ARTICLE |
Correspondence to: Michel Claret, Signalisation Cellulaire et Calcium, INSERM U442, IFR-FR 46, Université Paris Sud, bât. 443, 91405 Orsay, France.
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Summary |
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In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8-PVA. The Gq/G
11, PLCß3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. G
q/G
11, PLCß3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of G
q/G
11 with F-actin. (J Histochem Cytochem 47:601616, 1999)
Key Words:
hepatocyte multiplets, fluorescent antagonist, V1a receptors, antibodies, Gq/G
11, cytoskeleton, fluorescence and confocal micoscopy, Western blotting
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Introduction |
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The hepatocyte is a highly polarized epithelial cell. Its plasma membrane consists of three morphologically and functionally different regions: the basal domain, which is in contact with the blood; the lateral domain, which segregates gap and tight junctions; and the canalicular domain, which is involved in the secretion of native bile (
The hepatocyte doublet system has been used to demonstrate that the intracellular Ca2+ wave begins in the pericanalicular (apical) region and propagates peripherally (
Here we investigated the subcellular localization of the signaling molecules involved in vasopressin-mediated Ca2+ signals in multiplets of rat hepatocyte. This cellular model was used because the morphological and functional properties typical of liver tissues are preserved. Thus, vasopressin-induced calcium oscillation had been observed both on intact perfused liver (q/
11 G-protein subunits (G
q/ G
11) and InsP3 receptor were associated with both the basolateral membrane and intracellular structures.
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Materials and Methods |
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Primary and Secondary Antibodies
The purified polyclonal antibody directed against the carboxy terminal decapeptide of Gq/G
11 has previously been described (
q/G
11 and inhibits vasopressin-stimulated PLC activity. Because the carboxy terminal decapeptide is common to
q and
11 G-protein subunits, the antibody recognizes both molecules. The purified polyclonal antibody directed against a 20-amino-acid C-terminal peptide from rat PLCß3 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The specific mouse monoclonal antibody directed against PIP2 (IgG2b subclass), has been described elsewhere (
-tubulin antibody and the Cy3-labeled goat anti-rabbit IgG were obtained from Amersham (Arlington Heights, IL). The polyclonal anti-actin antibody was purchased from Sigma (St Louis, MO). FITCanti-mouse IgG2b was supplied by Nordic Immunological Laboratories (Tilburg, The Netherlands), TRITCanti-mouse IgG and biotinylated anti-rabbit IgG were obtained from Jackson Immunoresearch Laboratories (West Grove, PA), and peroxidase-conjugated anti-rabbit and anti-mouse IgG were purchased from Sanofi Diagnostics Pasteur (Marnes-la-Coquette, France).
Properties of the Fluorescent Vasopressin Antagonist of the V1a Receptor
A fluorescent vasopressin antagonist, 4-HO-Ph(CH2)2-CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5-carboxytetramethylrhodamyl]-NH2 (Rhm8-PVA), which binds to the V1a vasopressin receptor, has been synthesized (
Preparation of Rat Hepatocytes
Single-cell and multicellular (doublets, triplets, quadruplets) hepatocyte systems were prepared by collagenase (Boehringer Mannheim; Meylan, France) perfusion of liver from female Wistar rats (Janvier; Le Genest-St-Isle, France) as previously described (
Cells (2.5 x 105 cells/ml) were plated on collagen-coated glass coverslips and incubated in Williams' medium E (Gibco) containing 10% fetal calf serum, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). They were incubated for 2 hr at 37C under an atmosphere containing 5% CO2 and 95% air.
Cells for treatment with cytochalasin D or colchicine were washed twice with MEM containing 5 mM NaHCO3, 20 mM Hepes, 1 g/liter glucose, pH 7.4, and were incubated for 1 hr at 37C in this medium with or without 2 µM cytochalasin D or 10 µM colchicine (Sigma).
