Lipid rafts establish calcium waves in hepatocytes - PubMed (original) (raw)

Lipid rafts establish calcium waves in hepatocytes

Jun Nagata et al. Gastroenterology. 2007 Jul.

Abstract

Background & aims: 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.

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Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1

The type II InsP3R is concentrated in the pericanalicular region of hepatocytes. Confocal immunofluorescence of rat liver shows that the type II InsP3 receptor (blue) colocalizes with submembraneous actin (red) rather than with the apical plasma membrane protein MRP2 (green) in rat hepatocytes.

Figure 2

The endoplasmic reticulum is distributed throughout the cell in isolated hepatocyte couplets. (A) Isolated rat hepatocyte couplet labeled with the ER membrane dye ER-Tracker visualized by 2-photon microscopy shows that the ER is distributed throughout the apical and basolateral region. (B) Confocal image of an isolated rat hepatocyte couplet loaded with the low-affinity calcium dye mag-fluo-4, which selectively labels ER Ca2+stores. This shows that ER Ca2+stores also are distributed throughout the apical and basolateral region.

Figure 3

m_β_CD depeletes cholesterol and redistributes caveolin in isolated rat hepatocyte couplets. Isolated rat hepatocyte couplets were double-labeled with the cholesterol dye filipin (green) and immunofluorescent staining of caveolin (red). Filipin was visualized by 2-photon microscopy while caveolin was visualized simultaneously by confocal microscopy. (A) Under control conditions filipin staining is present in a punctuate pattern throughout each hepatocyte, while caveolin is localized to the plasma membrane and is most concentrated along the canalicular membrane. (B) After treatment with m_β_CD there is little residual filipin staining, and caveolin staining redistributes along the entire plasma membrane. Resultsarerepresentativeof what was observed in 5 couplets under each condition. (C) Rat liver section double-labeled for caveolin-1 (green) and the actin stain phalloidin (red) examined by confocal immunofluorescence. Phalloidin staining is specific for filamentous actin, which outlines the plasma membrane in hepatocytes. The staining is most intense along the canalicular membrane, where actin is most concentrated. Areas of colocalization (yellow) in the merged image confirm that caveolin is most concentrated along the canalicular membrane of hepatocytes under normal conditions.

Figure 4

The type II InsP3R is in a cholesterol-dependent fraction of the ER. Membranes were collected from isolated rat hepatocytes, and then separated by density gradient into 10 fractions. (A) Distribution of types I and II InsP3R, along with calreticulin, MRP2, and the Na+/K+-ATPase. The type II InsP3R is in fractions 6–8 under control conditions but spreads into fractions 9–10 after cells have been treated with m_β_CD (5 mmol/L) for 30 minutes. The distribution of the type I InsP3R (fractions 7–10) is not affected by treatment with m_β_CD. Calreticulin is used to identify ER fractions, while MRP2 and Na+/K+-ATPase are markers for the apical and basolateral plasma membrane, respectively. (B) Distribution of type II InsP3R, along with SERCA 2b and caveolin-1. The type II InsP3R is in fractions 6–8 under control conditions but spreads into fractions 9–10 after cells have been treated with m_β_CD (5 mmol/L) for 30 minutes. SERCA 2b is used to identify ER fractions, while caveolin is a marker for plasma membrane lipid rafts. Note that caveolin is in (plasma membrane) fractions 3–4 under control conditions but spreads into fraction 5 after treatment with m_β_CD.

Figure 5

The type II InsP3R redistributes to the basolateral region after cholesterol depletion. Confocal fluorescence image of an isolated rat hepatocyte couplet labeled with antibody against type II InsP3R receptor (green), plus Alexa 647 phalloidin (red) to label actin, which is most concentrated along the apical membrane. The type II InsP3R is concentrated in the apical region of hepatocytes under control conditions. After treatment with m_β_CD, the InsP3R is more diffusely distributed throughout the couplet. The InsP3R receptor was labeled with the same antibody used for immunoblots. No InsP3R labeling was detected in cells stained with secondary antibody alone (not shown).

Figure 6

Quantification of the redistribution of the type II InsP3R. (A) Line scan analysis was performed to measure fluorescence intensity along the (red) line crossing each couplet (top panels). Fluorescence intensity from InsP3R staining (green tracing) and actin staining (red tracing) along the scan line is graphically depicted in the bottom panels. (B) Apical/basolateral fluorescence intensity ratio reveals that basolateral fluorescence labeling of type II InsP3R increases significantly relative to apical fluorescence after treatment with m_β_CD (*P < .0001; n = 15 couplets). Fluorescence ratios were used so that measurements would be independent of individual fluorescence intensity values, to account for variations in labeling among cells.

