Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland

Yasuko Ishikawa,1 Zhenfang Yuan,1 Noriko Inoue,1 Mariusz T. Skowronski,2,3 Yoshiko Nakae,4 Masayuki Shono,5 Gota Cho,6 Masato Yasui,7,8 Peter Agre,7,8 and Søren Nielsen2,3

1Department of Medical Pharmacology, 4Department of Oral and Maxillofacial Anatomy, 5Support Center for Advanced Medical Sciences, and 6Dental Anesthesiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; 7Department of Biological Chemistry and 8Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 2Water and Salt Research Center and 3Institute of Anatomy, University of Aarhus, Aarhus, Denmark

Submitted 3 May 2005 ; accepted in final form 2 July 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M3 muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M3 mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca2+ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

translocation; aquaporin-5


AQUAPORINS (AQPs) form water channels that selectively transport water across the plasma membrane (19). Thirteen mammalian AQPs, AQP0–AQP12, have been identified (1, 27). AQP5, initially cloned from rat submandibular glands (32), is an apical membrane water channel that is distributed to epithelial cells in several secretory glands, such as salivary glands (10). Salivary fluid secretion is defective in transgenic mice lacking AQP5, indicating that AQP5 has an important role in fluid secretion (24).

The parotid glands are innervated by both sympathetic and parasympathetic nerves (2). The activation of M3 muscarinic acetylcholine receptors (mAChRs) and {alpha}1-adrenoceptors increases intracellular Ca2+ concentration ([Ca2+]i) and induces salivary fluid secretion (2). In vitro experiments using rat parotid slices demonstrated that ACh and epinephrine acting at M3 mAChRs and {alpha}1-adrenoceptors, respectively, induce a rapid increase in the AQP5 levels in the apical plasma membrane (APM) by increasing [Ca2+]i (14, 15). We previously investigated (16) the possible role of Ca2+-mediated intracellular signal transduction in the M3 mAChR agonist-induced increase in AQP5 levels in the APM and demonstrated that activation of endogenous nitric oxide synthase and protein kinase G in the cells is coupled with the increase in AQP5 levels in the APM. M3 mAChR agonist- or cGMP-induced increases in AQP5 levels in the parotid cells disappear with block of the increase in [Ca2+]i (16). These findings strongly suggest that intracellular trafficking of AQP5 is regulated in response to agonist-induced stimulation of M3 mAChRs. In vitro, SNI-2011 (cevimeline) induces long-lasting salivation with a persistent increase in AQP5 levels in the APM of parotid glands (12, 17, 26). These findings, however, have not been supported by in vivo experiments.

Several lines of evidence indicate that some AQPs are regulated by membrane trafficking in response to hormonal stimuli (8, 25, 29). Furthermore, exposure to hypertonic solution triggers a reversible translocation of AQP1 between the subplasmalemmal fraction and the cytosol in cardiac myocytes (30). In lung (31, 34) and cardiomyocytes (30), AQP1 is located in a particular subtype of lipid raft called the caveola, which is a glycosphingolipid-, cholesterol-, and caveolin-enriched microdomain. In keratinocytes, AQP3 is located in caveolae containing caveolin-1 (41). In addition to caveolins, the involvement of several other protein families (e.g., flotillins/reggies, stomatins) is implicated in structural and functional modifications of lipid rafts (7). The lipid rafts are involved in various cellular events, such as signaling transduction (38), membrane sorting and recycling (6, 36, 37), and cell polarization (35).