Detection of Rhm8-PVA Binding to the V1a Vasopressin Receptor in Rat Hepatocytes
Cells were rinsed twice and incubated for 15 min at 4C in MEM with (autofluorescence and nonspecific binding) or without (autofluorescence and total binding) 5 µM nonfluorescent antagonist V4253 ([ß-mercapto-ß-ß-cyclopenta-methylenepropionyl1,O-Et-Tyr2, Val4,Arg8]-vasopressin) or 100 nM vasopressin (Sigma). The cells were then incubated for 30 min at the same temperature with 10 nM fluorescent antagonist, Rhm8-PVA, in MEM containing 1 mg/ml bovine serum albumin (BSA) (Miles Laboratories; Kankakee, IL). Cells were washed twice with MEM at 4C, fixed by incubation for 15 min with 4% formaldehyde (FA) (Merck; Darmstadt, Germany), and mounted in buffered glycerin (Sanofi Diagnostics Pasteur). In some experiments, fixed cells were further incubated with FITCphalloidin (Sigma) (1 µg/ml) for 15 min at room temperature (RT) to label F-actin. Cells were then observed either with an Axioskop epifluorescence photomicroscope (Carl Zeiss; Le Pecq, France) equipped with a sensitive 3CCD-cooled camera LH750RC3 (Lhesa; Cergy Pontoise, France) or with a confocal microscope (BIO-RAD MRC 600; Ivry-sur-Seine, France) equipped with an argonkrypton mixed gas laser. The excitation wavelengths were 546 nm for Rhm8-PVA and 490 nm for FITCphalloidin. Images were processed using Photomat, Photoshop (epifluorescent microscope), or BIO-RAD (confocal microscope) software.
Immunodetection of Gq/G
11, PLCß3, PIP2, the InsP3 Receptor, Tubulin and F-actin by Fluorescence and Confocal Microscopy
Cells were fixed in 4% FA in PBS for 15 min at RT and stored in 0.4% FA at 4C or were fixed in acetone at -20C for 5 min, then air-dried and stored desiccated at -20C. FA fixation was essential for labeling of PIP2 and Gq/G
11, whereas acetone fixation was better for PLCß3. Both techniques were suitable for the InsP3 receptor, tubulin, and F-actin.
One of three methods was used to permeabilize FA-fixed cells: (a) 0.1% Triton X-100 (Sigma) in PBS for 2 min; (b) acetone:PBS (1:1) for 5 min; or (c) freezing and thawing after incubation with 1 M sucrose for cryoprotection (essential for PIP2).
Cells were washed with PBS and nonspecific binding sites were blocked by incubation for 15 min with 50 mM NH4Cl, then for a further 15 min with PBS containing 0.5% ovalbumin (Sigma). Cells were incubated overnight at 4C with the specific primary antibodies [anti-Gq/G
11 1:100, anti-PLCß3 1:100, anti-PIP2 1:1000, anti-InsP3 receptor (InsP3R) 1:10 or anti-tubulin 1:400], then for 30 min to 1 hr with the appropriate secondary antibody (for details see figure legends). A second fixation step with 4% FA was generally performed after incubation with the secondary antibody to stabilize the immune complex. Cells were finally incubated for 20 min with 0.1% sodium borohydride (Sigma) in PBS to reduce autofluorescence.
Negative controls were carried out systematically by omitting the specific primary antibodies and, if possible, by incubating the primary antibodies with an excess (500- to 1000-fold molar excess) of the corresponding antigen before use.
The cells were incubated with fluorescent secondary antibodies or biotinylated secondary antibodies followed by FITCstreptavidin (Immunotech; Marseille, France). TRITC or FITCphalloidin (Sigma) was used at a concentration of 0.51 µg/ml for 515 min for detection of F-actin. This staining of F-actin made it easier to identify the bile canaliculus between adjacent hepatocytes.
Cells were mounted using the Slow Fade-Light antifade kit (Molecular Probes; Leiden, The Netherlands) or buffered glycerin. They were then observed and the images recorded with epifluorescence and confocal microscopes equipped with appropriate software, as described above.
