Lipid rafts establish calcium waves in hepatocytes.
OBJECTIVE
Polarity is critical for hepatocyte function. Ca(2+) 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 Ca(2+) 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 (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy.


RESULTS
Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD.


CONCLUSIONS
Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) 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.
Nagata
Jun
J
Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8019, USA.
Guerra
Mateus T
MT
Shugrue
Christine A
CA
Gomes
Dawidson A
DA
Nagata
Naoki
N
Nathanson
Michael H
MH
D000818:Animals; D001648:Bile Canaliculi / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D046790:Pancreas, Exocrine / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
D053496:Inositol 1,4,5-Trisphosphate Receptors; D014662:Vasoconstrictor Agents; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014667:Vasopressins; D002784:Cholesterol; D002118:Calcium