The aim of the present study was to directly visualize the distribution and translocation of AQP5 in rat parotid glands with immunohistochemistry. Multiphoton laser confocal microscopy revealed that AQP5 is located in intracellular sites with lipid rafts containing flotillin-2 and GM1 in the interlobular ducts of parotid glands under unstimulated conditions. These findings were further confirmed by detergent insolubility and buoyant density experiments. Activation of M3 mAChRs induced AQP5 translocation via a [Ca2+]i increase from the cytosol to the APM with lipid rafts, and then some of the AQP5 was moved to nonrafts in the APM of the interlobular ducts. In the first stage, saliva is formed initially in the lumen of the acinar cells (3). This saliva is called primary saliva and is plasmalike in concentration of Na+, Cl, and HCO3. In the second stage, the primary saliva is modified in ductal cells. The activation of parasympathetic nerves inhibited Na+ reabsorption and K+ secretion and stimulated HCO3 secretion across the ductal cells. Our findings suggest that the interlobular ducts and acini have an important role in saliva secretion.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
 RESULTS
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Preparation and incubation of rat parotid tissue. Male Wistar rats (8 wk old) were provided with laboratory chow (MF; Oriental Yeast, Tokyo, Japan) and water ad libitum and were maintained in a temperature-controlled environment (22 ± 2°C) with a 12:12-h light-dark cycle (lights on at 0600). All procedures were approved by the animal care committee of the University of Tokushima Graduate School. Rats were killed by a blow to the head, and the parotid glands were rapidly removed and transferred to ice-cold Krebs-Ringer-Tris (KRT) solution [in mM: 120 NaCl, 4.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, 16 Tris·HCl (pH 7.4), 5 glucose] that was aerated with O2 gas. Tissue slices (0.4 mm thick) were prepared from the parotid glands with a McIlwain tissue chopper (Mickle Laboratory Engineering, Guildford, UK) and equilibrated with the KRT solution for 20 min at 37°C with shaking, as described previously (16). The slices (wet wt 300 mg) were then incubated at 37°C in 10 ml of KRT solution in the presence or absence of cevimeline or other agents as indicated.

Immunohistochemistry. Cevimeline (5.0 mg/kg) or A-23187 (1.0 mg/kg) (Calbiochem, Nottingham, UK) was intravenously injected into the tail. At 0, 10, and 60 min after the injection, parotid glands were quickly removed from the rats, embedded in Jung tissue freezing medium (Leica Instruments, Heidelberg, Germany), and rapidly frozen with liquid nitrogen. Frozen sections (7 µm thick) were cut, mounted on poly-L-lysine-coated glass slides, and immediately fixed for 10 min with 5% neutral buffered formalin (40). The sections were permeabilized in prechilled (–20°C) methanol for 30 min. After being washed in PBS (pH 7.5), the sections were blocked in avidin and biotin solutions (Zymed Laboratories, South San Francisco, CA) and then in 1% membrane blocking agent (Amersham Biosciences, Little Chalfont, UK) for 1 h and double-stained as follows. The sections were incubated at 4°C overnight with primary antibodies: rabbit anti-AQP5 antibody (1:1,000 dilution) and goat anti-flotillin-2 antibody (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-AQP5 antibody (Santa Cruz Biotechnology) and rabbit anti-ganglioside GM1 antibody (1:1,000 dilution; Calbiochem-Novabiochem, Darmstadt, Germany). These antibodies were raised to COOH-terminal amino acid sequence rat and rabbit anti-AQP5 antibody was generated in response to a synthetic peptide (KGTYEPEEDWEDHREERKKTI) (32). The sections were followed by incubation for 1 h with biotinylated goat anti-rabbit antibody or horse anti-goat IgG antibody (Vector Laboratories, Burlingame, CA). The labeling was visualized with streptavidin-conjugated Alexa Fluor 488 or Alexa Fluor 568 (1:1,000 dilution; Molecular Probes, Leiden, The Netherlands).

Fluorescence images of sections excited at 488 nm and at 568 nm and excited simultaneously at both wavelengths were captured with a confocal laser scanning microscope (Leica TCS NT) equipped with an Ar-Kr laser and a x40 dry objective (Leica Plan Apochromat) (28).

Preparation of Triton X-100-insoluble and -soluble membrane domains. Tissues were suspended in cold lysis buffer containing (in mM) 10 KCl, 1.5 MgCl2, and 10 HEPES with 5 µM phenylmethylsulfonyl fluoride (pH 7.4) and were incubated on ice for 30 min. The tissues were homogenized with a Dounce homogenizer (50 strokes) on ice, and the lysate was centrifuged at 5,600 g for 10 min at 4°C to remove the nuclei and debris. The supernatant was centrifuged at 100,000 g for 1 h at 4°C (35, 39). The resulting pellet was collected as the membrane fraction as described in Smith et al. (39) and solubilized with (in mM) 25 Tris·HCl (pH 7.4), 150 NaCl, 5 EDTA (TNE solution) containing 1% Triton X-100 on ice for 30 min. Subsequently, soluble and insoluble fractions were obtained by centrifuging at 200,000 g for 30 min at 4°C. The soluble sample and insoluble fraction were suspended with solubilizing buffer (20) and analyzed by SDS-PAGE and Western blotting.