Preparation of Subcellular Fractions of Rat Hepatocytes
Hepatocytes plated and incubated for 2 hr on collagen-coated coverslips were rinsed twice with 20 mM Hepes buffer, pH 7.4, containing 50 mM NaCl, 5.6 mM KCl, 1.2 mM NaH2PO4, 5.1 mM NaHCO3, and were collected by scraping. The following steps were all performed at 4C. The collected cells were suspended in homogenization buffer [10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (Sigma)], subjected to sonication four times for 10 sec each, and centrifuged at 100,000 x g for 1 hr. The pellet, containing total cell membranes (M), was resuspended in homogenization buffer. Cytosolic proteins (Cy) in the supernatant were precipitated overnight at -80C by adding 9 volumes of acetone and were collected by centrifugation at 21,000 x g for 6 min.
The cytoskeleton (CK) was prepared as described by
All fractions were stored at -80C and their protein content was determined by the Bio-Rad protein assay reagent using BSA as a standard.
Western Blotting of Gq/G
11, PLCß3, F-actin, and Tubulin
Proteins (1020 µg per lane) were resolved by SDS-PAGE (7.5% acrylamide for PLCß3 and 13% for actin, tubulin, and Gq/G
11) and transferred to nitrocellulose membranes. Blots were incubated for 1 hr at 37C in blocking medium (PBS containing 5% skimmed milk powder and 0.1% Tween-20). They were then incubated for 2 hr at RT with polyclonal anti-PLCß3 (1:200), anti-G
q/G
11 (1:500), anti-F-actin (1:200), or monoclonal anti-
-tubulin (1:1000) antibodies in the blocking medium. The blots were washed with PBS and incubated for 1 hr at RT with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:2000). Blots were washed with PBS and detected using the ECL kit (Amersham).
Fluorescence Intensity Profiles for Signaling Molecules Involved in the Vasopressin Response
Digitized confocal images of signaling molecules labeled with fluorescent probes were used to generate a radial line fluorescence intensity profile from the basal membrane to the nucleus using Matrox MAGIC software. One profile per cell was obtained for a total of more than 20 cells from two or three separate hepatocyte preparations. Fluorescence intensity profiles were normalized as a percentage of maximal fluorescence (arbitrary units) and were averaged to form a mean profile. For the X-axis, pixels were transformed into µm (1 pixel = 0.25 µm) and the cell membrane was positioned at point O. For double labeling (Gq/G
11F-actin, G
q/G
11tubulin, InsP3RF-actin), pseudocolor images were generated from digitized confocal images by Photoshop software using green for FITC and red for TRITC. Red (F-actin or tubulin) and green images (G
q/G
11 or InsP3R) from double labeling experiments were superimposed. Fluorescence intensity profiles from the basal membrane to the nucleus were obtained as described above.
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Results |
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Subcellular Distribution of the V1a Vasopressin Receptor as Detected by the Fluorescent Antagonist Rhm8-PVA
We used a new specific fluorescent vasopressin antagonist, Rhm8-PVA (
Rhm8-PVA bound to the surface of rat hepatocytes (Figure 1A). There was also significant labeling within the cell, probably due to the high autofluorescence of rat hepatocytes (
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Subcellular Immunodetection of q/
11 G-protein Subunits
The polyclonal anti-Gq/G
11 antibody used in this study was characterized in WRK1 cells (
q/G
11 from rat liver. This antibody has been shown to be suitable for immunocytochemistry experiments (
q/G
11 antibody (Table 1).
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Association of q/
11 G-protein Subunits with F-actin and Tubulin
Gq/G
11 is associated with the cellular cytoskeleton of many cell lines (
q/G
11F-actin and G
q/G
11tubulin) to investigate the effect of drugs known to disorganize the actin or tubulin networks on G
q/G
11 distribution in rat hepatocyte multiplets. Anti-G
q/G
11 antibodies, biotinylated anti-rabbit IgG, and FITCstreptavidin were used to detect G
q/G
11 (Figure 3B) and TRITCphalloidin to detect F-actin (Figure 3A). There was clear but partial co-localization of F-actin and G
q/G
11. Both were detected under the bile canaliculus and basolateral membranes. Intracellular labeling for F-actin and G
q/G
11 was less intense. Cytochalasin D treatment strongly affected the actin network in both the basolateral membrane and bile canaliculus domains (Figure 3C), with no consistent peripheral labeling. Separate aggregates were labeled for F-actin. Cytochalasin D treatment also disorganized G
q/G
11 labelings (Figure 3D). However, co-localization of these two molecules was preserved, their distributions being almost identical. The effect of cytochalasin D on F-actin and G
q/G
11 distribution was specific, with no change in tubulin labeling observed (not shown). The close association of G-proteins and actin filaments was confirmed by biochemical experiments (see Figure 5). In the total cell membrane fraction, G
q/G
11 and actin were detected by Western blotting. By contrast, only G
q was present with actin in the cytoskeleton fraction. G
11 was not detected even if we doubled the amount of proteins.
