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CELLULAR AND MOLECULAR
Department of Pathology, Tsurumi University School of Dental Medicine, Yokohama, Japan (T.Ni., K.O., H.I., K.Mis., I.S.); Sjogren's Syndrome Project, Shinanomachi Research Park, Keio University, Tokyo, Japan (T.Ni., I.S.); Calcium Oscillation Project, International Cooperative Research Project, Japan Science and Technology Agency, Minato-ku, Tokyo, Japan (T.Na., K.Mik.); and Divisions of Molecular Neurobiology (N.M., K.Mik.) and Neuronal Network (M.M., T.M.,), Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Received September 1, 2006; accepted October 30, 2006.
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Abstract |
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-galactosidase staining in PAR-2KO mice, in which the -galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca2+ response were enhanced in mice lacking M3 or both M1 and M3 mAChRs, in which mAChR-stimulated secretion and Ca2+ response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M3-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca2+-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.
), belongs to a large superfamily of G protein-coupled receptors. PAR-2 is activated by proteolytic unmasking of the N-terminal extracellular tethered ligand that binds to the extracellular loop 2 of the receptor itself (Lerner et al., 1996; Al-Ani et al., 1999; Al-Ani and Hollenberg, 2003). Serine proteases, including trypsin and tryptase, are endogenous activators of PAR-2, and the cleavage mediated by these proteases generated a distinct N-terminal tethered ligand sequence (SLIGRL and SLIGKV for murine and human PAR-2, respectively) (Nystedt et al., 1994; Kawabata and Kuroda, 2000; Macfarlane et al., 2001). Synthetic peptides based on the receptor-activating sequence of the tethered ligand (SLIGRL and SLIGKV) are also capable of activating PAR-2 by direct binding to the receptor (Kawabata et al., 2000a,b, 2001, 2002b; Oshiro et al., 2002). Since its cloning, the expression and functional role of PAR-2 have been elucidated in various tissues (Kawabata et al., 2000a, 2001, 2002b; Macfarlane et al., 2001). Various physiological/pathophysiological roles of PAR-2 have been identified. PAR-2 seems to play a role in inflammation (Kawagoe et al., 2002), and it has also been shown that PAR-2 present in sensory neurons causes pain sensation and hyperalgesia (Kawabata et al., 2002a). Recent studies regarding the effect of PAR-2 activation on exocrine tissues have reported that agonist peptides for PAR-2 induce exocrine secretion in the salivary gland, the lacrimal gland, the gastrointestinal tract, and the pancreas (Kawabata et al., 2000a, 2001, 2002b). The results demonstrate a novel secretory mechanism in these exocrine tissues and, thus, suggest that PAR-2 is a possible molecular target for the treatment of exocrine gland dysfunctions.
Xerostomia is a condition caused by lack of saliva in the oral cavity, and the primary causes are medications, Sjogren's syndrome (SS), irradiation of the head and neck, and aging (Bivona, 1998). Living with dry mouth conditions can be a harrowing experience for the sufferer; therefore, stimulating salivary output is a clinical goal for the treatment of xerostomia. From a molecular basis, salivary secretion is mainly regulated by M3-mediated cholinergic stimulation in the salivary gland cells (Maeda et al., 1988; Nakamura et al., 2004).
In fact, pilocarpine and cevimeline, which both stimulate mAChRs, have been clinically used. However, some SS patients have autoantibodies against the mAChR (Bacman et al., 2001; Nagaraju et al., 2001). Furthermore IgG from SS patients and anti-M3 antibody reduced Ca2+ signaling in both human and mouse submandibular acinar cells (Bacman et al., 2001; Nagaraju et al., 2001; Dawson et al., 2006). In addition, side effects, such as sweating, flushing, and urinary frequency, are common in mAChR stimulants (Wiseman and Faulds, 1995). Furthermore, use of pilocarpine is contraindicated in patients with uncontrolled asthma, narrow-angle glaucoma, or acute iritis, and caution is advised with use in patients with cardiovascular disease (Wiseman and Faulds, 1995). Therefore, development of other types of drugs to stimulate salivary flow with different mechanisms would be of clinical relevance in terms of the proper choice of drugs suitable for individual patients.
