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Am J Physiol Cell Physiol 294: C1454-C1464, 2008. First published April 2, 2008; doi:10.1152/ajpcell.00151.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Alan L. Lin,1 Bing Zhu,1 WanKe Zhang,3 Howard Dang,2 Bin-Xian Zhang,3,4 Michael S. Katz,3,4 and Chih-Ko Yeh1,2,4

Departments of 1Dental Diagnostic Science, 2Community Dentistry, and 3Medicine, University of Texas Health Science Center at San Antonio, and 4Geriatric Research, Education and Clinical Center, Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas

Submitted 11 April 2007 ; accepted in final form 25 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i) by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms—pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC—converging on the ERK pathway.

muscarinic receptor; epidermal growth factor receptor; protein kinase C

CHOLINERGIC-MUSCARINIC RECEPTOR signaling pathways play an essential role in salivary fluid secretion (3). Pilocarpine, a parasympathomimetic agent with muscarinic action, is used clinically as a sialogogue. Specifically, pilocarpine has been employed to alleviate symptoms of oral dryness in patients undergoing head and neck radiation therapy or with Sjögren's syndrome, an autoimmune disease of salivary and lacrimal glands (43). Increased salivary secretion in response to systemic administration of pilocarpine is thought to be mediated by actions on muscarinic receptors in the central nervous system and salivary gland tissue (43). Despite the wide clinical use of pilocarpine, the mechanisms by which this agent exerts its action on salivary gland tissue have not been clarified.

The muscarinic receptor is a member of the G protein-coupled receptor (GPCR) family and consists of five subtypes, of which M1 and M3 are coupled to the Gq protein and are present in salivary cells (26, 40). Stimulation of M1 and M3 receptors by agonists classically induces Gq-mediated phospholipase Cβ (PLCβ) activation, leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 then evokes an increase in intracellular Ca2+ ([Ca2+]i) mobilization, whereas DAG directly activates PKC. The signaling intermediates, i.e., [Ca2+]i and activated PKC, subsequently elicit an array of physiological responses in salivary cells, resulting in electrolyte and water secretion (3). Recent evidence further demonstrates that activation of muscarinic receptors can stimulate MAPK pathways classically linked to receptor tyrosine kinases, e.g., receptors for epidermal growth factor (EGF) and platelet-derived growth factor, mediating cell proliferation, differentiation, and other cellular functions (27).

Among MAPK signaling pathways, which comprise a series of serine/threonine kinases, the ERK pathway consists of a three-tiered cascade composed of an MAPK kinase kinase (MAPKKK; e.g., c-Raf1 or B-Raf), an MAPK kinase (e.g., MEK1 and MEK2), and an MAPK (e.g., ERK1 and ERK2) (27). In a number of systems, ERK activation by muscarinic agonists has been attributed to [Ca2+]i- and PKC-mediated signaling events, as well as transactivation of EGF receptors. The involvement of individual signaling intermediates in muscarinic receptor-induced ERK activation appears to be organ, tissue, and cell specific (6, 13, 20, 21, 32). One preliminary study suggested that the muscarinic agonist carbachol elevates ERK activities in mouse salivary acinar cells. Activation of ERKs was reported to be independent of [Ca2+]i and EGF receptors and dependent on PKC (8). However, the ERK signaling pathways and downstream targets mediating salivary cell responses to muscarinic receptor agonists are yet to be clearly defined.

The signaling responses to pilocarpine in salivary cells have generally been assumed to mimic the signal transduction pathways elicited by carbachol. However, recent evidence suggests that different ligands acting on the same GPCR may activate distinct signaling pathways (10). In the present study, we have examined the cellular signals linking muscarinic receptor stimulation to the activation of ERK1/2 in a human salivary cell line (HSY) (46). Evaluation of the signals elicited by pilocarpine and carbachol reveals two distinct pathways for muscarinic receptor-mediated ERK1/2 activation in HSY cells. Whereas pilocarpine induces ERK activation via EGF receptor transactivation, carbachol activates ERKs by an EGF receptor-independent, PKC-dependent pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Chemicals, including pilocarpine hydrochloride, carbamoylcholine chloride (carbachol), protease inhibitor cocktails (catalog no. p8340), phosphatase inhibitors, EGTA, atropine sulfate, PMA, HEPES, and pirenzepine, were purchased from Sigma Chemical (St. Louis, MO). AG-1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline], 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3), GF-109203X, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U-0126), and N-(3-chloropropyl)-4-piperidnyl diphenylacetate (4-DAMP) were obtained from Calbiochem (San Diego, CA). Antibodies against EGF receptor, phosphotyrosine (pY), and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against p44/p42MAPK (ERK1/2), phospho-p44/p42MAPK (Thr202/Tyr204; pERK1/2), and phospho-EGF receptor (Y1068) were purchased from Cell Signaling Technology (Beverly, MA). Goat anti-rabbit IgG Fc conjugated to horseradish peroxidase was obtained from Jackson Immuno Research Laboratories (West Grove, PA). Small interfering RNA (siRNA) against muscarinic receptor subtype 1 [M1; 5'-GUGGCCUUCAUUGGGAUCAtt-3' (sense) and 5'-UGAUCCCAAUGAAGGCCACtt-3' (antisense)] and muscarinic receptor subtype 3 [M3; 5'-GGUCAACAAGCAGCUGAAGtt-3' (sense) and 5'-CUUCAGCUGCUUGUUGACCtt-3' (antisense)] were obtained from Ambion (Austin, TX).

