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Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands
Life Sciences 80 (2007) 842 – 851 www.elsevier.com/locate/lifescie
Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands Lucila Busch a,⁎, Enri Borda a,b a
Pharmacology Unit, School of Dentistry, University of Buenos Aires, Marcelo T. de Alvear 2142 (C1122AAH), Buenos Aires, Argentina b National Scientific and Technical Research Council (CONICET), Rivadavia 1917 (C1033AAJ) Buenos Aires, Argentina Received 25 August 2006; accepted 3 November 2006
Abstract We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE2 levels and a positive (r = 0.8870) and significant (p = 0.0077) correlation between PGE2 production and mucin released was determined. The participation of PGE2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M1 and M4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M1 and M4 mAChR subtypes induces an increase of intracellular Ca2+ concentration ([Ca2+]I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE2 released induces cAMP accumulation which, together with PKC are involved in the PGE2 increased Ca2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production. © 2006 Elsevier Inc. All rights reserved. Keywords: Submandibular gland; Mucin; Pilocarpine
Introduction The saliva is a complex secretion originating from the parotid, submandibular, sublingual and so-called minor glands in the oral cavity. The major functions of saliva include lubrication of the oral mucosal surfaces, protection against microorganism and neutralization of acids produced by bacterial plaque. The defense factors of saliva include both immune and non-immune systems as well as a broad range of cytokines and growth factors (Slomiany et al., 1989). Mucins are predominant among the components of salivary non-immune defense system. These large, highly glycosylated
⁎ Corresponding author. Cátedra de Farmacología, Facultad de Odontología, Universidad de Buenos Aires, Marcelo T. de Alvear 2142 4to “B”, (C1122AAH) Buenos Aires, Argentina. Fax: +54 11 4963 2767. E-mail address: [email protected] (L. Busch). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.11.010
proteins play a major role in the maintenance of viscoelastic properties of saliva, participate in the formation of the protective coat of the oral mucosa and tooth enamel pellicle (Slomiany et al., 1989). Mucins promote also bacterial aggregation and clearance from the oral cavity (Tabak, 1990). Oral diseases, including periodontal infections, can thus occur when such defense mechanisms are impaired. Microorganisms, such as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, may evade these protective mechanisms and can act as etiologic agents of periodontitis (Slots and Listgarten, 1988; Van Winkelhoff and De Graaff, 1991). Salivary components that interact with A. actinomycetemcomitans, include fractions of SIgA, lactoferrin and the low molecular weight mucin species MG2 (Alugupalli et al., 1995; Groenink et al., 1996). These proteins might therefore participate in the salivary defense against this bacterium. In submandibular glands mucin secretion is regulated by both the adrenergic and the muscarinic cholinergic systems.
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Mucin released by stimulation of β-adrenoceptors is independent of extracellular Ca 2+ (Taylor and Mc Whorter, 1991) while the stimulation caused by activation of cholinergic receptors is dependent on extracellular Ca 2+ (Dohi et al., 1991). Mucins are synthesized in the Golgi apparatus and stored in intracellular granules, which are transported to the luminal surface of the cell (Forstner and Forstner, 1994). These mucin granules finally discharge their contents to the lumen of the duct in an exocytotic fashion. The first event in exocytosis is the fusion of the granule with the plasma membrane at the fusion pore, an event mediated by exocytosisrelated proteins (Südhof, 1995) whose activities are regulated by intracellular Ca2+ concentrations ([Ca2+ ]I), protein kinase A (PKA), protein kinase C (PKC) and G protein activities, and ATP levels (Padfield and Panesar, 1997; Williams et al., 1997). In antral mucus cells, acetylcholine (Ach) increases [Ca2+ ]I by promoting Ca2+ release from intracellular stores followed by the store-operated Ca2+ entry. The increase in [Ca2+ ]I results in increased exocytosis and also in the stimulation of cyclooxygenase-1 (COX-1) activity, which in its turn results in prostaglandin E2 (PGE2) synthesis and release from cells. The released PGE2 acts on EP4 receptors and further potentiates Ca 2+ -regulated exocytosis in a cAMP-mediated process (Shimamoto et al., 2005). Apart from its protective role in oral mucosal surfaces, salivary mucin serves also as a lubricant and protective factor against refluxed gastric acid and pepsin within the esophageal lumen (Sarosiek et al., 1994). PGE2 is known to act as a local protective mediator of the gastric mucosa from acid-peptic injury probably through its participation in mucin release (Ohnishi et al., 2001). The goal of the present study was thus to study the signal transduction pathways activated after cholinergic stimulation of mucin release in submandibular gland
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Table 1 Effect of indomethacin and acetyl salicylic acid (ASA), on pilocarpine-induced mucin release in submandibular gland slices Experiment
Mucin release (% of total)
% inhibition #
Control Indomethacin (5 × 10− 7 M) Indomethacin (1 × 10− 6 M) Indomethacin (5 × 10− 6 M) Control ASA (5 × 10− 5 M) ASA (1 × 10− 4 M) ASA (5 × 10− 4 M)
8.9 ± 0.7 7.7 ± 0.6 6.6 ± 0.5⁎ 5.3 ± 0.3⁎⁎ 8.6 ± 0.8 7.5 ± 0.6 6.4 ± 0.4 5 ± 0.4⁎⁎
0 27.3 47.7 81.8 0 26.8 53.6 87.8
Submandibular gland slices were preincubated in the absence or the presence of the indicated concentrations of indomethacin or ASA, further incubated with 10− 6 M pilocarpine and mucin produced was determined as described in Methods. Results are the mean ± SEM of four independent experiments. ⁎ p b 0.05; ⁎⁎ p b 0.01 versus pilocarpine alone. #: extracting basal values: 4.5.
and to examine the participation of PGE2 in the secretory process. Methods Drugs Pilocarpine, carbachol, atropine, pirenzepine, staurosporine, N G-methyl-L-Arginine (L-NMMA), thapsigargin, verapamil, trifluperazine, A 23187, indomethacin, acetyl-salicylic acid and PGE2 are from Sigma Chemical Company (St. Louis, MO, U.S.A.). U-73122, SQ 22536 and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were purchased from Research Biochemicals International (Natick, MA, U.S.A.). AF-DX 116 and tropicamide were supplied by Tocris Cookson Inc (Ellisville, MO, U.S.A.).
Fig. 1. Effect of carbachol and pilocarpine on mucin release in submandibular glands. Submandibular slices were incubated with the indicated concentrations of carbachol (A) or pilocarpine (B) alone (●) and in the presence of 5 × 10− 6 M atropine (○), Results are expressed as percentage release from total mucin content of the gland. Each point represents the mean ± SEM of four independent experiments.
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Fig. 2. Effect of indomethacin and of PGE2 on pilocarpine-induced mucin release from submandibular glands. Slices were incubated with the indicated concentrations of pilocarpine or preincubated with 5×10− 6 M indomethacin or indomethacin+ 10− 8 M PGE2 and further treated with pilocarpine. Results are expressed as percentage release from total mucin content of the gland. Each point represents the mean±SEM of four experiments. ⁎pb 0.05; ⁎⁎pb 0.01 versus pilocarpine alone.