As mentioned above, recent studies have shown that the PAR-2 agonist mediated the exocrine secretions, including secretion of mucus in rat stomach (Kawabata et al., 2001), N-acetylneuraminic acid in rat sublingual gland (Kawabata et al., 2000a), and pancreatic and salivary amylase in rats (Kawabata et al., 2002b). Salivary fluid secretion, also induced by PAR-2 activation in mice and rats, was not inhibited by antagonists for mAChR, 
-adrenergic, or -adrenergic receptors (Kawabata et al., 2000b), indicating that the PAR-2 agonist does not stimulate these secretion-related receptors known to date in the salivary glands. Investigation of PAR-2 expression by reverse transcription-polymerase chain reaction or immunohistochemistry showed that it was expressed in rat pancreas, submandibular gland, parotid gland, and sublingual gland (Kawabata et al., 2000a,b, 2002b). However, it has not been investigated whether salivary fluid secretion can be induced by direct stimulation of PAR-2 present in the salivary glands. Although PAR-2-mediated generation of Ca2+ signaling was reported in some cells (Oshiro et al., 2002; Kawabata et al., 2004b), no examination has been conducted in salivary gland cells.
The purpose of the present study is to characterize the salivary secretion stimulated by PAR-2 and to explore the [Ca2+]i increase using WT, PAR-2KO and mAChRKO mice (M1KO, M3KO, and M1/M3KO mice). Specifically, the following points were investigated in these mice: 1) amylase and protein concentrations in PAR-2-induced saliva; 2) the amount and time course of PAR-2-induced salivary secretion; 3) generation of intracellular Ca2+ signaling in response to PAR-2 activation in salivary gland acinar cells, and 4) distribution of PAR-2 in the salivary glands. The results indicate that PAR-2 mediates Ca2+-dependent fluid secretion in the salivary glands and is potentially useful as a molecular target for dry mouth treatment.
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Materials and Methods |
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Mutant Mice. Generation and characterization of mAChRKO mice (M1KO, M3KO, and M1/M3KO mice) have been described previously (Matsui et al., 2000; Ohno-Shosaku et al., 2003), and the mice used were maintained by backcrossing for at least 7 (M1KO), 10 (M3KO), or 3 (M1/M3KO) generations with C57BL/6J mice (CLEA Japan, Tokyo, Japan) that were used as WT mice. Generation and characterization of PAR-2KO mice have been described previously (Ferrell et al., 2003). A bacterial LacZ was inserted downstream of the PAR-2 promoter. PAR-2KO mice were maintained by backcrossing for eight generations with C57BL/6J mice (CLEA Japan) and then supplied by Kowa Co. Ltd. (Tokyo, Japan). -galactosidase transgenic (TG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). This mutant was made by a retroviral insertion into embryonic stem cells. LacZ was under an unknown endogenous promoter and expressed in most tissues of adult mouse (strain name: B6.129S7-Gt(ROSA)26Sor/J, stock number: 001292). The lights in the animal room were turned on between 7:00 AM and 7:00 PM. The mice were fed standard dry pellets, CA-1 (CLEA Japan), and water ad libitum. To improve the growth of M3KO and M1/M3KO mice, hydrated paste food was prepared by mixing the powder form of CA-1 (CLEA Japan) with twice the weight of sterilized tap water and was given to the litters from the age of 2 weeks until the age of 8 weeks.
Histologic Analysis. WT (n = 7), M1KO (n = 18), M3KO (n = 6), M1/M3KO (n = 6), and PAR-2KO (n = 24) mice were anesthetized with a mixture of 36 mg/kg ketamine (Sigma, St. Louis, MO) and 16 mg/kg xylazine (Sigma) and sacrificed. SMGs were removed and fixed with 4% phosphate-buffered formaldehyde and embedded in paraffin. The sections (4 µm) were prepared and stained with hematoxylin and eosin (HE) for histologic examination.
SMGs freshly isolated from PAR-2KO mice, anesthetized and sacrificed as described above, were embedded in Optimal Cutting temperature compound (OCT; Sakura Finetechnical, Tokyo, Japan) and frozen in liquid nitrogen. Cryostat sections (5 µm) were made, and the sections were stained for -galactosidase activity. The sections were then fixed in phosphate-buffered saline (pH 7.2) containing 0.25% glutaraldehyde for 10 min at 4°C and stained in 5 mM K4Fe (CN)6·3H2O, 5 mM K3Fe(CN)6, 0.2 mM MgCl2, and 1 mg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)/ml in phosphate-buffered saline (pH 7.2) overnight at 37°C. They were then rinsed in phosphate-buffered saline (pH 7.2) and stained with hematoxylin at room temperature for better visualization of the staining. The sections were examined by light microscopy, and pictures were taken with original magnification of 40x objective lens.