Cell culture and rat parotid cells. The HSY cell line was originally established by Yanagawa et al. (45) and was kindly provided by Dr. James Turner (National Institute of Dental and Craniofacial Research, Bethesda, MD). Cells were plated at a density of ~2 x 104 cells/cm2 in 100-mm culture plates and cultured in DMEM supplemented with 10% FBS and penicillin (100 U/ml)-streptomycin (100 µg/ml) at 37°C in a humidified 5% CO2-atmosphere incubator. Cells grown to near confluence were washed three times with PBS and continued in culture with serum-free medium for 18–20 h. Cultured cells were treated with pilocarpine, carbachol, or other reagents for various time periods as indicated for immunoblot or immunoprecipitation analysis. For reagents (U-0126, AG-1478, 4-DAMP, pirenzepine, PP2, and PP3) dissolved in DMSO, the appropriate concentration of DMSO was added as solvent control.

Rat parotid acinar cell aggregates were prepared by a procedure previously utilized in our laboratory (28). Briefly, parotid glands dissected from male Sprague-Dawley rats (250 g body wt; Harlan, Indianapolis, IN) were minced and digested in collagenase-hyaluronidase (100 U/ml and 200 µg/ml, respectively) in modified Hanks’ balanced salt solution (HBSS) containing 33 mM HEPES and 0.1% FBS with gentle dispersion and gassing (95% O2-5% CO2) every 20 min for 80 min. After the cells were washed, the parotid cell aggregates were resuspended in modified HBSS containing 100 µg/ml trypsin inhibitor until use.

Western blot analysis. Immunoblot (Western blot) analysis was performed as described previously (46). HSY cells were washed three times with cold PBS, scraped, and lysed in a buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease and phosphatase inhibitor cocktails (RIPA buffer) at 4°C for 30 min. After centrifugation of the cell lysates at 10,000 g for 15 min at 4°C, supernatant protein samples (50 µg) were added to 15 µl of 4x sample buffer [150 mM Tris·HCl (pH 8.8), 1% SDS, and 40% glycerol] with β-mercaptoethanol and then diluted with RIPA buffer to a total volume of 60 µl. The protein samples were separated on 8% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were immuno blotted with primary antibody [1:500 dilution for p44/p42MAPK, phospho-p44/p42MAPK, and EGF receptor; 1:250 dilution for phospho-EGF receptor (Y1068)] and then with a secondary horseradish peroxidase-conjugated antibody (1:10,000 dilution). MAPKs/ERKs and EGF receptor were visualized by an enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate, Pierce Biotechnology, Rockford, IL), and phospho-EGF receptor was visualized using the ECL Advance Western Blotting Kit (Amersham Bioscience, Little Chalfont, Buckinghamshire, UK).

Immunoprecipitation assay of EGF receptor. EGF receptor phosphorylation was detected by EGF receptor immunoprecipitation from cell lysates followed by immunoblotting with anti-pY antibodies as described previously (46). Briefly, whole cell lysates (750 µg protein) were precleared by incubation with 50% protein A-Sepharose beads in RIPA buffer containing 1 µg/ml BSA for 1 h, followed by incubation of the supernatants with EGF receptor antibody (1 µg/sample) overnight at 4°C. Then 20 µl of protein A-Sepharose was added, and the incubation was continued at 4°C for 1 h. The immune complexes were washed three times with RIPA buffer containing protease and phosphatase inhibitors. Samples were dissolved in RIPA buffer containing gel loading buffer and β-mercaptoethanol and separated by SDS-PAGE. Proteins were detected by Western blotting using anti-pY monoclonal antibody (1:500 dilution) as described above (see Western blot analysis).

Transfection of siRNA. HSY cells were plated at a density of 2 x 105/ml in 35-mm plates and cultured in DMEM overnight. The cells were washed three times with DMEM and then incubated with 800 µl of DMEM containing siRNA (60 nM) and Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according the manufacturer's recommendation for 4–5 h at 37°C. Cells were then washed twice with PBS and cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin for an additional 60 h. At the end of the culture period, muscarinic agonist (pilocarpine or carbachol) was added to individual wells for 10 min, and the cells were harvested and prepared for Western blot analysis.