Animals Male Wistar rats weighing 250–300 g were used throughout the study. Animals had free access to food and water until the night before experiments when food, but not water was withdrawn. For surgical removal of submandibular glands, the animals were sacrificed using ether. Animal care was provided according to “The Guide to the Care and Use of Experimental Animals” (DHEW Publication, NIH 80-23). Measurement of mucin secretion Extirpated submandibular glands were detached from free connective tissue and fat and sliced into pieces approximately
2–3 mm thick and 15 mg wet weight with a razor blade. Gland slices were incubated for 30 min in 500 μl of Krebs Ringer bicarbonate medium (KRB), pH 7.4 bubbled with 5% CO2 in O2 at 37 °C. When used, inhibitors were included from the beginning of the incubation and stimuli were added in the last 15 min of the incubation time. Placing the tubes on ice then stopped the reaction. Slices were homogenized in sodium acetate buffer 25 mM Cl2Mg, pH 5.8, supplemented with protease inhibitors (0.1 mM phenylmethyl-sulfonyl fluoride, 1 mM sodium ethylenediaminetetra-acetate and 1 mM iodoaceamide) at 4 °C and centrifuged at 900 ×g for 15 min. Mucin was determined in the supernatants (total mucin content in the gland) and in the incubation medium (mucin released) using the Alcian Blue method described by Hall et al. (1980) and modified by Sarosiek et al. (1994). Briefly, aliquots of diluted supernatants (1:100) or medium (1:10) were incubated for 30 min in a 1% solution of Alcian Blue in 50 mM sodium acetate buffer 25 mM MgCl2, pH 5.8 under constant agitation at room temperature. Following incubation, the samples were centrifuged for 20 min at 3000 rpm pellets washed in 95% ethanol, vortexed gently for 10 s and after 5 min, centrifuged for 20 min at 3000 rpm. Mucin–dye complexes were dissociated by the addition of a 1:2 dilution of Aerosol OT in distilled water, brief mixing and sonication. Subsequently, samples were extracted with equal volumes of ethyl ether under vigorous shaking. The resulting solution was centrifuged for 15 min at 3000 rpm and the dye concentration determined spectrophotometrically at 605 nm in the aqueous layer. Mucin released is expressed as percentage released from total mucin content in the gland (% of total). Determination of prostaglandin production Submandibular gland slices (55 mg) were incubated for 30 min in 160 μl of KRB solution, pH 7.4, gassed with 5% CO2 in O2 at 37 °C. When used, inhibitors were added at the beginning of the incubation and pilocarpine in the last 15 min.
Fig. 3. Effect of pilocarpine on PGE2 production in submandibular slices. Submandibular gland slices were incubated in the absence (▴) or the presence (●) of increasing concentrations of pilocarpine, homogenized as described in Methods and the amount of produced PGE2 determined by ELISA. (A) Data are expressed as pg/mg wet weight and represent the mean ± SEM of four independent experiments. (B) Correlation analysis between PGE2 production and mucin release. Data are derived from Figs. 1B and 3A, and mucin release was plotted as a function of the PGE2 production. r value = 0.8870, p = 0.0077.
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After homogenization all procedures employed were those indicated in the protocol of Prostaglandin E2 Biotrak Enzyme Immuno Assay (ELISA) System (Amersham Biosciences, Piscataway, NJ, U.S.A.). Results are expressed as picogram PGE2/mg of tissue wet weight (pg/mg wet wt).
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Cyclic AMP was then determined using the EIA Kit (Cayman Chemical Company, Ann Arbor, MI, U.S.A.) according to the instruction provided by the supplier. Results are expressed as picomol cAMP/mg of tissue wet weight (pmol/mg wet wt). Determination of nitric oxide synthase activity (NOS)
Determination of cyclic AMP levels Submandibular slices (50 mg) were incubated for 30 min in 500 μl of KRB medium, pH 7.4, with 5% CO2 in O2 at 37 °C in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX). Antagonists were present from the beginning while pilocarpine was present during the final 15 min of incubation. After incubation, the tissue was homogenized in 400 μl of 5% trichloroacetic acid (TCA) and centrifuged for 10 min at 1500 ×g at 4 °C. Following centrifugation, the supernatant was transferred to a clean tube and TCA was extracted from the samples using water-saturated ether.
NOS activity was measured in submandibular glands using L[U-14C] arginine as substrate as described by Bredt and Snyder (1990). Briefly, 50–60 mg of submandibular slices were incubated for 30 min with 0.4 μCi L-[U-14C] arginine (Amersham Pharmacia Biotech, Buckinghamshire, England, about 300 mCi/mmol) in 500 μl of KRB solution pH 7.4 gassed with 5% CO2 in O2 at 37 °C. The tissue was then homogenized, centrifuged for 10 min at 10,000 ×g and [14C]citrulline formed in the supernatants was separated by ion exchange chromatography on AG 50 W resin (Biorad). When used, inhibitors were added at the beginning of the
Fig. 4. Effect of various selective muscarinic receptor antagonist on pilocarpine-induced mucin release. Submandibular gland slices were incubated with the indicated concentrations of pilocarpine in the absence (●) or the presence of varying concentrations of pirenzepine (A), AF-DX 116 (B), 4-DAMP (C) and tropicamide (D) and the amount of mucin produced was determined as described in Methods. Results are expressed as percentage release from total mucin content of the gland. Each point represents the mean ± SEM of four independent experiments.
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incubation and pilocarpine was present in the last 15 min. Nitric oxide production (measured as pmol of [14C]citrulline) is expressed as picomol [14C]citrulline/g of tissue wet weight (pmol/g wet wt). Statistical analysis Statistical significance of differences was determined by analysis of variance (ANOVA) followed by Tukey's test. Differences between means were considered significant at p b 0.05. Correlation and fitting dose–response curves were done using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego CA, USA). Results First, we studied the effect of cholinergic stimulation on mucin release in submandibular glands. Stimulation of the glands with pilocarpine and carbachol resulted in a dose-dependent increase in mucin production as shown in Fig. 1. The secretory ability of both cholinergic agonists showed identical EC50, (1.2 × 10− 6 M) and nearly equal maximal effect. Atropine (5 × 10− 6 M) antagonized the effect of both pilocarpine and carbachol and consequently produced a right shift of the dose–response curves as seen in panels A and B of Fig. 1. Finally, in the absence of stimulation, submandibular glands released 4.5 ± 0.5% of total mucin content during the 30 min of incubation (Fig. 1). Next, we analyzed the effect of varying concentrations of the cyclooxygenase (COX) inhibitors indomethacin and acetyl salicylic acid (ASA). Table 1 shows that both drugs inhibited pilocarpine-induced mucin release in a dose-dependent manner, with an approximately 80 and 90% maximal inhibition for indomethacin and ASA respectively. The effect of indometh-
acin on mucin release was partially abrogated in the presence of 10− 8 M PGE2, a concentration of PGE2 that was ineffective in inducing mucin release per se (Fig. 2 and data not shown). On the other hand, pilocarpine produced a dose-dependent increase of PGE2 production in rat submandibular gland as evidenced in Fig. 3A. A significant correlation was observed between PGE2 production and mucin release by the submandibular glands (Fig. 3B). In order to determine the subtype of muscarinic acetylcholine receptor (mAChR) responsible for mucin secretion pilocarpineinduced mucin release was assayed in the presence of pirenzepine, AF-DX 116, 4-DAMP and tropicamide (M1, M2, M3 and M4 receptor antagonists respectively). Of the compounds employed, the M1, and M4 antagonists pirenzepine and tropicamide blunted the effect of pilocarpine thus producing a right shift of the dose–response curve (Fig. 4A and D). The potencies of pirenzepine and tropicamide, expressed as pA2 after Schild plots, were 6.1 ± 0.5 and 5.7 ± 0.4 for pirenzepine and tropicamide respectively. Surprisingly, the M3 mAChR subtype selective antagonist 4-DAMP, failed to inhibit pilocarpine-induced mucin release (Fig. 4C) even at concentrations up to 10− 5 M (data not shown). The M2 mAChR subtype selective antagonist, AF-DX 116, failed also to inhibit the effect of pilocarpine on mucin release (Fig. 4B). The participation of different second messengers in the signal transduction pathway triggered after Ach receptor activation was then explored by measuring the effect of selective inhibitors on pilocarpine-induced mucin release. Inhibition of adenylate cyclase (AC) activity by SQ 22536 (5 × 10− 6 M) produced a significant decrease of mucin release while inhibition of phospholipase C (PLC) activity by U-73122 (5 × 10− 6 M) had no effect on mucin release (Fig. 5A) thus
Fig. 5. Effect of U-73122, SQ 22536, verapamil, thapsigargin, trifluoperazine, staurosporine, and of L-NMMA on pilocarpine-induced mucin release in submandibular glands. Submandibular slices were preincubated with 5 × 10− 6 M U-73122, 5 × 10− 6 M SQ 22536, 5 × 10− 6 M verapamil 5 × 10− 6 M thapsigargin, 5 × 10− 6 M trifluoperazine, 5 × 10− 9 M staurosporine or 5 × 10− 5 M L-NMMA, further incubated with 10− 6 M pilocarpine and mucin released was determined as described in Methods. Data are expressed as percentage release from total mucin content of the gland, and bars represent the mean ± SEM of four independent experiments. Basal values and the effect of 10− 6 M pilocarpine are also shown. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 versus pilocarpine alone.