Measurement of Salivary Secretion. Salivary secretion was measured in vivo in 10 to 15-week-old male WT (n = 7), M1KO (n = 5), M3KO (n = 5), M1/M3KO (n = 5), and PAR-2KO (n = 5) mice. The mice were anesthetized i.p. with a mixture of 36 mg/kg ketamine and 16 mg/kg xylazine and then injected with either 18 mg/kg SLIGRL-OH (Peptide Institute Inc., Osaka, Japan) i.v., 5 mg/kg pilocarpine-HCl (sanpilo 1%; Santen Pharmaceutical Co. Ltd., Osaka, Japan) i.p., or 0.5 mg/kg isoproterenol (Sigma) i.p. The saliva secreted into the oral cavity during each 1-min period following an injection of either of the above stimulants was carefully collected using capillaries for 15 min after the injection (ringcaps; Hirschmann Laborgerate GmbH & Co. KG, Eberstadt, Germany).
Analysis of Amylase Activity and Total Protein Concentration in Secreted Saliva. The secreted saliva was collected for a period of 15 min after administration of the stimulant as described above and was subjected to amylase activity assay. The amount of amylase in the collected saliva was measured by the colorimetric method using Amylase Test Wako (Wako Pure Chemicals, Osaka, Japan). Total protein concentration in the secreted saliva was determined using bicinchoninic acid protein assay kit (Pierce, Rockford, IL).
SMG Cell Preparation. Three-to-four-month-old mice were used for the experiments. Each mouse was anesthetized with a mixture of 36 mg/kg ketamine and 16 mg/kg xylazine and sacrificed. The bilateral SMGs were immediately removed and placed in ice-cold balanced salt solution (BSS) containing 115 mM NaCl, 5.4 mM KCl, 2 mM Ca2+, 1 mM Mg2+, 20 mM HEPES, and 10 mM glucose, pH 7.4, supplemented with 1.25% bovine serum albumin (BSS-BSA), and rapidly minced. The material was then digested for 20 min at 37°C with 2 mg/ml collagenase type-2 (Worthington, Malvern, PA) in BSS-BSA. The suspension was gently passed through a pipette 20 times every 10 min. After digestion, the preparation was centrifuged at 70 g for 2 min and then the pellet was resuspended in 10 ml of BSS-BSA, rinsed twice, and filtered through a 100-µm nylon mesh (Cell Strainer 100 µm; BD Biosciences, Bedford, MA) to generate a batch of SMG cells.
Measurement of the Intracellular Ca2+ Concentration. The isolated SMG cell preparation was loaded with fura-2 by incubation for 1 h at room temperature with 3 µM fura-2/acetoxymethyl ester (Dojindo, Kumamoto, Japan) suspended in BSS-BSA, rinsed twice, resuspended in 4 ml of BSS-BSA, and stored at 4°C. Ratiometric measurement of fura-2 fluorescence was made using a spectrofluorometer (CAF-110; Jasco, Tokyo, Japan). A 500-µl sample of fura-2-loaded SMG cells was transferred to a glass cuvette and alternately illuminated with 340- and 380-nm excitation light; the resultant fluorescence (510 ± 10 nm) was collected at 25 Hz. SLIGRL-OH (10, 30, 100, and 300 µM) and 30 µM carbachol (CCh) were added directly to the cell suspension during fluorescence recordings. At the end of each experiment, the Rmax was determined by adding 0.2% Triton X-100 to the cuvette, and then the Rmin was determined by adding 10 mM EGTA; these values were then used to calculate the [Ca2+]i using Grynkiewicz's equation (Grynkiewicz et al., 1985). The fluorescence intensities excited by 340- and 380-nm wavelength light (F340 and F380, respectively) and the ratios (F340/F380) were digitized with 12-bit resolution and stored and displayed in a personal computer using the MacLab4/s system (AD Instruments-Japan, Tokyo, Japan).
For two-dimensional measurement of [Ca2+]i changes, a small aliquot (20–50 µl) of fura-2-loaded SMG cell suspension was dispersed on the Cell-Tak coated glass (BD Biosciences, Bedford, MA) that formed the bottom of the recording chamber and then mounted on the stage of an inverted fluorescence microscope (IX70; Olympus, Tokyo, Japan) and perfused with BSS at a rate of 2 ml/min at room temperature. Excitation of fura-2 was made every 5 s by an alternate illumination of 340- and 380-nm light, and the resultant fluorescence (510–550 nm; F340 and F380) was collected using an objective lens (UPlanApo 20x/340; Olympus) and silicon-intensified target camera (Hamamatsu Photonics, Hamamatsu, Japan), processed to obtain pseudo-colored images of F340/F380, and stored in a personal computer using the software ARGUS50/CA (Hamamatsu Photonics).