Gradient density floatation assay. The tissue homogenate was solubilized with TNE solution containing 1% Triton X-100 and protease inhibitors (chymostatin, leupeptin, antipain, and pepstatin A; final concentration 10 µg/ml each) for 30 min and brought to 45% sucrose (wt/vol) in a final volume of 1 ml and sequentially overlaid with 1.5 ml of 45%, 35%, and 5% sucrose (11). Gradients were centrifuged at 240,000 g for 18 h. Six fractions (0.75 ml) were collected from the top (fraction 1) to the bottom (fraction 6) of the gradients and immediately supplemented with protease inhibitors. The pellet was resuspended with a Dounce homogenizer in 1 ml of Tris·HCl buffer (pH 7.4) and designated as fraction 7.

The tissue homogenate described above was adjusted to 40% OptiPrep and overlaid with 1.5 ml of 30%, 20%, and 5% OptiPrep in TNE solution (21, 22). Gradients were centrifuged at 240,000 g for 18 h. Eight fractions were collected from the top of the gradient [designated fractions 1 (top)–8 (bottom)], and proteins were methanol-chloroform precipitated before separation on SDS-PAGE.

Immunoblot analysis. The APM fraction was treated with solubilizing buffer (20) and subjected to SDS-PAGE on a 12.5% gel. After SDS-PAGE, the separated proteins were electrophoretically transferred from the unstained gel to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech UK) with a Trans-Blot apparatus (Bio-Rad, Hercules, CA). The blots were probed with antibodies to AQP5 (1:1,500 dilution). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and electrochemiluminescence reagents (Amersham Pharmacia Biotech UK).

Immunoelectron microscopy. Tissue was prepared for electron microscopy by freeze substitution as described previously (9). Ultrathin Lowicryl HM20 sections were incubated overnight at 4°C with anti-AQP5 antibodies and visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles at 1:50 (GAR.EM10; BioCell Research Laboratories, Cardiff, UK). Sections stained with uranyl acetate and lead citrate were examined with a Philips CM100 or Philips Morgagni electron microscope (Philips, Eindhoven, The Netherlands). Semiquantitative assessment of immunogold labeling at the APM and the intracellular compartments from interlobular ducts of rat parotid glands from three different animals within each group was performed.

Statistical analysis. Data are presented as means ± SE and were analyzed for statistical significance with Student's t-test or analysis of variance at all time points. A P value of <0.05 was considered to be statistically significant.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Subcellular localization of AQP5 in interlobular ducts and acinar cells of rat parotid glands. We previously demonstrated that AQP5 is translocated from intracellular vesicle fractions to APM fractions in response to M3 mAChR (14) or {alpha}1-adrenoceptors (15) by biochemical methods. In the present study we used a histological approach to directly visualize the cellular distribution and translocation of AQP5 in rat parotid glands. Confocal microscopy revealed that the cellular distribution of AQP5 in the duct cells is different from that in the acinar cells (Fig. 1). In acinar cells, AQP5 was enriched in the APM under control conditions (Fig. 1B-1), as reported by Gresz et al. (9). In contrast, in interlobular duct cells under control conditions, AQP5 was localized in the intracellular structures as well as in the APM (Fig. 1A-1). Preadsorption of the primary antibody with the immunizing peptides blocked labeling of AQP5 (Fig. 1C).



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Fig. 1. Changes in confocal immunofluorescence microscope images of aquaporin (AQP)5 in interlobular duct cells and acinar cells of parotid glands of rat injected with cevimeline. Parotid glands were obtained from rats injected with physiological saline (A-1, B-1) and cevimeline (5.0 mg/kg; A-2, B-2, C-2, A-3, B-3). The parotid glands were removed and embedded 0 (A-1, B-1), 10 (A-2, B-2, C-2), or 60 (A-3, B-3) min after injection. The section was immunostained to detect AQP5 with Alexa Fluor 488 (green). Nuclei were stained with ethidium bromide. Confocal fluorescence images are shown for interlobular duct cells (A and C) and acinar cells (B). The section in C was incubated with an anti-AQP5 antibody that had been preadsorbed with immunizing peptides. Bars, 10 µm.