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Table 2 Effect of verapamil, indomethacin and L-NMMA on pilocarpine-stimulated PGE2 and cAMP production and NOS activity in submandibular glands Group
PGE2 (pg/mg cAMP (pmol/mg NOS activity wet wt) wet wt) (pmol/g wet wt)
Basal Pilocarpine (10− 6 M) Pilocarpine + verapamil (5 × 10− 6 M) Pilocarpine + indo (5 × 10− 6 M) Pilocarpine + L-NMMA (5 × 10− 5 M)
38 ± 2.2⁎⁎⁎ 74 ± 5.1 38 ± 2.6⁎⁎⁎
0.35 ± 0.04⁎⁎⁎ 2.62 ± 0.17 0.98 ± 0.10⁎⁎⁎
391 ± 20⁎⁎ 586 ± 45 600 ± 48
43 ± 4.4⁎⁎⁎
1.33 ± 0.11⁎⁎⁎
572 ± 36
73 ± 4.3
2.54 ± 0.21
187 ± 12⁎⁎⁎
Submandibular gland slices were preincubated in the absence or the presence of the indicated concentrations of verapamil, indomethacin or L-NMMA, further incubated with 10− 6 M pilocarpine and the amounts of PGE2 and of cAMP produced as well as the activity of NOS determined as described in Methods. Data are expressed as pmol/mg wet weight (for PGE2 and cAMP) or pmol/g wet weight (NOS activity). Values are the mean ± SEM of four independent experiments. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 versus pilocarpine alone. Fig. 6. Effect of calcium ionophore A 23187 on mucin release. Submandibular gland slices were incubated with the indicated concentrations of A23187 and the amount of mucin produced determined as described under Methods. Data are expressed as percentage release from total mucin content of the gland. Each point represents the mean ± SEM of four independent experiments.
suggesting the participation of cAMP but not of PLC metabolites. On the other hand, the participation of calcium from both intracellular stores and extracellular sources was confirmed by the inhibitory effect of 5 × 10− 6 M thapsigargin and of 5 × 10− 6 M verapamil respectively (Fig. 5B). Pilocarpine-induced mucin release was not modified by 5 × 10− 6 M trifluoperazine, a calcium-calmodulin (CaM) inhibitor (Fig. 5B). Finally, L-NMMA (5 × 10 − 5 M), and staurosporine (10− 9 M), produced a significant inhibition of mucin release, indicating the participation of nitric oxide synthase (NOS) and
PKC in mediating the effect of cholinergic receptor regulation of mucin release from submandibular glands. In order to confirm the ability of calcium in inducing mucin release, we studied the effect of the calcium ionophore, A 23187, in submandibular slices. Fig. 6 shows the concentrationdependent increase in mucin release elicited by this calciummobilizing agent. In view of the inhibitory effect of SQ 22536 and L-NMMA on pilocarpine-induced mucin release, we studied the ability of pilocarpine in inducing cAMP accumulation and NOS activity. As shown in Fig. 7, pilocarpine produced a dose-dependent effect on cAMP production and NOS activity, an effect that was expectedly blocked in the presence of atropine.
Fig. 7. Effect of pilocarpine on cAMP production and NOS activity in submandibular slices. The slices were incubated in the absence or the presence of the indicated concentrations of pilocarpine, homogenized and the amount of cAMP produced was determined by ELISA (A), or the activity of NOS determined radiometrically as described in Methods (B). Results are expressed as pmol/mg wet weight (A) or pmol/g wet weight (B). Each point represents the mean ± SEM of four independent experiments. (▴) Effect of 10− 6 M atropine.
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Fig. 8. Correlation analysis between PGE2 production and cAMP accumulation (A) and between cAMP accumulation and mucin release (B) in submandibular glands. (A) Data are derived from Figs. 3A and 7A, and cAMP accumulation was plotted as a function of PGE2 production. r value = 0.9474, p = 0.0012. (B) Data are derived from Figs. 1B and 7A and mucin release was plotted as a function of cAMP accumulation. r value = 0.9773, p b 0.001.
The effect of verapamil, indomethacin and L-NMMA on pilocarpine-stimulated PGE2, cAMP and NOS activity in submandibular glands was also analyzed and is described in Table 2. As observed, verapamil (5 × 10− 6 M) and indomethacin (5 × 10− 6 M) significantly inhibited pilocarpine-induced PGE2 production and cAMP accumulation. Neither verapamil nor indomethacin inhibited NOS activity suggesting a common pathway for PGE2 and cAMP without the participation of NO. Finally, highly significant correlations were observed when cAMP production was plotted against PGE2 production (Fig. 8A) and when mucin release was studied as a function of cAMP produced (Fig. 8B). Together, these results confirm the role of cAMP in mucin exocytosis and would suggest that the elevations of cAMP levels are, in part, a consequence of prostaglandin receptor stimulation. Discussion In this paper we have studied the cholinergic regulation of mucin secretion from submandibular glands. The experimental model used comprised the effect of the muscarinic agonist pilocarpine on slices of rat submandibular glands. Our selection of pilocarpine was based on the fact that it is the drug of choice in the treatment of xerostomia. The advantage over than that offered by mucin-based artificial saliva in the management of patients with radiation-induced xerostomia (Davies and Singer, 1994) and in patients with advanced cancer (Davies et al., 1998) justifies the therapeutic use of pilocarpine. In our work, pilocarpine and carbachol showed the same ability to induce mucin release from rat submandibular gland. Consequently, the EC50 of both agonists was increased to a similar extent in the presence of atropine. Pilocarpine-stimulated mucin release was decreased in the presence of the COX inhibitors indomethacin and ASA. Expectedly, the inhibitory effect of indomethacin was reversed
by the addition of exogenous PGE2. In line with those results, pilocarpine increased the levels of PGE2 in submandibular slices. Moreover, a significant correlation was observed when the levels of PGE2 were plotted against mucin release. Our results demonstrate that PGE2 is part of the cascade of events triggered by the activation of Ach receptors during cholinergic regulation of mucin release in submandibular glands. This is in agreement with previous reports that link PGE2 with AChstimulated mucin release in antral mucous cells (Shimamoto et al., 2005). We also explored here the subtypes of muscarinic acetylcholine receptors involved in the regulation of mucin release in the submandibular gland. Our pharmacological approach identified the M1 and M4 muscarinic receptors as the subtypes involved in the control of mucin release. Although the M3 subtype is the most abundant mAChR expressed in submandibular gland (Pérez Leirós et al., 2000) it did not participate in the exocytotic process induced by pilocarpine. Our conclusion is sustained by the fact that neither blockade of the M3 receptor by a selective M3 antagonist, 4-DAMP, nor inhibition of the known downstream effector of the M3 receptor (phospholipase C by U-73122) affected mucin release. Conversely, the other mAChR subtype predominantly expressed in submandibular gland, M1, (Pérez Leirós et al., 2000) was involved in pilocarpine-induced exocytosis. The M1 and M3 mAChR subtypes, by coupling to Gq-protein, are known to increase intracellular Ca2+ through activation of various isoforms of PLC and the subsequent inositol 1,4,5-triphosphate (IP3)-induced release of Ca2+ from intracellular stores (Carrol and Peralta, 1998). It is reported that the control of salivary flow and amylase release are mediated by both, M1 and M3 mAChR subtypes in the salivary gland (Gautman et al., 2004; Busch and Borda, 2003). Our results show, however, that in cholinergicstimulated mucin release the mAChR subtypes involved are the M1 and M4. It is not clear why the M3 mAChR subtype does not
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participate in pilocarpine-induced mucin release. One possible explanation is that each receptor subtype and its corresponding downstream effectors are specifically compartmentalized, serving to form a segregated intracellular signaling network (Guderman et al., 1996). Two-dimensional Ca2+ -imaging analysis revealed the patchy distribution of M1 in submandibular gland acini, in contrast to the ubiquitous distribution of M3 (Nakamura et al., 2004). Another explanation could be that the conceptualization of signal transduction pathways in a linear fashion, i.e. one receptor coupling to one G protein that activates one effector, is inadequate to account for recent findings. The majority of heptahelical receptors interacts with diverse G proteins and elicits multiple intracellular signals. G protein-mediated signals transduction can be regarded as a complex, highly organized signaling network with diverging and converging transduction steps at the ligand–receptor, receptor–G protein, and G protein–effector interfaces (Guderman et al., 1997). Thus, M1 and M3 mAChR subtypes may be coupled to different G proteins and then, their participation in salivary glands activities may be different. As a matter of fact, it was suggested that M3 and M1 mACh receptors subtypes play different roles in salivation during eating (Nakamura et al., 2004). On the other hand, there are evidences that in rat parotid gland M3 muscarinic