Chemicals. PAR-2 agonist peptide SLIGRL-OH and 2-furoylated-LIGRL-NH2 were prepared by standard solid-phase synthesis procedure. The concentration, purity, and composition of the peptides were determined by high-performance liquid chromatography, mass spectrometry, and quantitative amino acid analysis. Pilocarpine-HCl was purchased as sanpilo 1% from Santen Pharmaceutical Co. Ltd. Isoproterenol and CCh were purchased from Sigma.
Statistics. Data were calculated as the mean ± S.E.M. in the text and figures, and p values were determined using Dunnett's multiple comparisons (Figs. 2 and 4) or Scheffe's test (Fig. 3). p < 0.05 was considered significant.
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Results |
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), M3KO mice showed normal histology. Furthermore, no histologic abnormalities were found in the M1/M3KO or PAR-2KO mice (Fig. 1). In this context, deficiency of M1, M3, and PAR-2 are unlikely to be involved in the morphogenesis of the SMG.
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, 2004a), PAR-2 activation in the salivary gland has not been shown yet. Therefore, the fact that M3 plays a central role in the control of salivary secretion (Nakamura et al., 2004) raises a possibility that systemic administration of SLIGRL-OH may induce cholinergic activation via PAR-2, which is expressed ubiquitously (Macfarlane et al., 2001). To examine this possibility, salivary secretion was measured in vivo in WT, PAR-2KO, and mAChRKO mice (M1KO, M3KO, and M1/M3KO), among which M3 and M1/M3 KO mice had impairment of cholinergically stimulated Ca2+ signaling and salivary secretion. Figure 2, a through c, shows the saliva output for each 1-min period after stimulant injection, and Fig. 2, d through f, shows the cumulative amounts of saliva secreted in 15 min. When 5 mg/kg pilocarpine was injected i.p. into anesthetized mice, the cumulative amount of saliva secreted in 15 min was 4.2 ± 1.3 µl/g in M3KO and 0 µl/g in M1/M3KO mice, whereas considerable salivary secretion was induced in WT (13.3 ± 1.0 µl/g), M1KO (13.2 ± 0.6 µl/g), and PAR-2KO (13.3 ± 0.7 µl/g) mice.
Although SLIGRL-OH evoked salivary secretion equally in WT (1.6 ± 0.2 µl/g) and M1KO mice (2.1 ± 0.3 µl/g), no saliva was detected in the PAR-2KO mice, as reported previously (Kawabata et al., 2004a). However, it was notable that SLIGRL-OH-induced salivary secretion relative to body weight was significantly increased in M3KO (3.0 ± 0.3 µl/g) and M1/M3KO mice (3.4 ± 0.1 µl/g) compared with WT mice (Fig. 2e, p < 0.001). The body weights of mice used were 25.0 ± 0.7 g (WT), 22.8 ± 0.6 g (M1KO), 23.0 ± 1.6 g (M3KO), 22.3 ± 1.2 g (M1/M3KO), and 23.8 ± 0.8 g (PAR-2KO). Statistical difference was not detected in the body weights. PAR-2-activated salivary secretion lasted only for 5 min after administration (Fig. 2b), and the amount of secreted saliva by pilocarpine was eight times larger than that by SLIGRL-OH. The difference in route of administration (i.p. versus i.v.) and rapid degradation of SLIGRL-OH (Hollenberg et al., 1993; Kawabata et al., 2000b) may explain the transient time course of PAR-2-mediated salivary secretion. Actually, SLIGRL-OH induced salivary secretion only by i.v. administration without amastatin. Since Kawabata et al. (2004a) reported 2-furoylated-LIGRL-NH2 as potent and metabolically stable modified agonists, 2-furoylated-LIGRL-NH2 was used to analyze the effect of administration route. 2-Furoylated-LIGRL-NH2 (0.6 mg/kg) induced salivary secretion in WT mice (3.6 ± 0.3 µl/g i.v., 5.9 ± 0.1 µl/g i.p., 6.0 ± 0.5 µl/g s.c., or 1.7 ± 0.5 µl/g p.o.). These results demonstrated that potent and metabolically stable agonists induce larger salivary secretion and that 2-furoylated-LIGRL-NH2 may be degraded by oral administration.