 
Effect of cevimeline on AQP5 trafficking in rat parotid glands. We previously found (12, 17), using in vitro approaches, that cevimeline induces translocation of AQP5 from intracellular vesicles to the APM, with a persistent increase in AQP5 levels in the APM of rat parotid glands. To confirm that cevimeline induces translocation of AQP5 in rat parotid glands, cevimeline was intravenously injected into the tail vein. Ten minutes after the injection, AQP5 immunofluorescence was associated predominantly with the APM domains of interlobular duct cells of rat parotid glands (Fig. 1, A-2 and C-2). The trafficking of AQP5 was more predominant in large-sized ducts (Fig. 1C-2) than in small-sized ducts (Fig. 1A-2). Conversely, 60 min after the injection there was diffuse AQP5 staining in the cell cytoplasm (Fig. 1A-3). In contrast, AQP5 immunolabeling in acinar cells was consistently associated with the APM domains regardless of the treatment (Fig. 1, B-2 and B-3).

Immunoelectron microscopy of AQP5 subcellular localization in interlobular ducts of rat parotid glands. To quantify the subcellular distribution of AQP5, we analyzed the interlobular duct cells in sections from control and cevimeline-treated rats with immunoelectron microscopy. An assessment of immunogold labeling at the APM and within the intracellular compartments was made on sections from three different animals from each group. Under control conditions, 89.7 ± 4.0% of the immunogold particles were associated with intracellular compartments; the remaining 10.3 ± 4.0% were associated with the APM (Fig. 2A). Thus, under control conditions, AQP5 was mainly localized in intracellular vesicular structures. After the administration of cevimeline, the percentage of gold particles in the APM rose to 70.4 ± 5.8% (Fig. 2B). The remaining 29.6 ± 5.8% localized intracellularly. Thus there was an increase in AQP5 immunolabeling in the APM in response to cevimeline treatment.



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Fig. 2. A: immunoelectron microscopy of AQP5 expression in interlobular duct cells of rat parotid glands treated for 10 min with saline. Labeling is mainly observed at the intracellular vesicles (arrows). Labeling is also observed in the apical plasma membrane (APM; arrows). Magnification: x2,000 (a), x16,000 (b, c). B: immunoelectron microscopy of AQP5 expression in interlobular duct cells of rat parotid glands 10 min after 10-min cevimeline administration. Compared with control tissues (A), changes are observed in subcellular localization of AQP5. Labeling is mainly associated with the APM (arrows). Labeling is also observed in vesicles (arrows). Magnification: x2,000 (a), x16,000 (b–d). L, lumen, N, nucleus, TJ, tight junction.

 
Association of AQP5 with lipid rafts. To characterize the intracellular vesicles where AQP5 was located in interlobular duct cells, we examined whether AQP5 is located in lipid rafts because lipid rafts are involved in sorting some apical resident proteins (4). Flotillin-2 and GM1 ganglioside are a lipid raft-associated integral membrane protein and glycosphingolipid, respectively (7). We used flotillin-2 and GM1 as raft markers in the duct cells of rat parotid glands. Under resting conditions, AQP5 (green) was colocalized with flotillin-2 (red) or flotillin-1 (data not shown) in the cytoplasm of the interlobular duct cells of rat parotid glands (Fig. 3A). Cevimeline induced translocation of AQP5 together with flotillin-2 or flotillin-1 toward the APM and was colocalized in the APM in the cells at 10 min after the injection (Fig. 3B). Sixty minutes after treatment, the labeling was mainly dispersed throughout the cell (Fig. 3C).



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Fig. 3. Changes in confocal immunofluorescence microscope images of tissue slices with AQP5 and flotillin-2 in interlobular duct cells of parotid glands of rats injected with cevimeline. Parotid glands were obtained from rats injected with physiological saline (A-1, A-2, A-3) and cevimeline (5.0 mg/kg; B-1, B-2, B-3, C-1, C-2, C-3). The parotid glands were rapidly fixed by immersion and then sectioned 0 (A-1, A-2, A-3), 10 (B-1, B-2, B-3), or 60 (C-1, C-2, C-3) min after injection. Immunostaining was then performed to detect AQP5 (green) and flotillin-2 (red). Bars, 10 µm.