receptors are coupled to two second messenger systems, the stimulation of calcium mobilization and the inhibition of cAMP accumulation (Dai et al., 1991). Thus, although pilocarpine-induced mucin exocytosis was inhibited by pirenzepine, its effect was independent of PLC and CaM. Pilocarpine-induced mucin release was also inhibited by tropicamide, the selective antagonist of M4 mAChR subtype. This receptor subtype was described in salivary glands (Wang et al., 1998; Reina et al., 2005) and was reported to be involved in salivary flow (Bymaster et al., 2003). Preferential coupling of M4 mAChR to Gi/o proteins allows it to mediate an array of effects on ion channels to modify K+ and Ca2+ fluxes as well as an inhibitory action on adenylyl cyclase activity (Caulfield, 1993). In addition, some reports have provided evidence for M4 mAChR linking to adenylyl cyclase stimulatory pathways (Jones et al., 1991; Dittman et al., 1994). Final confirmation about the identity of the subtypes of muscarinic receptors involved in the regulation of mucin release in the submandibular gland could be gained by radioligand competitive studies and by analyzing submandibular function in knockout mice models. Our results show that pilocarpine-induced exocytosis was decreased in the presence of the adenylyl cyclase inhibitor, SQ 22536, supporting the view that cAMP accumulation was involved in the secretory process. This event was also observed by Nakahari et al. (2000) in submandibular acinar cells where ACh promoted the accumulation of cAMP and where this cAMP accumulation modulates Ca2+-regulated exocytosis. Pilocarpine-induced mucin release was decreased in the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, and the calcium channel blocker, verapamil. These results indicate that Ca2+ derived from intracellular stores or from external sources is involved in the secretory process. Three intracellular calcium pools have been described: (i) an
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IP3- and thapsigargin-sensitive Ca2+ pool; (ii) an IP3-insensitive, thapsigargin-sensitive Ca2+ pool; and (iii) an ionomycinsensitive Ca2+ pool (Baumgarten et al., 1995). The presence of those three types of Ca2+ stores has been described in the submandibular cell line, SMG-C6 (Liu et al., 2000). Thus, the lack of effect of the PLC inhibitor U-73122 on pilocarpineinduced mucin release observed here, supports the view that an IP3-insensitive, thapsigargin-sensitive Ca2+ store is involved in the secretory process. Although thapsigargin induces a rapid release of Ca2+ from intracellular stores, which in turn activates Ca2+ influx (Liu et al., 2000), this may not suffice to induce mucin release. However, the consequent depletion of the intracellular Ca2+ stores could then explain why thapsigargin abrogates the effect of pilocarpine. In non-excitable cells, the transient release of intracellular Ca2+ is usually accompanied by the influx of calcium across the plasma membrane. Muscarinic receptors are known to interact with both voltage-dependent and independent types including receptor operated and store operated Ca 2+ channels (Felder, 1995). Our results are consistent with the participation of a verapamil-sensitive Ca2+ channel which will account for the Ca2+ influx that occurs during pilocarpine-induced mucin release. Evidence of the presence of voltage-dependent Ca2+ channels in non-excitable cells was suggested by the impairment of salivary glands function induced by nifedipine, diltiazem and verapamil (Dehpour et al., 1995). In addition, in the colon, mucin discharge induced by bethanechol requires extracellular calcium and the activation of voltage-dependent calcium channels of Ltype (Barcelo et al., 2001). Finally, the activation of both PKC and NOS during pilocarpine-induced mucin release could be mediated by increases in [Ca2+]I (Chung and Fleming, 1995; Watson et al., 1999).
Fig. 9. Model depicting pilocarpine's signaling pathway of mucin release in submandibular slices. ER: endoplasmic reticulum; COX: cyclooxygenase PGE2: prostaglandin E2; cAMP: cyclic adenosine monophosphate; NOS: nitric oxide synthase; NO: nitric oxide; PKC: protein kinase C.
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In conclusion, based on our results, we propose the following mechanism for pilocarpine-induced mucin release in the submandibular gland: pilocarpine, through activation of M1 and M4 receptors increases [Ca2+]I from intracellular stores (Noda et al., 1993) and from external sources and elevates cAMP levels The simultaneous increase of [Ca2+]I and cAMP stimulates exocytosis and results in the activation of COX, NOS and PKC. The activity of NOS is probably regulated by Ca2+ released from intracellular stores as evidenced by the lack of effect of verapamil on pilocarpine-induced NOS activity. PGE2 released through COX activation, stimulates cAMP accumulation, which potentiates cAMP and Ca2+-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production since both are inhibited by indomethacin and verapamil. PKC, together with cAMP, may participate in PGE2 regulatory events (Li et al., 2004). The product of NOS activity, NO, may be involved in capacitative Ca2+ entry (Watson et al., 1999). This conclusion is summarized in Fig. 9. Acknowledgements This work was supported by the Grants from University of Buenos Aires (UBACYT), from the Argentinean Agency for the Promotion of Science and Technology (ANPCyT) and from the Argentinean National Research Council (CONICET). We thank Mrs. Elvita Vannucchi and Mrs. Elena Vernet for their outstanding technical assistance. References Alugupalli, K.R., Kalfas, S., Edwardsson, S., Naidu, A.S., 1995. Lactoferrin interaction with Actinobacillus actinomycetemcomitans. Oral Microbiology and Immunology 10, 35–41. Barcelo, A., Claustre, J., Abello, J., Chayvialle, J.A., Plaisancie, P., 2001. Selective involvement of calcium and calcium channels in stimulated mucin secretion from rat colon. Scandinavian Journal of Gastroenterology 36, 1339–1343. Baumgarten, L.B., Lee, H.C., Villereal, M.L., 1995. Multiple intracellular Ca2+ pools exist in human foreskin fibroblast cells: the effect of BK on release and filling of the non-cytosolic Ca2+ pools. Cell Calcium 17, 41–52. Bredt, D.S., Snyder, S.H., 1990. Isolation of nitric oxide synthase, a calmodulinrequiring enzyme. Proceedings of the National Academy of Sciences of the United States of America 87, 682–685. Busch, L., Borda, E., 2003. Castration decreases amylase release associated with muscarinic acetylcholine receptor down regulation in rat parotid gland. British Journal of Pharmacology 139, 399–407. Bymaster, F.P., Carter, P.A., Yamada, M., Gomeza, J., Wess, J., Hamilton, S.E., Nathanson, N.M., McKinzie, D.L., Felder, C.C., 2003. Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis and pilocarpine-induced seizure activity. European Journal of Neuroscience 17, 1403–1410. Carrol, R.C., Peralta, E.G., 1998. The M3 muscarinic acetylcholine receptor differentially regulates calcium influx and release through modulation of monovalent cation channels. The EMBO Journal 17, 3036–3044. Caulfield, M.P., 1993. Muscarinic receptors—characterization, coupling and function. Pharmacology and Therapeutics 58, 319–379. Chung, H.C., Fleming, N., 1995. Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular cells. Pflugers Archiv 431, 161–168. Dai, Y., Ambudkar, I.S., Horn, V.J., Yeh, C., Kousvelari, E.E., Wall, S.J., Li, M., Yasuda, R.P., Wolfe, B.B., Baum, B.J., 1991. Evidence that M3 muscarinic
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L. Busch, E. Borda / Life Sciences 80 (2007) 842–851 Padfield, P.J., Panesar, N., 1997. MgATP acts before Ca2+ to prime amylase secretion from permeabilized rat pancreatic acini. American Journal of Physiology: Gastrointestinal and Liver Physiology 273, G655–G660. Pérez Leirós, C., Rosignoli, F., Genaro, A.M., Sales, M.E., Sterin-Borda, L., Borda, E.S., 2000. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands. Journal of the Autonomic Nervous System 9, 99–107. Reina, S., Sterin-Borda, L., Orman, B., Borda, E., 2005. Autoantibodies against submandibular gland muscarinic cholinoceptor subtypes in primary Sjögren syndrome. European Journal of Inflammation 3, 135–141. Sarosiek, J., Rourk, R.M., Piascik, R., Namiot, Z., Hetzel, D.P., McCallum, R.W., 1994. The effect of esophageal mechanical and chemical stimuli on salivary mucin secretion in healthy individuals. The American Journal of the Medical Sciences 308, 23–31. Shimamoto, Ch., Fujiwara, S., Kato, M., Ito, S., Katsu, K., Mori, H., Nakahari, T., 2005. Inhibition of Ach-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2. American Journal of Physiology-GI 288, 39–47. Slomiany, B.L., Murty, V.L.N., Mandel, I.D., Zalesna, G., Slomiany, A., 1989. Physochemical characteristics of mucus glycoproteins and lipids of the human oral mucosal mucus coat in relation to caries susceptibility. Archives of Oral Biology 34, 229–237. Slots, J., Listgarten, M.A., 1988. Bacteroides gingivalis, Bacteroides intermedius, and Actinobacillus actinomycetemcomitans in human periodontal diseases. Journal of Clinical Periodontology 15, 85–93.