Isoproterenol-induced salivary secretions were much less than pilocarpine-induced secretions: 1.6 ± 0.3 µl/g in WT, 1.7 ± 0.1 µl/g in M1KO, 1.0 ± 0.4 µl/g in M3KO, 1.3 ± 0.4 µl/g in M1/M3KO, and 1.8 ± 0.2 µl/g in PAR-2KO mice, which are consistent with the fact that -adrenergic, sympathetic stimulation evokes water secretion only slightly but mainly induces protein secretion after production of cyclic adenosine monophosphate (Slomiany et al., 1992).
The salivary response of mAChRKO mice caused by pilocarpine or isoproterenol administration concurs with a previous report (Nakamura et al., 2004). The results that the activation of PAR-2 caused salivary secretion in M1/M3KO mice strongly suggest that the secretion was not mediated by the activation of the cholinergic stimulation but caused by a direct activation of PAR-2.
Comparison of Saliva Composition Secreted in Response to SLIGRL-OH, Pilocarpine, and Isoproterenol. To characterize the secretory pathway activated by PAR-2, composition of the saliva secreted in response to SLIGRL-OH was compared with saliva secreted in response to pilocarpine or isoproterenol in WT mice. The activities of amylase in concentration (1684 ± 220 U/µl; Fig. 3a, n = 14) in the saliva secreted in response to SLIGRL-OH were closer to those secreted in response to pilocarpine (2443 ± 220 U/µl, n = 15) than those in the saliva secreted in response to isoproterenol, which showed much higher activity (17137 ± 887 U/µl, n = 11, p < 0.001, comparing isoproterenol to pilocarpine or SLIGRL-OH). In the point of total amylase activity secreted in 15 min, the activities of amylase (1697 ± 300 U/15 min g body weight) in saliva secreted in response to SLIGRL-OH were lower than those in response to pilocarpine (40813 ± 6070 U/15 min g body weight, p < 0.001, comparing pilocarpine to SLIGRL-OH) or isoproterenol (23876 ± 5180 U/15 min g body weight, p < 0.05, comparing isoproterenol to SLIGRL-OH). This high amylase activity in the saliva secreted in response to pilocarpine is dependent on the larger volume of saliva induced by pilocarpine.
There was no statistically significant difference between protein concentration in the saliva secreted in response to SLIGRL-OH (0.98 ± 0.1 mg/ml; Fig. 3b n = 17) and that in response to pilocarpine (0.94 ± 0.2 mg/ml, n = 10). The saliva secreted in response to isoproterenol demonstrated significantly high protein concentration (26.6 ± 2.5 mg/ml, n = 17, p < 0.01, comparing isoproterenol to pilocarpine or SLIGRL-OH). Regarding the mass of protein secretion in 15 min, total protein in saliva secreted in response to isoproterenol (47.0 ± 12.2 µg/15 min g body weight, p < 0.05 or p < 0.01, comparing isoproterenol to pilocarpine or SLIGRL-OH) was much higher than that in response to pilocarpine (18.4 ± 3.9 µg/15 min g body weight) or SLIGRL-OH (3.3 ± 1.1 µg/15 min g body weight). In summary, composition of SLIGRL-OH-induced saliva was similar to that of pilocarpine-induced saliva rather than that of -adrenergic stimulation (isoproterenol)-induced saliva, which contains abundant proteins (Slomiany et al., 1992). However, the ability of SLIGRL-OH to induce salivary secretion was lower than that of pilocarpine as shown in the total amylase activity.
Increase in the Intracellular Ca2+ Concentration. PAR-2-mediated Ca2+ signaling in some cells was demonstrated similar to other G protein-coupled receptors (Oshiro et al., 2002; Kawabata et al., 2004a). As shown above, activation of PAR-2, similarly to mAChRs, seems to lead to fluid secretion in the salivary glands but not to significant amylase secretion. Taken together with the fact that the mAChR-mediated secretion is Ca2+-dependent (Nakamura et al., 2004), we considered that PAR-2-induced salivary secretion is also mediated by Ca2+-dependent mechanism. Therefore, we examined the Ca2+ signaling induced by cholinergic stimulation and SLIGRL-OH in the SMG cells. The [Ca2+]i in enzymatically dispersed SMG cells from WT, M3KO, M1/M3KO, and PAR-2KO mice was measured ratiometrically using fura-2. Figure 4, a through d, shows typical [Ca2+]i increases in the SMG cells isolated from each specific genotype mouse in response to stimulation with SLIGRL-OH at 10, 30, 100, and 300 µM, and CCh, a nonselective cholinergic agonist, at 30 µM. Figure 4, e and f, shows the summarized results. CCh-induced [Ca2+]i increases were similar to those reported previously (Nakamura et al., 2004); CCh (30 µM)-induced [Ca2+]i increases were significantly reduced (8.3% WT mice, p < 0.001) in M3KO mice (32.4 ± 5.1 nM) compared with WT mice (325.4 ± 14.0 nM), and no [Ca2+]i increase was seen in M1/M3KO mice (1.0 ± 1.0 nM, p < 0.001). In PAR-2KO mice, CCh induced a large [Ca2+]i increase (224.6 ± 17.9 nM); however, the magnitude of the [Ca2+]i increase was significantly smaller than that in WT mice (p < 0.001). So far, we do not know the clear reason for this smaller response in PAR-2 KO SMG cells.
