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
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ABSTRACT |
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translocation; aquaporin-5
The parotid glands are innervated by both sympathetic and parasympathetic nerves (2). The activation of M3 muscarinic acetylcholine receptors (mAChRs) and 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
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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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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DISCUSSION |
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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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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