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Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands Lucila Busch a,⁎, Enri Borda a,b a
Pharmacology Unit, School of Dentistry, University of Buenos Aires, Marcelo T. de Alvear 2142 (C1122AAH), Buenos Aires, Argentina b National Scientific and Technical Research Council (CONICET), Rivadavia 1917 (C1033AAJ) Buenos Aires, Argentina Received 25 August 2006; accepted 3 November 2006
Abstract We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE2 levels and a positive (r = 0.8870) and significant (p = 0.0077) correlation between PGE2 production and mucin released was determined. The participation of PGE2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M1 and M4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M1 and M4 mAChR subtypes induces an increase of intracellular Ca2+ concentration ([Ca2+]I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE2 released induces cAMP accumulation which, together with PKC are involved in the PGE2 increased Ca2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production. © 2006 Elsevier Inc. All rights reserved. Keywords: Submandibular gland; Mucin; Pilocarpine
Introduction The saliva is a complex secretion originating from the parotid, submandibular, sublingual and so-called minor glands in the oral cavity. The major functions of saliva include lubrication of the oral mucosal surfaces, protection against microorganism and neutralization of acids produced by bacterial plaque. The defense factors of saliva include both immune and non-immune systems as well as a broad range of cytokines and growth factors (Slomiany et al., 1989). Mucins are predominant among the components of salivary non-immune defense system. These large, highly glycosylated
⁎ Corresponding author. Cátedra de Farmacología, Facultad de Odontología, Universidad de Buenos Aires, Marcelo T. de Alvear 2142 4to “B”, (C1122AAH) Buenos Aires, Argentina. Fax: +54 11 4963 2767. E-mail address: [email protected] (L. Busch). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.11.010
proteins play a major role in the maintenance of viscoelastic properties of saliva, participate in the formation of the protective coat of the oral mucosa and tooth enamel pellicle (Slomiany et al., 1989). Mucins promote also bacterial aggregation and clearance from the oral cavity (Tabak, 1990). Oral diseases, including periodontal infections, can thus occur when such defense mechanisms are impaired. Microorganisms, such as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, may evade these protective mechanisms and can act as etiologic agents of periodontitis (Slots and Listgarten, 1988; Van Winkelhoff and De Graaff, 1991). Salivary components that interact with A. actinomycetemcomitans, include fractions of SIgA, lactoferrin and the low molecular weight mucin species MG2 (Alugupalli et al., 1995; Groenink et al., 1996). These proteins might therefore participate in the salivary defense against this bacterium. In submandibular glands mucin secretion is regulated by both the adrenergic and the muscarinic cholinergic systems.
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Mucin released by stimulation of β-adrenoceptors is independent of extracellular Ca 2+ (Taylor and Mc Whorter, 1991) while the stimulation caused by activation of cholinergic receptors is dependent on extracellular Ca 2+ (Dohi et al., 1991). Mucins are synthesized in the Golgi apparatus and stored in intracellular granules, which are transported to the luminal surface of the cell (Forstner and Forstner, 1994). These mucin granules finally discharge their contents to the lumen of the duct in an exocytotic fashion. The first event in exocytosis is the fusion of the granule with the plasma membrane at the fusion pore, an event mediated by exocytosisrelated proteins (Südhof, 1995) whose activities are regulated by intracellular Ca2+ concentrations ([Ca2+ ]I), protein kinase A (PKA), protein kinase C (PKC) and G protein activities, and ATP levels (Padfield and Panesar, 1997; Williams et al., 1997). In antral mucus cells, acetylcholine (Ach) increases [Ca2+ ]I by promoting Ca2+ release from intracellular stores followed by the store-operated Ca2+ entry. The increase in [Ca2+ ]I results in increased exocytosis and also in the stimulation of cyclooxygenase-1 (COX-1) activity, which in its turn results in prostaglandin E2 (PGE2) synthesis and release from cells. The released PGE2 acts on EP4 receptors and further potentiates Ca 2+ -regulated exocytosis in a cAMP-mediated process (Shimamoto et al., 2005). Apart from its protective role in oral mucosal surfaces, salivary mucin serves also as a lubricant and protective factor against refluxed gastric acid and pepsin within the esophageal lumen (Sarosiek et al., 1994). PGE2 is known to act as a local protective mediator of the gastric mucosa from acid-peptic injury probably through its participation in mucin release (Ohnishi et al., 2001). The goal of the present study was thus to study the signal transduction pathways activated after cholinergic stimulation of mucin release in submandibular gland
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Table 1 Effect of indomethacin and acetyl salicylic acid (ASA), on pilocarpine-induced mucin release in submandibular gland slices Experiment
Mucin release (% of total)
% inhibition #
Control Indomethacin (5 × 10− 7 M) Indomethacin (1 × 10− 6 M) Indomethacin (5 × 10− 6 M) Control ASA (5 × 10− 5 M) ASA (1 × 10− 4 M) ASA (5 × 10− 4 M)
8.9 ± 0.7 7.7 ± 0.6 6.6 ± 0.5⁎ 5.3 ± 0.3⁎⁎ 8.6 ± 0.8 7.5 ± 0.6 6.4 ± 0.4 5 ± 0.4⁎⁎
0 27.3 47.7 81.8 0 26.8 53.6 87.8
Submandibular gland slices were preincubated in the absence or the presence of the indicated concentrations of indomethacin or ASA, further incubated with 10− 6 M pilocarpine and mucin produced was determined as described in Methods. Results are the mean ± SEM of four independent experiments. ⁎ p b 0.05; ⁎⁎ p b 0.01 versus pilocarpine alone. #: extracting basal values: 4.5.
and to examine the participation of PGE2 in the secretory process. Methods Drugs Pilocarpine, carbachol, atropine, pirenzepine, staurosporine, N G-methyl-L-Arginine (L-NMMA), thapsigargin, verapamil, trifluperazine, A 23187, indomethacin, acetyl-salicylic acid and PGE2 are from Sigma Chemical Company (St. Louis, MO, U.S.A.). U-73122, SQ 22536 and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were purchased from Research Biochemicals International (Natick, MA, U.S.A.). AF-DX 116 and tropicamide were supplied by Tocris Cookson Inc (Ellisville, MO, U.S.A.).
Fig. 1. Effect of carbachol and pilocarpine on mucin release in submandibular glands. Submandibular slices were incubated with the indicated concentrations of carbachol (A) or pilocarpine (B) alone (●) and in the presence of 5 × 10− 6 M atropine (○), Results are expressed as percentage release from total mucin content of the gland. Each point represents the mean ± SEM of four independent experiments.
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Fig. 2. Effect of indomethacin and of PGE2 on pilocarpine-induced mucin release from submandibular glands. Slices were incubated with the indicated concentrations of pilocarpine or preincubated with 5×10− 6 M indomethacin or indomethacin+ 10− 8 M PGE2 and further treated with pilocarpine. Results are expressed as percentage release from total mucin content of the gland. Each point represents the mean±SEM of four experiments. ⁎pb 0.05; ⁎⁎pb 0.01 versus pilocarpine alone.
