Ca2+ signaling. This may be analogous to what is observed in cholangiocytes, in which InsP3R3 rather than InsP3R2 is the predominant isoform.11 InsP3R3

expression is concentrated subapically in cholangiocytes and is lost in animal models of ductular cholestasis, as well as in patients with a variety of cholestatic disorders.27 This decreased InsP3R3 expression impairs polarized Ca2+ signaling27 and ductular bicarbonate secretion.43 Therefore, loss or redistribution of apical InsP3Rs may be a common basis for MCE impaired secretion in polarized epithelia. Cells in various tissues use raft-based platforms to spatially coordinate membrane proteins with the Ca2+ release machinery that regulates them.44 Rafts can promote physical

and functional interactions between ER channels and membrane proteins,45-48 and also can cluster Ca2+ signaling proteins, including PLC49 and PIP244; downstream effectors such as PKCα49 and store operated Ca2+ entry proteins,44 such as TRPC1 and Orai; and the plasma membrane Ca2+ ATPase.44 Thus, lipid rafts help generate large amplitude localized Ca2+ transients in specific membrane microdomains, and this may be ideal for regulating Bsep insertion. It is interesting to note that InsP3R2 labeling can be punctate, perhaps indicating focal densities of release channels coordinated with sites of exocytosis. The localized nature of Ca2+ signaling microdomains would also help explain the apparent inconsistency between the present results, which support a choleretic effect of Ca2+, and earlier studies, which suggest a cholestatic effect.