 
AQP5 (green) was also colocalized with GM1 (red) in the cytoplasm in the cells under resting conditions (Fig. 4A). AQP5 translocated with GM1 to the APM 10 min after the injection of cevimeline (Fig. 4B) and then, conversely, 60 min after the injection with GM1 toward the cytoplasm diffusely in the cells (Fig. 4C). These results indicate that AQP5 is located in the lipid rafts and translocates with lipid rafts to the APM.



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Fig. 4. Changes in confocal immunofluorescence microscope images of tissue slices with AQP5 and GM1 in interlobular duct cells of parotid glands of rats injected with cevimeline. Parotid glands, obtained from rats as described in Fig. 1, were rapidly fixed by immersion and then sectioned. Immunostaining was then performed to detect AQP5 (green) and GM1 (red). Bars, 10 µm.

 
Effects of cevimeline on subcellular localization of AQP5 in rat parotid glands. We further examined the localization of AQP5 in lipid rafts by gradient density flotation. After separation on sucrose density gradients, AQP5 fractionated to fractions 2 and 3 (Fig. 5A). GM1 and flotillin-2 fractionated to the same fractions containing AQP5, further supporting the view that AQP5 is located in lipid rafts under resting conditions (Fig. 5, B and C).



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Fig. 5. Subcellular distribution of AQP5, GM1, and flotillin-2 in rat parotid glands. Rat parotid tissue homogenate solubilized with Tris-NaCl-EDTA (TNE) buffer containing 1% Triton X-100 and protease inhibitors was brought to 45% sucrose (wt/vol) in a final volume of 1 ml and subsequently overlaid with 1.5 ml of 45%, 35%, and 5% sucrose. The gradients were centrifuged at 240,000 g for 18 h. Fractions (0.75 ml) were collected from the top (fraction 1) to the bottom (fraction 6) of the gradients, and proteins were methanol-chloroform precipitated before separation on SDS-PAGE. The pellet (Ppt) was solubilized and designated as fraction 7. The separated proteins were transferred to nitrocellulose membranes, and then the blots were stained, using anti-AQP5 (A), anti-GM1 (B), and anti-flotillin-2 (C) antibodies. Immune complexes were detected with chemiluminescence reagents.

 
Lipid rafts float to the lighter density fractions in an OptiPrep discontinuous density gradient. After separation on OptiPrep gradients, the amount of AQP5 in the tissues under resting conditions was highest in fractions 1–6 (Fig. 6). On the other hand, the amount of AQP5 in the tissue incubated with cevimeline for 10 min was highest in fractions 4–8. These results further support the view that cevimeline induced translocation of AQP5 from lipid rafts to nonrafts in rat parotid glands.



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Fig. 6. Effect of cevimeline on the distribution of AQP5 in OptiPrep gradients of rat parotid glands. After incubation without (A) or with (B) cevimeline for 10 min, rat parotid tissue slices were homogenized and solubilized with TNE buffer (pH 7.4) containing 1% Triton X-100 and protease inhibitors. The lysates were adjusted to 40% OptiPrep and overlaid with 1.2 ml of 30%, 20%, and 5% OptiPrep in TNE buffer (pH 7.4). The gradients were centrifuged at 240,000 g for 18 h. Fractions (0.6 ml) were collected from the top (fraction 1) to the bottom (fraction 8). The pellet was solubilized and designated as fraction 9. Protein (5 µg) in each fraction was subjected to immunoblot analysis with antibodies to AQP5.

 
It is well established that lipid rafts are insoluble in 1% Triton X-100 at 4°C (7, 38); therefore, we next tested solubility in Triton X-100. After incubation of rat parotid tissue slices with or without 10 µM cevimeline for 10 min, the slices were treated with TNE solution containing 1% Triton X-100 for 30 min at 4°C. The Triton X-100-insoluble and -soluble fractions were then separated by centrifugation. Immunoblot analysis of these fractions revealed that, under unstimulated conditions, the major portion of AQP5 was present in the Triton X-100-insoluble fraction (Fig. 7A, lane 1). Conversely, after incubation with cevimeline, the amount of AQP5 decreased in the Triton X-100-insoluble fraction (Fig. 7A, lane 2) and increased in the Triton X-100-soluble fraction (Fig. 7A, lane 4). In contrast, cevimeline did not change the amount of GM1 and flotillin-2 in the Triton X-100-insoluble fraction (Fig. 7, B and C, lanes 1 and 2). These results suggest that cevimeline induced translocation of AQP5 from lipid rafts to nonrafts in rat parotid glands.