Animals Male Wistar rats weighing 250–300 g were used throughout the study. Animals had free access to food and water until the night before experiments when food, but not water was withdrawn. For surgical removal of submandibular glands, the animals were sacrificed using ether. Animal care was provided according to “The Guide to the Care and Use of Experimental Animals” (DHEW Publication, NIH 80-23). Measurement of mucin secretion Extirpated submandibular glands were detached from free connective tissue and fat and sliced into pieces approximately
2–3 mm thick and 15 mg wet weight with a razor blade. Gland slices were incubated for 30 min in 500 μl of Krebs Ringer bicarbonate medium (KRB), pH 7.4 bubbled with 5% CO2 in O2 at 37 °C. When used, inhibitors were included from the beginning of the incubation and stimuli were added in the last 15 min of the incubation time. Placing the tubes on ice then stopped the reaction. Slices were homogenized in sodium acetate buffer 25 mM Cl2Mg, pH 5.8, supplemented with protease inhibitors (0.1 mM phenylmethyl-sulfonyl fluoride, 1 mM sodium ethylenediaminetetra-acetate and 1 mM iodoaceamide) at 4 °C and centrifuged at 900 ×g for 15 min. Mucin was determined in the supernatants (total mucin content in the gland) and in the incubation medium (mucin released) using the Alcian Blue method described by Hall et al. (1980) and modified by Sarosiek et al. (1994). Briefly, aliquots of diluted supernatants (1:100) or medium (1:10) were incubated for 30 min in a 1% solution of Alcian Blue in 50 mM sodium acetate buffer 25 mM MgCl2, pH 5.8 under constant agitation at room temperature. Following incubation, the samples were centrifuged for 20 min at 3000 rpm pellets washed in 95% ethanol, vortexed gently for 10 s and after 5 min, centrifuged for 20 min at 3000 rpm. Mucin–dye complexes were dissociated by the addition of a 1:2 dilution of Aerosol OT in distilled water, brief mixing and sonication. Subsequently, samples were extracted with equal volumes of ethyl ether under vigorous shaking. The resulting solution was centrifuged for 15 min at 3000 rpm and the dye concentration determined spectrophotometrically at 605 nm in the aqueous layer. Mucin released is expressed as percentage released from total mucin content in the gland (% of total). Determination of prostaglandin production Submandibular gland slices (55 mg) were incubated for 30 min in 160 μl of KRB solution, pH 7.4, gassed with 5% CO2 in O2 at 37 °C. When used, inhibitors were added at the beginning of the incubation and pilocarpine in the last 15 min.
Fig. 3. Effect of pilocarpine on PGE2 production in submandibular slices. Submandibular gland slices were incubated in the absence (▴) or the presence (●) of increasing concentrations of pilocarpine, homogenized as described in Methods and the amount of produced PGE2 determined by ELISA. (A) Data are expressed as pg/mg wet weight and represent the mean ± SEM of four independent experiments. (B) Correlation analysis between PGE2 production and mucin release. Data are derived from Figs. 1B and 3A, and mucin release was plotted as a function of the PGE2 production. r value = 0.8870, p = 0.0077.
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After homogenization all procedures employed were those indicated in the protocol of Prostaglandin E2 Biotrak Enzyme Immuno Assay (ELISA) System (Amersham Biosciences, Piscataway, NJ, U.S.A.). Results are expressed as picogram PGE2/mg of tissue wet weight (pg/mg wet wt).
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Cyclic AMP was then determined using the EIA Kit (Cayman Chemical Company, Ann Arbor, MI, U.S.A.) according to the instruction provided by the supplier. Results are expressed as picomol cAMP/mg of tissue wet weight (pmol/mg wet wt). Determination of nitric oxide synthase activity (NOS)
Determination of cyclic AMP levels Submandibular slices (50 mg) were incubated for 30 min in 500 μl of KRB medium, pH 7.4, with 5% CO2 in O2 at 37 °C in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX). Antagonists were present from the beginning while pilocarpine was present during the final 15 min of incubation. After incubation, the tissue was homogenized in 400 μl of 5% trichloroacetic acid (TCA) and centrifuged for 10 min at 1500 ×g at 4 °C. Following centrifugation, the supernatant was transferred to a clean tube and TCA was extracted from the samples using water-saturated ether.
NOS activity was measured in submandibular glands using L[U-14C] arginine as substrate as described by Bredt and Snyder (1990). Briefly, 50–60 mg of submandibular slices were incubated for 30 min with 0.4 μCi L-[U-14C] arginine (Amersham Pharmacia Biotech, Buckinghamshire, England, about 300 mCi/mmol) in 500 μl of KRB solution pH 7.4 gassed with 5% CO2 in O2 at 37 °C. The tissue was then homogenized, centrifuged for 10 min at 10,000 ×g and [14C]citrulline formed in the supernatants was separated by ion exchange chromatography on AG 50 W resin (Biorad). When used, inhibitors were added at the beginning of the
Fig. 4. Effect of various selective muscarinic receptor antagonist on pilocarpine-induced mucin release. Submandibular gland slices were incubated with the indicated concentrations of pilocarpine in the absence (●) or the presence of varying concentrations of pirenzepine (A), AF-DX 116 (B), 4-DAMP (C) and tropicamide (D) and the amount of mucin produced was determined as described in Methods. Results are expressed as percentage release from total mucin content of the gland. Each point represents the mean ± SEM of four independent experiments.
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incubation and pilocarpine was present in the last 15 min. Nitric oxide production (measured as pmol of [14C]citrulline) is expressed as picomol [14C]citrulline/g of tissue wet weight (pmol/g wet wt). Statistical analysis Statistical significance of differences was determined by analysis of variance (ANOVA) followed by Tukey's test. Differences between means were considered significant at p b 0.05. Correlation and fitting dose–response curves were done using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego CA, USA). Results First, we studied the effect of cholinergic stimulation on mucin release in submandibular glands. Stimulation of the glands with pilocarpine and carbachol resulted in a dose-dependent increase in mucin production as shown in Fig. 1. The secretory ability of both cholinergic agonists showed identical EC50, (1.2 × 10− 6 M) and nearly equal maximal effect. Atropine (5 × 10− 6 M) antagonized the effect of both pilocarpine and carbachol and consequently produced a right shift of the dose–response curves as seen in panels A and B of Fig. 1. Finally, in the absence of stimulation, submandibular glands released 4.5 ± 0.5% of total mucin content during the 30 min of incubation (Fig. 1). Next, we analyzed the effect of varying concentrations of the cyclooxygenase (COX) inhibitors indomethacin and acetyl salicylic acid (ASA). Table 1 shows that both drugs inhibited pilocarpine-induced mucin release in a dose-dependent manner, with an approximately 80 and 90% maximal inhibition for indomethacin and ASA respectively. The effect of indometh-
acin on mucin release was partially abrogated in the presence of 10− 8 M PGE2, a concentration of PGE2 that was ineffective in inducing mucin release per se (Fig. 2 and data not shown). On the other hand, pilocarpine produced a dose-dependent increase of PGE2 production in rat submandibular gland as evidenced in Fig. 3A. A significant correlation was observed between PGE2 production and mucin release by the submandibular glands (Fig. 3B). In order to determine the subtype of muscarinic acetylcholine receptor (mAChR) responsible for mucin secretion pilocarpineinduced mucin release was assayed in the presence of pirenzepine, AF-DX 116, 4-DAMP and tropicamide (M1, M2, M3 and M4 receptor antagonists respectively). Of the compounds employed, the M1, and M4 antagonists pirenzepine and tropicamide blunted the effect of pilocarpine thus producing a right shift of the dose–response curve (Fig. 4A and D). The potencies of pirenzepine and tropicamide, expressed as pA2 after Schild plots, were 6.1 ± 0.5 and 5.7 ± 0.4 for pirenzepine and tropicamide respectively. Surprisingly, the M3 mAChR subtype selective antagonist 4-DAMP, failed to inhibit pilocarpine-induced mucin release (Fig. 4C) even at concentrations up to 10− 5 M (data not shown). The M2 mAChR subtype selective antagonist, AF-DX 116, failed also to inhibit the effect of pilocarpine on mucin release (Fig. 4B). The participation of different second messengers in the signal transduction pathway triggered after Ach receptor activation was then explored by measuring the effect of selective inhibitors on pilocarpine-induced mucin release. Inhibition of adenylate cyclase (AC) activity by SQ 22536 (5 × 10− 6 M) produced a significant decrease of mucin release while inhibition of phospholipase C (PLC) activity by U-73122 (5 × 10− 6 M) had no effect on mucin release (Fig. 5A) thus
Fig. 5. Effect of U-73122, SQ 22536, verapamil, thapsigargin, trifluoperazine, staurosporine, and of L-NMMA on pilocarpine-induced mucin release in submandibular glands. Submandibular slices were preincubated with 5 × 10− 6 M U-73122, 5 × 10− 6 M SQ 22536, 5 × 10− 6 M verapamil 5 × 10− 6 M thapsigargin, 5 × 10− 6 M trifluoperazine, 5 × 10− 9 M staurosporine or 5 × 10− 5 M L-NMMA, further incubated with 10− 6 M pilocarpine and mucin released was determined as described in Methods. Data are expressed as percentage release from total mucin content of the gland, and bars represent the mean ± SEM of four independent experiments. Basal values and the effect of 10− 6 M pilocarpine are also shown. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 versus pilocarpine alone.