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Double labeling for tubulin and Gq/G
11 was performed in rat hepatocyte multiplets. The specific anti-
-tubulin antibody labeled the typical filamentous tubulin network, which was abundant in regions near the nucleus and around the bile canaliculi (Figure 4A). Sparse punctate labeling of the tubulin network, was detected associated with the basolateral membrane areas (Figure 4A), in contrast to the continuous labeling of G
q/G
11 proteins (Figure 4B). In contrast, tubulin and G
q/G
11 were both located under the bile canaliculus membrane (96% of 43 bile canaliculi observed). Prior treatment with colchicine (10 µM for 1 hr at 37C) completely disrupted the intracellular microtubule network and significantly reduced the labeling in the bile canalicular area (Figure 4C). This colchicine effect was specific because no change in the F-actin labeling was observed (not shown). The labeling of G
q/G
11 in the basolateral membrane and cytoplasm was not affected by colchicine treatment, but the treatment did reduce labeling around the bile canaliculus area (90% of bile canaliculi observed) (Figure 4D). On Western blots (Figure 5), G
q/G
11 and tubulin were detected in the total membrane and cytoskeleton fractions. In contrast, tubulin was abundant in the cytosolic fraction in which G
q/G
11 was not detected.
Subcellular Distribution of PLCß3
The antibody used to detect PLCß3 in rat hepatocytes was specific. On Western blots, it recognized only one band, with a molecular weight (152 kD) corresponding to that of PLCß3 (Figure 5). PLCß3 was present mostly in the cytosol and the total membrane fractions after cell fractionation. It was not detected in the cytoskeleton preparation. Immunofluorescence experiments showed similar results. There was heterogeneous labeling of vesicles in the cytoplasm, mainly under the basal membrane as shown by optical sections at a tangent to the cell surface (Figure 6A). To a lesser extent, labeling vesicles were also associated with the plasma membrane in equatorial sections (Figure 6B). The nuclear area was not specifically labeled, whereas an intense signal was obtained with an antibody directed against PLCß1 (UBI; Lake Placid, NY) in rat hepatocytes (not shown). All labeling was abolished if the primary antibody was omitted from the first incubation medium or if it was incubated before the experiment with an excess of the antigen used to produce it (Figure 6C). More than 100 cells were examined and no labeling was observed near the bile canaliculi (Table 1).