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Fig. 7. Effect of cevimeline on the amount of AQP5 in Triton X-100-soluble and -insoluble membrane domains. After incubation with or without cevimeline, parotid tissues were suspended in cold lysis buffer. The tissues were homogenized with a Dounce homogenizer (50 strokes) on ice, and the lysate was centrifuged at 5,600 g for 10 min at 4°C to remove the nuclei and debris. The supernatant was centrifuged at 100,000 g for 1 h at 4°C. The resulting pellet was solubilized with 1% Triton X-100-containing TNE buffer (pH 7.4) on ice for 30 min. Subsequently, soluble and insoluble fractions were obtained by centrifuging at 100,000 g for 1 h at 4°C. Each fraction was analyzed by SDS-PAGE and Western blotting with anti-AQP5 (A), anti-GM1 (B), and anti-flotillin-2 (C) antibodies.

 
Effects of [Ca2+]i on AQP5 levels in rafts. In parotid glands, the activation of M3 mAChRs generates inositol trisphosphate and diacylglycerol through the stimulation of Gq protein and phospholipase C. Inositol trisphosphate is then involved in the subsequent elevation of [Ca2+]i (2). Previous studies indicated that a rise in [Ca2+]i is involved in M3 mAChR agonist-induced increases in AQP5 levels in the APM in vitro (14, 16). To investigate the involvement of Ca2+ in the translocation of AQP5, the Ca2+ ionophore A-23187 was injected intravenously into the tail vein. Ten minutes after the injection, AQP5 fluorescence was predominantly associated with the APM of interlobular duct cells of rat parotid glands (Fig. 8D). Moreover, treatment of the tissues with 10 µM A-23187 decreased the amount of AQP5 in the Triton X-100-insoluble fraction (Fig. 8, A and B). Together, these results suggest that [Ca2+]i mediates the effects of cevimeline on the movement of AQP5 with the lipid rafts from the cell cytoplasm to the APM and subsequently from the lipid rafts to nonrafts within the APM.



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Fig. 8. Effect of A-23187 on the amount of AQP5 in Triton X-100-insoluble membrane domains. A: rat parotid tissue slices were incubated for 0, 1, 3, and 10 min at 37°C in the presence of 10 µM A-23187. The Triton X-100-insoluble fraction (10 µg protein) was then prepared and subjected to immunoblot analysis with antibody to AQP5. B: immunoblots similar to those shown in A were subjected to densitometric analysis, and the amount of AQP5 was expressed as a % of the value for cells incubated in the absence of ACh. Data are means ± SE of 3 independent experiments. ***P < 0.001 vs. value for control cells. C and D: parotid glands were obtained from rats injected with physiological saline (C) or A-23187 (1.0 mg/kg; D). Parotid glands were removed and embedded 10 min after injection. The sections were immunostained to detect AQP5 with Alexa Fluor 568 (red). Bars, 10 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Salivary secretion is rapidly controlled by autonomic nervous activity. The rate of fluid secretion is regulated by parasympathetic activity through M3 mAChRs as well as through {alpha}1-adrenoceptors (2). AQP5 is likely to be a target molecule for parasympathetic control of saliva production (14, 16). We previously demonstrated (12, 17) the translocation of AQP5 from intracellular vesicles to the APM in response to cevimeline, an M3 mAChR agonist, using the membrane fraction from rat parotid gland tissues. In the present study, we directly visualized the expression and translocation of AQP5 in interlobular duct cells as well as intercalated duct cells and acinar cells of rat parotid glands by confocal microscopy and immunoelectron microscopy. Under unstimulated conditions AQP5 was mainly localized in the intracellular structures of the interlobular duct cells (Figs. 13 and 5). In contrast, in acinar cells AQP5 was very abundant in the APM (Fig. 1), consistent with previous observations (9). Importantly, in the interlobular duct cells, activation of M3 mAChRs by cevimeline treatment induced translocation of AQP5 from intracellular structures to the APM (Figs. 13 and 5). This observation raises the possibility that regulation of duct cells represents an important mechanism for regulation of fluid secretion in salivary glands.