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Table 2 Effect of verapamil, indomethacin and L-NMMA on pilocarpine-stimulated PGE2 and cAMP production and NOS activity in submandibular glands Group
PGE2 (pg/mg cAMP (pmol/mg NOS activity wet wt) wet wt) (pmol/g wet wt)
Basal Pilocarpine (10− 6 M) Pilocarpine + verapamil (5 × 10− 6 M) Pilocarpine + indo (5 × 10− 6 M) Pilocarpine + L-NMMA (5 × 10− 5 M)
38 ± 2.2⁎⁎⁎ 74 ± 5.1 38 ± 2.6⁎⁎⁎
0.35 ± 0.04⁎⁎⁎ 2.62 ± 0.17 0.98 ± 0.10⁎⁎⁎
391 ± 20⁎⁎ 586 ± 45 600 ± 48
43 ± 4.4⁎⁎⁎
1.33 ± 0.11⁎⁎⁎
572 ± 36
73 ± 4.3
2.54 ± 0.21
187 ± 12⁎⁎⁎
Submandibular gland slices were preincubated in the absence or the presence of the indicated concentrations of verapamil, indomethacin or L-NMMA, further incubated with 10− 6 M pilocarpine and the amounts of PGE2 and of cAMP produced as well as the activity of NOS determined as described in Methods. Data are expressed as pmol/mg wet weight (for PGE2 and cAMP) or pmol/g wet weight (NOS activity). Values are the mean ± SEM of four independent experiments. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 versus pilocarpine alone. Fig. 6. Effect of calcium ionophore A 23187 on mucin release. Submandibular gland slices were incubated with the indicated concentrations of A23187 and the amount of mucin produced determined as described under Methods. Data are expressed as percentage release from total mucin content of the gland. Each point represents the mean ± SEM of four independent experiments.
suggesting the participation of cAMP but not of PLC metabolites. On the other hand, the participation of calcium from both intracellular stores and extracellular sources was confirmed by the inhibitory effect of 5 × 10− 6 M thapsigargin and of 5 × 10− 6 M verapamil respectively (Fig. 5B). Pilocarpine-induced mucin release was not modified by 5 × 10− 6 M trifluoperazine, a calcium-calmodulin (CaM) inhibitor (Fig. 5B). Finally, L-NMMA (5 × 10 − 5 M), and staurosporine (10− 9 M), produced a significant inhibition of mucin release, indicating the participation of nitric oxide synthase (NOS) and
PKC in mediating the effect of cholinergic receptor regulation of mucin release from submandibular glands. In order to confirm the ability of calcium in inducing mucin release, we studied the effect of the calcium ionophore, A 23187, in submandibular slices. Fig. 6 shows the concentrationdependent increase in mucin release elicited by this calciummobilizing agent. In view of the inhibitory effect of SQ 22536 and L-NMMA on pilocarpine-induced mucin release, we studied the ability of pilocarpine in inducing cAMP accumulation and NOS activity. As shown in Fig. 7, pilocarpine produced a dose-dependent effect on cAMP production and NOS activity, an effect that was expectedly blocked in the presence of atropine.
Fig. 7. Effect of pilocarpine on cAMP production and NOS activity in submandibular slices. The slices were incubated in the absence or the presence of the indicated concentrations of pilocarpine, homogenized and the amount of cAMP produced was determined by ELISA (A), or the activity of NOS determined radiometrically as described in Methods (B). Results are expressed as pmol/mg wet weight (A) or pmol/g wet weight (B). Each point represents the mean ± SEM of four independent experiments. (▴) Effect of 10− 6 M atropine.
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Fig. 8. Correlation analysis between PGE2 production and cAMP accumulation (A) and between cAMP accumulation and mucin release (B) in submandibular glands. (A) Data are derived from Figs. 3A and 7A, and cAMP accumulation was plotted as a function of PGE2 production. r value = 0.9474, p = 0.0012. (B) Data are derived from Figs. 1B and 7A and mucin release was plotted as a function of cAMP accumulation. r value = 0.9773, p b 0.001.
The effect of verapamil, indomethacin and L-NMMA on pilocarpine-stimulated PGE2, cAMP and NOS activity in submandibular glands was also analyzed and is described in Table 2. As observed, verapamil (5 × 10− 6 M) and indomethacin (5 × 10− 6 M) significantly inhibited pilocarpine-induced PGE2 production and cAMP accumulation. Neither verapamil nor indomethacin inhibited NOS activity suggesting a common pathway for PGE2 and cAMP without the participation of NO. Finally, highly significant correlations were observed when cAMP production was plotted against PGE2 production (Fig. 8A) and when mucin release was studied as a function of cAMP produced (Fig. 8B). Together, these results confirm the role of cAMP in mucin exocytosis and would suggest that the elevations of cAMP levels are, in part, a consequence of prostaglandin receptor stimulation. Discussion In this paper we have studied the cholinergic regulation of mucin secretion from submandibular glands. The experimental model used comprised the effect of the muscarinic agonist pilocarpine on slices of rat submandibular glands. Our selection of pilocarpine was based on the fact that it is the drug of choice in the treatment of xerostomia. The advantage over than that offered by mucin-based artificial saliva in the management of patients with radiation-induced xerostomia (Davies and Singer, 1994) and in patients with advanced cancer (Davies et al., 1998) justifies the therapeutic use of pilocarpine. In our work, pilocarpine and carbachol showed the same ability to induce mucin release from rat submandibular gland. Consequently, the EC50 of both agonists was increased to a similar extent in the presence of atropine. Pilocarpine-stimulated mucin release was decreased in the presence of the COX inhibitors indomethacin and ASA. Expectedly, the inhibitory effect of indomethacin was reversed
by the addition of exogenous PGE2. In line with those results, pilocarpine increased the levels of PGE2 in submandibular slices. Moreover, a significant correlation was observed when the levels of PGE2 were plotted against mucin release. Our results demonstrate that PGE2 is part of the cascade of events triggered by the activation of Ach receptors during cholinergic regulation of mucin release in submandibular glands. This is in agreement with previous reports that link PGE2 with AChstimulated mucin release in antral mucous cells (Shimamoto et al., 2005). We also explored here the subtypes of muscarinic acetylcholine receptors involved in the regulation of mucin release in the submandibular gland. Our pharmacological approach identified the M1 and M4 muscarinic receptors as the subtypes involved in the control of mucin release. Although the M3 subtype is the most abundant mAChR expressed in submandibular gland (Pérez Leirós et al., 2000) it did not participate in the exocytotic process induced by pilocarpine. Our conclusion is sustained by the fact that neither blockade of the M3 receptor by a selective M3 antagonist, 4-DAMP, nor inhibition of the known downstream effector of the M3 receptor (phospholipase C by U-73122) affected mucin release. Conversely, the other mAChR subtype predominantly expressed in submandibular gland, M1, (Pérez Leirós et al., 2000) was involved in pilocarpine-induced exocytosis. The M1 and M3 mAChR subtypes, by coupling to Gq-protein, are known to increase intracellular Ca2+ through activation of various isoforms of PLC and the subsequent inositol 1,4,5-triphosphate (IP3)-induced release of Ca2+ from intracellular stores (Carrol and Peralta, 1998). It is reported that the control of salivary flow and amylase release are mediated by both, M1 and M3 mAChR subtypes in the salivary gland (Gautman et al., 2004; Busch and Borda, 2003). Our results show, however, that in cholinergicstimulated mucin release the mAChR subtypes involved are the M1 and M4. It is not clear why the M3 mAChR subtype does not
