Lipid rafts establish calcium waves in hepatocytes

Background & Aims: Polarity is critical for hepatocyte function. Ca2+ waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca2+ waves depends on lipid rafts. Methods: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (m beta CD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca2+ waves were examined by confocal microscopy. Results : Type H lnsP3Rs initially were localized to only some endoplasmic reticultun fractions in hepatocytes, but redistributed into all fractions in m beta CD-treated cells. This lnsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after m beta CD treatment. Vasopressin-induced Ca2+ signals began as apical-to-basal Ca2+ waves, and m beta CD slowed the wave speed and prolonged the rise time. M beta CD had a similar effect on Ca2+ waves in acinar cells but did not affect Ca2+ signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of m beta CD. Conclusions: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca2+ waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.