SLIGRL-OH elicited [Ca2+]i increase in a dose-dependent manner at concentrations of 10 to 300 µM in the SMG cells from M3KO, M1/M3KO and WT mice, whereas SLIGRL-OH did not induce any [Ca2+]i changes in the SMG cells from PAR-2KO mice. These results show that PAR-2 present in the SMG is responsible for the [Ca2+]i increase in response to SLIGRL-OH, regardless of the presence of mAChRs. These results are consistent with the in vivo salivary secretion (Fig. 2). SLIGRL-OH induced significantly larger [Ca2+]i increases in M3KO (48.6 ± 8.9 nM) and M1/M3KO (44.6 ± 3.9 nM) SMG cells, compared with those in WT (26.4 ± 4.7 nM) SMG cells at a concentration of 300 µM (statistical significance determined using Dunnett's multiple comparisons, p < 0.05, respectively), which may explain the enhanced salivary secretion by SLIGRL-OH in M3KO and M1/M3KO mice (Fig. 2).
PAR-2 Distribution in the SMG. We next monitored [Ca2+]i changes in the SMG acinar cells using a two-dimensional fluorescent digital videomicroscopy technique. Figure 5, a and b, shows the [Ca2+]i changes in response to 300 µM SLIGRL-OH and 30 µM CCh in individual acinar cell clusters, each containing a few tens of acinar cells prepared from WT (Fig. 5a) or PAR-2KO (Fig. 5b) mice. The two-dimensional analysis in the WT SMG revealed that stimulation with SLIGRL-OH caused [Ca2+]i increase in a punctate fashion in single acinar clusters. Moderate [Ca2+]i increase was induced in limited regions of the clusters (e.g., regions of interest 1–3 in Fig. 5a) in response to 300 µM SLIGRL-OH, whereas little or very slight [Ca2+]i increase was seen in some regions (e.g., regions of interest 4 and 5). SLIGRL-OH-induced [Ca2+]i increase was, overall, smaller in magnitude than CCh-induced [Ca2+]i increase in the WT SMG acinar cells and was not seen at all in PAR-2KO SMG acinar cells (Fig. 5b). By contrast, CCh at 30 µM induced a large [Ca2+]i increase in almost the entire region of the acinar clusters in both WT and PAR-2KO mice. These results suggest that the expression level of PAR-2 is not equalized among the acinar cells in the SMG but that a certain population of acinar cells expresses PAR-2 enough to cause [Ca2+]i signaling on activation.
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-galactosidase reporter, LacZ, under independent translational control; detection of enzyme activity in cryosections allows us to anticipate the pattern and level of transcription of the endogenous PAR-2 gene in tissues. Using this PAR-2KO mouse line, PAR-2 distribution in the SMG was examined by -galactosidase staining (Rose et al., 2001). -Galactosidase TG mice were used as positive controls under the same experimental condition to indicate the sensitivity of this staining in the PAR-2KO SMG. In contrast to the ubiquitous positive staining pattern for -galactosidase in the positive control SMG, a scattered positive staining pattern was obtained in the PAR-2KO SMG, indicating that PAR-2 is not ubiquitously expressed in the SMG (Fig. 5c). This is consistent with the result shown by Ca2+ imaging in WT mice (Fig. 5a).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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,b, 2002b), and there are two possibilities for the secretion mechanism. The first possibility is that PAR-2 in the SMG directly induces salivary secretion, and the second is that PAR-2 causes autonomic nervous system activation, which secondarily induces salivary secretion. In the rat gastrointestinal tract, SLIGRL-NH2 induced gastric mucus secretion that was mediated by calcitonin gene-related peptide receptor and neurokinin-2 receptor activation (Kawabata et al., 2001).