Figure 7

Cholesterol depletion slows Ca2+ waves in hepatocytes. (A) Serial confocal images of an isolated rat hepatocyte couplet loaded with the Ca2+ dye fluo-4 and then stimulated with vasopressin (10 nmol/L). Fluorescence was monitored in the apical (A) and basolateral (B) regions. Region of interest is outlined in yellow; serial images demonstrate that a Ca2+ wave begins in the apical region of the cell (white arrow in middle panel). This and subsequent Ca2+ images are pseudocolored according to the scale shown at bottom. (B) Graphical representation of the fluorescence increase detected in the apical and basolateral region. Note the time lag between the apical increase in Ca2+ and the basolateral increase; the Ca2+ wave speed is calculated by dividing the distance between the apical and basolateral reference points by the time lag. The rise time is the time required for the Ca2+ signal to increase from 20% to 80% of its maximum value. (C) The Ca2+ wave speed is slowed in couplets treated with m_β_CD (*P < 10−6, n = 15 couplets in each group). (D) The rise time is prolonged in hepatocytes treated with m_β_CD (*P < .01, n = 15).

Figure 8

Cholesterol depletion slows Ca2+ waves in pancreatic acinar cells. (A) Transmission image and serial confocal images of a pancreatic acinus loaded with fluo-4 and then stimulated with ACh (1 _μ_mol/L). Fluorescence was monitored in the apical (A) and basolateral (B) region of individual acinar cells. Region of interest in a particular cell is outlined in red; serial images demonstrate that a Ca2+ wave begins in the apical region of the cell. (B) Graphical representation of the fluorescence increase detected in the apical and basolateral region of a typical acinar cell. As in hepatocytes, the apical increase in Ca2+ precedes the basolateral increase, reflecting an apical-to-basal Ca2+ wave. (C) Pretreatment with m_β_CD significantly slows Ca2+ wave speed (*P < .001, n = 15). This effect reverses in acinar cells that have been reloaded with cholesterol (n = 8). (D) The rise time of Ca2+ signals increases in cells treated with m_β_CD (*P < .05, n = 15). This effect reverses in acinar cells that have been reloaded with cholesterol (n = 8).

Figure 9

Cholesterol depletion does not affect Ca2+ signals in SKHep1 cells. (A) Confocal immunofluorescence shows that the InsP3R does not redistribute in SKHep1 cells after treatment with m_β_CD. SKHep1 cells were used as a control of m_β_CD treatment because they are a nonpolarized liver cell line. Cells were triple labeled to reveal actin (red) and the type II (green) and type III (blue) InsP3R; these are the 2 InsP3R isoforms expressed in this cell line. (B) Serial confocal images of an SKHep1 cell loaded with fluo-4 and then stimulated with vasopressin (100 nmol/L). (C) The rise time of vasopressin-induced Ca2+ signals is not prolonged by treatment with m_β_CD (P = .52, n = 15).

References

1. Leite MF, Nathanson MH. Calcium signaling in the liver. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Schachter D, Shafritz DA, editors. The liver biology and pathobiology. 4. Philadelphia, PA: Lippincott Willliams & Wilkins; 2001. pp. 537–554.
1. Pusl T, Wu JJ, Zimmerman TL, Zhang L, Ehrlich BE, Berchtold MW, Hoek JB, Karpen SJ, Nathanson MH, Bennett AM. Epidermal growth factor-mediated activation of the ETS domain transcription factor Elk-1 requires nuclear calcium. J Biol Chem. 2002;277:27517–27527. - PubMed
1. Mendes CC, Gomes DA, Thompson M, Souto NC, Goes TS, Goes AM, Rodrigues MA, Gomez MV, Nathanson MH, Leite MF. The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria. J Biol Chem. 2005;280:40892–40900. - PubMed
1. Dolmetsch RE, Xu K, Lewis RS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature. 1998;392:933–936. - PubMed
1. Li W, Llopis J, Whitney M, Zlokarnik G, Tsien RY. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature. 1998;392:936–941. - PubMed

Lipid rafts establish calcium waves in hepatocytes - PubMed (original) (raw)