This study demonstrates that AQP5 is subjected to intracellular trafficking in duct cells in response to M3 mAChR agonist treatment. Thus AQP5 and several other AQPs are subjected to trafficking between the plasma membrane and intracellular vesicles in response to hormone or neurotransmitter stimulation; for example, vasopressin induces translocation of AQP2 in renal collecting duct cells (29), secretin induces translocation of AQP1 in rat cholangiocytes (25), and glucagons induce translocation of AQP8 in hepatocytes (8).

Our findings suggest that AQP5 is regulated by different mechanisms in acinar cells and in interlobular duct cells. Because AQP5 is already expressed in the APM (Fig. 1), AQP5 might be trafficked from lipid rafts to nonrafts on the APM and/or regulated by gating in the acinar cells. In contrast, AQP5 is regulated by trafficking from intracellular lipid rafts to the APM in the interlobular duct cells (Figs. 13 and 5). According to the classic two-stage hypothesis, a primary fluid containing a plasmalike electrolyte concentration is generated by the acinar cells, and the fluid is subsequently modified by solute reabsorption and secretion as it passes along the ducts, resulting in the final hypotonic solutions (3). This hypothesis therefore suggests that the principal site of water transport is likely to be the acini and that relatively little transepithelial water movement occurs in the ducts. On the other hand, several lines of evidence support an important role for the ductal system in fluid secretion in the pancreas (13, 18). Our present findings suggest that large fluid movement occurs in the interlobular ducts of the parotid glands as well as the pancreas. Detailed physiological studies are required to assess the role of the ductal system in saliva production.

In the present study, we demonstrated that AQP5 is present in the lipid rafts with immunohistochemistry, detergent insolubility, and buoyant density (floating) experiments. AQP5 colocalized with the lipid raft markers flotillin-2 and GM1 in the interlobular duct cells of unstimulated parotid glands of rats (Figs. 3 and 4). Lipid rafts are defined as glycosphingolipid- and cholesterol-enriched microdomains that are insoluble in cold Triton X-100 (7, 38) or that are detected in the high-buoyancy fraction when separating membranes of differing densities with discontinuous sucrose-density centrifugation (11, 38). Lipid rafts are proposed to function as domains that interact with apically designated sorting vesicles (4, 6, 7, 21). We demonstrated that AQP5 was located in the lipid rafts of rat parotid glands and translocated to the APM in response to cevimeline, although it was previously reported that AQP5 is not located in lung lipid rafts (31). Intracellular distributions of AQP5 might be controlled in a tissue-specific manner.

The translocation of AQP5 by cevimeline is mediated by the rise in [Ca2+]i because an M3 mAChR agonist does not induce an increase in the amount of AQP5 in the absence of Ca2+ (16). The importance of the increase in [Ca2+]i has been demonstrated for parasympathetic control of salivation (2). The downstream pathways, however, remain largely unknown. We propose the translocation of AQP5 to the APM as one of the final target events, at least in interlobular duct cells. Interestingly, type 2 inositol 1,4,5-trisphosphate receptors are localized in subapical sites of duct cells in submandibular glands (5, 23). The functional coupling of AQP5 translocation and the type 2 inositol 1,4,5-trisphosphate receptor-mediated [Ca2+]i increase requires further study. AQP5 dissociated from lipid rafts to nonrafts as it arrived at the APM via the increase in [Ca2+]i (Figs. 7 and 8). As lipid rafts are small and highly dynamic structures (33), further experiments are necessary to identify whether AQP5 plays a role in lipid rafts or nonrafts.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, The Nordic Centre for Research in Water Imbalance Related Disorders (WIRED), the Danish National Research Foundation, and Karen Elise Jensens Foundation. Cevimeline (SNI-2011) was kindly provided by the Daiichi Pharmaceutical Company (Tokyo, Japan).


    ACKNOWLEDGMENTS
 
We thank Dr. Hajime Ishida, Professor Emeritus at Tokushima University, for many insightful comments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Ishikawa, Dept. of Medical Pharmacology, Inst. of Health Biosciences, Univ. of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima, 770-8504, Japan (e-mail:isikawa{at}dent.tokushima-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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