Therefore, in our present study, we used mAChRKO (M1KO, M3KO, and M1/M3KO) mice to investigate the involvement of the parasympathetic nervous system in PAR-2-mediated salivary secretion. The results in Fig. 2 showed that SLIGRL-OH administration induced salivary secretion in mAChRKO mice, including M1/M3KO mice, in which no salivary secretion was induced by parasympathetic activation (Nakamura et al., 2004), and this is further confirmed by the observation that IP3 production is required for the exocrine secretion (Futatsugi et al., 2005). Our result is consistent with the anecdotal report that atropine (7.2 µmol/kg) did not attenuate salivary secretion evoked by PAR-2-activating peptide (Kawabata et al., 2000b). In addition, SLIGRL-OH administration did not induce any salivary secretion in PAR-2KO mice, which is also consistent with a previous report (Kawabata et al., 2004a). Because there are some nonacetylcholine substances that activate the parasympathetic nervous system, such as substance P, calcitonin-gene-related peptide, and vasoactive intestinal peptide (Ekstrom, 1987), these nonacetylcholine substances might induce some salivary secretion, even in mAChRKO mice. However, they are not major salivary secretion activators (Ekstrom, 1987), and the PAR-2-induced salivary secretion was significant. Therefore, we consider that these results are sufficient to indicate that parasympathetic nervous system activity is not essential for PAR-2-mediated salivary secretion.
As shown in Fig. 3, we found that the composition of SLIGRL-OH-induced saliva was similar to that of mAChR activation-induced saliva; amylase activity in concentration and total protein concentration in secreted saliva induced by SLIGRL-OH or pilocarpine were much lower than those induced by isoproterenol, and protein concentration in SLIGRL-OH-induced saliva was comparable to that of pilocarpine-induced saliva. However the significant difference in total amylase secretion between SLIGRL-OH stimulation and pilocarpine stimulation demonstrates the higher activity of pilocarpine to induce salivary secretion and that the intracellular signaling of mAChR and PAR-2 is not completely the same. From these results, we consider that stimulation with SLIGRL-OH induces fluid secretion rather than protein secretion, which is mainly induced by -adrenergic stimulation (Slomiany et al., 1992), although some amount of amylase can be secreted as reported previously (Kawabata et al., 2000b, 2002b). The results also indicate that sympathetic nervous activity is not involved in PAR-2-mediated salivary secretion.
PAR-2 activation induces [Ca2+]i increase in guinea pig tracheal epithelial cells (Oshiro et al., 2002), rat longitudinal muscle cells (Mule et al., 2002), and some cell lines (Kawabata et al., 2004a) or activates phospholipase C in longitudinal muscle (Mule et al., 2002). Considering these studies, PAR-2, as with M1, M3 and M5, is likely to be coupled to Gq/11 and increase [Ca2+]i in salivary glands. Therefore, in the present study, we investigated [Ca2+]i changes induced by SLIGRL-OH using enzymatically dispersed SMG cells and found that SLIGRL-OH induced dose-dependent [Ca2+]i increase in the SMG acinar cells. The [Ca2+]i increase was mediated by PAR-2, because this [Ca2+]i increase was abolished in PAR-2KO acinar cells. Because [Ca2+]i increase in acinar cells triggers fluid secretion in salivary glands, the above results indicate that direct activation in the salivary glands to evoke [Ca2+]i increase in acinar cells causes PAR-2-mediated salivary secretion. As shown in a previous report (Nakamura et al., 2004), activation of mAChRs also induced [Ca2+]i increases in the SMG cells. Taken together with the composition of saliva, PAR-2 mediates salivary secretion by a Ca2+-dependent mechanism that is similar to mAChRs-mediated salivary secretion.