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participate in pilocarpine-induced mucin release. One possible explanation is that each receptor subtype and its corresponding downstream effectors are specifically compartmentalized, serving to form a segregated intracellular signaling network (Guderman et al., 1996). Two-dimensional Ca2+ -imaging analysis revealed the patchy distribution of M1 in submandibular gland acini, in contrast to the ubiquitous distribution of M3 (Nakamura et al., 2004). Another explanation could be that the conceptualization of signal transduction pathways in a linear fashion, i.e. one receptor coupling to one G protein that activates one effector, is inadequate to account for recent findings. The majority of heptahelical receptors interacts with diverse G proteins and elicits multiple intracellular signals. G protein-mediated signals transduction can be regarded as a complex, highly organized signaling network with diverging and converging transduction steps at the ligand–receptor, receptor–G protein, and G protein–effector interfaces (Guderman et al., 1997). Thus, M1 and M3 mAChR subtypes may be coupled to different G proteins and then, their participation in salivary glands activities may be different. As a matter of fact, it was suggested that M3 and M1 mACh receptors subtypes play different roles in salivation during eating (Nakamura et al., 2004). On the other hand, there are evidences that in rat parotid gland M3 muscarinic receptors are coupled to two second messenger systems, the stimulation of calcium mobilization and the inhibition of cAMP accumulation (Dai et al., 1991). Thus, although pilocarpine-induced mucin exocytosis was inhibited by pirenzepine, its effect was independent of PLC and CaM. Pilocarpine-induced mucin release was also inhibited by tropicamide, the selective antagonist of M4 mAChR subtype. This receptor subtype was described in salivary glands (Wang et al., 1998; Reina et al., 2005) and was reported to be involved in salivary flow (Bymaster et al., 2003). Preferential coupling of M4 mAChR to Gi/o proteins allows it to mediate an array of effects on ion channels to modify K+ and Ca2+ fluxes as well as an inhibitory action on adenylyl cyclase activity (Caulfield, 1993). In addition, some reports have provided evidence for M4 mAChR linking to adenylyl cyclase stimulatory pathways (Jones et al., 1991; Dittman et al., 1994). Final confirmation about the identity of the subtypes of muscarinic receptors involved in the regulation of mucin release in the submandibular gland could be gained by radioligand competitive studies and by analyzing submandibular function in knockout mice models. Our results show that pilocarpine-induced exocytosis was decreased in the presence of the adenylyl cyclase inhibitor, SQ 22536, supporting the view that cAMP accumulation was involved in the secretory process. This event was also observed by Nakahari et al. (2000) in submandibular acinar cells where ACh promoted the accumulation of cAMP and where this cAMP accumulation modulates Ca2+-regulated exocytosis. Pilocarpine-induced mucin release was decreased in the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, and the calcium channel blocker, verapamil. These results indicate that Ca2+ derived from intracellular stores or from external sources is involved in the secretory process. Three intracellular calcium pools have been described: (i) an
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IP3- and thapsigargin-sensitive Ca2+ pool; (ii) an IP3-insensitive, thapsigargin-sensitive Ca2+ pool; and (iii) an ionomycinsensitive Ca2+ pool (Baumgarten et al., 1995). The presence of those three types of Ca2+ stores has been described in the submandibular cell line, SMG-C6 (Liu et al., 2000). Thus, the lack of effect of the PLC inhibitor U-73122 on pilocarpineinduced mucin release observed here, supports the view that an IP3-insensitive, thapsigargin-sensitive Ca2+ store is involved in the secretory process. Although thapsigargin induces a rapid release of Ca2+ from intracellular stores, which in turn activates Ca2+ influx (Liu et al., 2000), this may not suffice to induce mucin release. However, the consequent depletion of the intracellular Ca2+ stores could then explain why thapsigargin abrogates the effect of pilocarpine. In non-excitable cells, the transient release of intracellular Ca2+ is usually accompanied by the influx of calcium across the plasma membrane. Muscarinic receptors are known to interact with both voltage-dependent and independent types including receptor operated and store operated Ca 2+ channels (Felder, 1995). Our results are consistent with the participation of a verapamil-sensitive Ca2+ channel which will account for the Ca2+ influx that occurs during pilocarpine-induced mucin release. Evidence of the presence of voltage-dependent Ca2+ channels in non-excitable cells was suggested by the impairment of salivary glands function induced by nifedipine, diltiazem and verapamil (Dehpour et al., 1995). In addition, in the colon, mucin discharge induced by bethanechol requires extracellular calcium and the activation of voltage-dependent calcium channels of Ltype (Barcelo et al., 2001). Finally, the activation of both PKC and NOS during pilocarpine-induced mucin release could be mediated by increases in [Ca2+]I (Chung and Fleming, 1995; Watson et al., 1999).
Fig. 9. Model depicting pilocarpine's signaling pathway of mucin release in submandibular slices. ER: endoplasmic reticulum; COX: cyclooxygenase PGE2: prostaglandin E2; cAMP: cyclic adenosine monophosphate; NOS: nitric oxide synthase; NO: nitric oxide; PKC: protein kinase C.
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In conclusion, based on our results, we propose the following mechanism for pilocarpine-induced mucin release in the submandibular gland: pilocarpine, through activation of M1 and M4 receptors increases [Ca2+]I from intracellular stores (Noda et al., 1993) and from external sources and elevates cAMP levels The simultaneous increase of [Ca2+]I and cAMP stimulates exocytosis and results in the activation of COX, NOS and PKC. The activity of NOS is probably regulated by Ca2+ released from intracellular stores as evidenced by the lack of effect of verapamil on pilocarpine-induced NOS activity. PGE2 released through COX activation, stimulates cAMP accumulation, which potentiates cAMP and Ca2+-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production since both are inhibited by indomethacin and verapamil. PKC, together with cAMP, may participate in PGE2 regulatory events (Li et al., 2004). The product of NOS activity, NO, may be involved in capacitative Ca2+ entry (Watson et al., 1999). This conclusion is summarized in Fig. 9. Acknowledgements This work was supported by the Grants from University of Buenos Aires (UBACYT), from the Argentinean Agency for the Promotion of Science and Technology (ANPCyT) and from the Argentinean National Research Council (CONICET). We thank Mrs. Elvita Vannucchi and Mrs. Elena Vernet for their outstanding technical assistance. References Alugupalli, K.R., Kalfas, S., Edwardsson, S., Naidu, A.S., 1995. Lactoferrin interaction with Actinobacillus actinomycetemcomitans. Oral Microbiology and Immunology 10, 35–41. Barcelo, A., Claustre, J., Abello, J., Chayvialle, J.A., Plaisancie, P., 2001. Selective involvement of calcium and calcium channels in stimulated mucin secretion from rat colon. Scandinavian Journal of Gastroenterology 36, 1339–1343. Baumgarten, L.B., Lee, H.C., Villereal, M.L., 1995. Multiple intracellular Ca2+ pools exist in human foreskin fibroblast cells: the effect of BK on release and filling of the non-cytosolic Ca2+ pools. Cell Calcium 17, 41–52. Bredt, D.S., Snyder, S.H., 1990. Isolation of nitric oxide synthase, a calmodulinrequiring enzyme. Proceedings of the National Academy of Sciences of the United States of America 87, 682–685. Busch, L., Borda, E., 2003. Castration decreases amylase release associated with muscarinic acetylcholine receptor down regulation in rat parotid gland. British Journal of Pharmacology 139, 399–407. Bymaster, F.P., Carter, P.A., Yamada, M., Gomeza, J., Wess, J., Hamilton, S.E., Nathanson, N.M., McKinzie, D.L., Felder, C.C., 2003. Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis and pilocarpine-induced seizure activity. European Journal of Neuroscience 17, 1403–1410. Carrol, R.C., Peralta, E.G., 1998. The M3 muscarinic acetylcholine receptor differentially regulates calcium influx and release through modulation of monovalent cation channels. The EMBO Journal 17, 3036–3044. Caulfield, M.P., 1993. Muscarinic receptors—characterization, coupling and function. Pharmacology and Therapeutics 58, 319–379. Chung, H.C., Fleming, N., 1995. Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular cells. Pflugers Archiv 431, 161–168. Dai, Y., Ambudkar, I.S., Horn, V.J., Yeh, C., Kousvelari, E.E., Wall, S.J., Li, M., Yasuda, R.P., Wolfe, B.B., Baum, B.J., 1991. Evidence that M3 muscarinic
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