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Subcellular Distribution of Phosphatidyl Inositol 4,5-bisphosphate
We used the previously characterized specific antibody directed against PIP2 (
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Subcellular Distribution of the InsP3 Receptor
The polyclonal antibody used in this study was directed against the entire InsP3 receptor isoform 1 molecule purified from sheep cerebellum. This antibody recognized only one band of 260 kD in a plasma membrane-rich preparation of rat hepatocytes and was shown to be suitable for immunocytochemical experiments (
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Discussion |
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The cellular response to a given hormonal stimulus depends on many factors, the most important of which is the molecular equipment of the cell. Thus, for the hormonal G-protein-coupled receptors (GPCRs), the cell's specific patterns of response and regulation depend on the hormonal receptor subtype, the combination of heterotrimeric G-protein subunits (
The principal molecules involved in the generation of vasopressin-stimulated calcium waves were not evenly distributed within the cells (Table 1, Figure 9 and Figure 10). The V1a receptor detected with Rhm8-PVA was associated with the basolateral membrane and was absent from the bile canaliculi and cytoplasm. Phospholipase Cß1 interacts with the q/
11 G-protein (
q/
11 G-protein subunits (
q/G
11 and InsP3 receptors is more homogeneous. Both are associated with the plasma membrane and the cytoskeleton (
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Transducing molecules may also be rapidly redistributed within the hepatocyte, from the basolateral pole to the bile canaliculus, during hormonal stimulation. Such a redistribution has been described for V1a and V2 vasopressin receptors in A7r5 and A10 smooth muscle and LLC-PK1 cell lines (-subunits of G-proteins (
Previous studies have demonstrated the involvement of the cytoskeleton in the transduction mechanisms involving GPCRs. Drugs, such as cytochalasin D and colchicine, which disorganize actin or tubulin networks, inhibit second messenger production in a variety of cell types, including rat adrenal glomerulosa (i,
q,
11,
s, ß, or
12 have been found to be associated with actin and/or tubulin networks (
1 adrenergic, and P2Y purinergic receptors, are coupled to PLC via
q/
11 G-proteins (
q/
11 G-proteins are associated with the cytoskeleton, presumably particularly with actin filaments in rat hepatocytes because (a) cytochalasin D treatment disorganizes both F-actin and G
q/G
11 labeling, whereas colchicine does not (Figure 3), (b) Western blot analysis showed that G
q was associated with an actin-rich Triton X-100-insoluble cytoskeleton preparation (Figure 5), and (c) F-actin and G
q/G
11 double labeling gave similar profiles (Figure 11A) for both molecules, whereas double labeling with tubulin and G
q/G
11 did not (Figure 11B). These data are consistent with results obtained with WRK1 cells (
q in Sf9 cells (
s and G
i in rat cerebral cortex synaptic membranes (
q/G
11 were not identical, however (Figure 11A), so we cannot exclude the possibility that G
q/G
11 also interacts with tubulin or other intracellular filaments, as observed in rat adrenocortical cells (
q/G
11 with cytoskeleton fibers appears to be a general phenomenon but its functional significance is unknown. Actin filaments rapidly depolymerize and repolymerize on hormonal stimulation (
q/G
11. Remodeling of actin cytoskeleton networks may thus alter the generation of second messengers and regulate the physiological effects associated with GPCRs.
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Further evidence for the involvement of the cytoskeleton in hepatocyte signal transduction is provided by the following facts. (a) Both F-actin and V1a receptor-specific labeling are concentrated (Figure 10 and Figure 11A) and co-localized (results not shown) in the basolateral membrane. (b) InsP3 receptors are associated with cytoskeleton structures in the liver (
We also found a difference in the distribution of the two G-protein subunits studied. The plasma membrane contained both
q and
11 but in unequal proportions (73 ± 4 and 27 ± 4% respectively). The cytoskeleton fraction contained only the
q-subunit (Mr 42 kD). The
11 band was undetectable even if the amount of cytoskeleton proteins in the gel well was increased (Figure 5). This is one of the first examples of a cell preparation exhibiting only the
q G-protein subunit. However, the significance of this is unclear. It may reflect a preferential coupling of the V1a vasopressin receptor and the
q G-protein subunit in rat hepatocytes. Indeed, only the receptor-coupled G-protein subunit can be phosphorylated on hormonal stimulation, as previously observed in a transfected CHO cell line (
q or
11 G-proteins are required to test this hypothesis. Unfortunately, none of the available antibodies is suitable for immunocytochemical studies (unpublished results).
This study has determined the subcellular distribution of all the main transducing molecules involved in vasopressin signaling in the rat hepatocyte. Experiments are now in progress to study the translocation of signaling molecules from one compartment to another on hormonal stimulation, to improve our understanding of the hormonal signals in the liver.
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Acknowledgments |
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We would like to thank R. Leuillet for excellent hepatocyte preparations, D. Villette for technical assistance, Dr K. Fukami for his gift of the anti-PIP2 antibody, and J. Knight for help in editing the manuscript.
Received for publication September 14, 1998; accepted January 12, 1999.
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