It is interesting that the total amount of SLIGRL-OH-induced saliva standardized by body weight in M1/M3KO mice was twice as large as that in WT mice. The small body size of mice lacking M3 receptors (Matsui et al., 2000) is unlikely to result in the apparent salivary increase, because significant difference was not detected between the body weights of the mice used, and isoproterenol-induced salivary secretion standardized by body weight was similar among all of the groups tested (Fig. 2f). Rather, it is likely that some compensatory mechanism involving PAR-2, but not the -adrenergic system, emerged in the salivary glands. Interestingly, we detected significant increase in the SLIGRL-OH-induced [Ca2+]i response at 300 µM between WT and M3KO mice or WT and M1/M3KO mice (Fig. 4e), showing the enhancement of PAR-2-mediated [Ca2+]i increase in the M3-deficient SMG. This enhanced [Ca2+]i increase presumably contributes to the increased salivary secretion. PAR-2 expression was analyzed using reverse transcription-polymerase chain reaction for the mechanism of this increased salivary secretion and [Ca2+]i increase; however, up-regulation of PAR-2 expression was not detected in M1/M3KO mice (data not shown). Some functional analysis may be needed to elucidate this mechanism. In PAR-2KO mice, a smaller [Ca2+]i increase was detected than that in WT mice with 30 µM CCh, whereas a similar volume of saliva was secreted in PAR-2KO and WT mice with 5 mg/kg pilocarpine. So far, we do not know the clear reason for this discrepancy in the in vitro and in vivo response; therefore, further study is needed regarding this problem. We demonstrated PAR-2 distribution in the submandibular glands using Ca2+ imaging in WT mice and -galactosidase staining in PAR-2KO mice in which the -galactosidase gene was inserted downstream of the PAR-2 promoter instead of the PAR-2 gene. Although it is reported that PAR-2 was expressed throughout the parotid acini and pancreatic acini in rats (Kawabata et al., 2002b), our results revealed that PAR-2 is expressed in a heterogeneously scattered fashion in the mouse SMG. This distribution pattern is similar to that of the M1-subtype and different from the ubiquitous expression of M3 in the SMG acinar cells (Nakamura et al., 2004). This is the first demonstration of the distribution pattern of PAR-2 in the salivary glands that presumably accounts for the lower productivity of saliva by PAR-2 stimulation than that by mAChR stimulation. Some ductal cells expressed PAR-2, as well as acinar cells, in the -galactosidase staining (Fig. 5c). Large ducts were removed in the SMG cell preparation for Ca2+ imaging; however, small ductal cells may have remained in the dispersed SMG, but those ductal cells can not be distinguished under microscopy. So far, the difference in the PAR-2 roles in acinar cells and ductal cells is still unclear.
The present study demonstrates that SLIGRL-OH directly activates PAR-2 in the salivary gland and induces [Ca2+]i increase and salivary secretion without mediation via M1 and M3 subtypes. However, it remains under question whether and how PAR-2 is activated in the salivary glands under physiological conditions. It was reported that nanomolar concentration of trypsin cleaves and activates PAR-2 and that pancreatic trypsin may be capable of activating PAR-2 in some tissues (Dery et al., 1998). In addition to pancreatic trypsin, other trypsin-like enzymes can activate PAR-2: trypsinogen-2 in endothelial cells (Koshikawa et al., 1997) and mast cell tryptase (Corvera et al., 1997; Molino et al., 1997). Coagulation factors VII and X (Camerer et al., 1996; Belting et al., 2004) are reported as candidates of PAR-2 activators. Trypsin-like esteroproteases that were previously reported to be present in the mouse SMG (Takuma and Kumegawa, 1981; Takuma et al., 1985) may be a PAR-2 activator candidate. Further studies are needed to understand the physiological activation and inhibition mechanism of PAR-2 in salivary secretion.
There are still some problems to be solved, such as the rapid decline of salivary secretion induction following administration of SLIGRL-OH, the small volume of salivary secretion (less than 25% than the secretion by pilocarpine), and possible side effects because of widespread distribution of PAR-2 in many tissues. However, potent and metabolically stable agonists can induce larger salivary secretion as described in our results and another report (Kawabata et al., 2004a), and facilitation of fluid secretion is important for relief of xerostomia from a clinical point of view, even if the induced secretion volume is small. Furthermore, activation of PAR-2 may be favorable especially for the treatment of SS patients whose salivary secretion is inhibited by antimuscarinic antibodies (Dawson et al., 2006). Therefore, our present data demonstrate PAR-2 to be a potential drug target for patients who suffer from exocrine dysfunction, particularly those resulting from deficiency of functional cholinergic activation.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PAR-2, protease-activated receptor-2; SMG, submandibular gland; WT, wild type; mAChRs, muscarinic acetylcholine receptors; PAR-2KO, PAR-2 knockout; KO, knockout; SS, Sjogren's syndrome; TG, transgenic mice; HE, hematoxylin and eosin; BSS, balanced salt solution; BSA, bovine serum albumin; CCh, carbachol.
Address correspondence to: Dr. Ichiro Saito, Department of Pathology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan. E-mail: saito-i{at}tsurumi-u.ac.jp
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