Intracellular calcium stores and inositol 1,4,5-trisphosphate receptor in rat liver cells

Abstract

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.

The type 3 inositol 1,4,5-trisphosphate receptor is concentrated at the tight junction level in polarized MDCK cells

“…Finally, the chosen cells must be easy to transfect, because one of our long term aims, is to perform in vivo dynamic studies, using cells stably expressing different versions of GFP tagged InsP3R. Despite our previous work in liver cells study (Decaens and Cassio, 2001;Lièvremont et al, 1996) and the fact that the restricted InsP3R localization in MDCK cells seems to differ from that in other epithelial cells (lateral versus subapical), we have chosen to first investigate this non-tumorigenic cell line (i.e.MDCK) because it readily fullfills all the criteria and is by far the best known polarized cell model. In the present work, the distribution of the InsP3R type 3 (InsP3R-3), the most abundant InsP3R expressed in MDCK cells, was followed during MDCK polarization and also when the polarity was disturbed and restored.…”

Distribution of Signaling Molecules Involved in Vasopressin-induced Ca2+ Mobilization in Rat Hepatocyte Multiplets

“…In contrast, the distribution of G␣q/G␣11 and InsP 3 receptors is more homogeneous. Both are associated with the plasma membrane and the cytoskeleton (Bourguignon et al 1993;Feng and Kraus-Friedmann 1993;Kraus-Friedmann 1994;Ibarrondo et al 1995;Lièvremont et al 1996;Côté et al 1997b) or vesicles called caveoles (Fujimoto et al 1992). Nathanson et al (1994a) demonstrated that calcium waves induced by vasopressin originate from the apical pole (bile canaliculus) and propagate to the basolateral domain.…”

“…The subcellular distribution of the transducing molecules may also be important. For example, aquaporin-3 (AQP3) is constitutively expressed at the basolateral membrane of renal collecting duct cells (Ecelbarger et al transduction pathway (Fukami et al 1988;Tran et al 1993;Ibarrondo et al 1995;Lièvremont et al 1996), and accurate confocal microscopy techniques to determine the distribution of vasopressin-linked signaling molecules in rat hepatocyte multicellular systems.…”

“…Therefore, this antibody was suitable for immunocytochemical experiments. It labeled the basolateral and canalicular domains of hepatocytes on rat liver cryostat sections (Lièvremont et al 1996). The monoclonal anti-␣-tubulin antibody and the Cy3-labeled goat anti-rabbit IgG were obtained from Amersham (Arlington Heights, IL).…”

Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+

“…The antibodies against the 14 C-terminal residues of InsP $ R1 have been described elsewhere [35,37]. A synthetic peptide (FLGSNTPHENHHMPPH) corresponding to the 16 C-terminal residues (2686-2701) of InsP $ R2 was prepared by Covalab (Oullins, France).…”