[Ca2+]i measurement. [Ca2+]i was determined by spectrofluorometric measurements in cell suspensions (47). HSY cells or rat parotid acinar aggregates were loaded with 1.2 µM fura 2-AM in a high salt-glucose (HNG) buffer [in mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 1.2 KH2PO4, 10 glucose, and 10 HEPES (pH 7.4)] at 37°C for 30–45 min with gentle shaking. Loaded cells were washed with fresh buffer to remove the extracellular dye and resuspended in HNG or Ca2+-free HNG at a density of 106 cells/ml. The Ca2+-free HNG medium contained 100 µM EGTA, instead of 1 mM CaCl2. Fura 2-loaded cells were then exposed to carbachol or pilocarpine at 37°C. Mobilization of [Ca2+]i was measured using a fluorometer (model QM-6, Photon Technology International, South Brunswick, NJ) and indexed as the ratio of fluorescence excited at 340 nm to fluorescence at 380 nm and detected at 510 nm.

Data analysis. The densities of Western blots were quantified with Scion Image analysis software (Scion, Frederick MD). Phosphorylation of ERK1/2, EGF receptor, or tyrosine (pY) induced by pilocarpine, carbachol, or other reagents was normalized to the total immunoreactive protein of interest for each sample and expressed as the fold increase relative to the normalized value in untreated (control) cells. Values are untransformed means ± SE. Single comparisons were conducted by Student's t-test; ANOVA followed by post hoc Bonferroni's or Dunnett's test was used for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Muscarinic agonists pilocarpine and carbachol induce activation of ERK1/2 in HSY cells. To determine whether mucarinic agonists can induce the activation of ERK1/2 in HSY cells, we used Western blot analysis to measure the effects of pilocarpine and carbachol on the phosphorylation of ERK1/2. Pilocarpine caused transient phosphorylation of ERK1/2 without affecting total ERK1/2 protein levels; activation of ERK1/2 by pilocarpine peaked at 10–15 min, with a return to basal levels after 30 min (Fig. 1A). Phosphorylation of ERK1/2 increased with increasing concentrations of pilocarpine, up to a maximum response at 10–5–10–4 M agonist (Fig. 1B). Carbachol also induced phosporylation of ERK1/2, with time course and apparent potency similar to pilocarpine (Fig. 1C; time course for carbachol stimulation not shown). Addition of a maximally stimulatory concentration of pilocarpine (5 x 10–5 M) for 10 min increased phosphorylation of ERK1/2 by 3.3 ± 0.3 fold (n = 26) relative to control level; under equivalent conditions, carbachol (10–4 M, 10 min) induced an even greater response (6.8 ± 0.8 fold, n = 26, P < 0.001 vs. pilocarpine; Fig. 1D). The stimulatory actions of pilocarpine and carbachol on ERK1/2 in HSY cells were abolished by pretreatment with the MEK1/2 inhibitor U-0126 (Fig. 1E).


Figure 1
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  		Fig. 1. Activation of ERKs by pilocarpine and carbachol. A: time course of pilocarpine-induced activation of ERK1/2. HSY cells were cultured in DMEM containing 10% FBS for 72 h and in serum-free medium for 18–20 h before treatment with pilocarpine (5 x 10–5 M) for 0–60 min. Cell lysates were subjected to Western blot analysis using antibodies against phosphorylated p44/p42MAPK (pERK1/2), p44/p42MAPK (ERK1/2), and actin. A representative time course of pilocarpine-induced pERK1/2 is shown. Actin and ERK1/2 were used to monitor loading of each lane. B: activation of ERK1/2 by increasing concentrations of pilocarpine. Cells cultured under conditions described in A were treated with increasing concentrations of pilocarpine for 10 min. Western blot represents results from 5 independent experiments. C: activation of ERK1/2 by carbachol. Cells were exposed to increasing concentrations of carbachol for 10 min. Western blot represents results from 3 experiments. D: ERK1/2 activation by maximally stimulatory concentrations of pilocarpine and carbachol. Cells were treated with pilocarpine (5 x 10–5 M) or carbachol (10–4 M) for 10 min, and densities of immunoblotted pERK1/2 and ERK1/2 were quantified with Scion Image analysis software. pERK1/2 induced by pilocarpine or carbachol was normalized to ERK1/2 for each sample and expressed as fold increase relative to the normalized value in untreated [control (CON)] cells. Values are means ± SE (n = 26). *P < 0.001 vs. CON. {dagger}P < 0.001 vs. pilocarpine. E: inhibition of pilocarpine- and carbachol-induced ERK1/2 activation by U-0126. Western blot represents results from cells pretreated with U-0126 (5 x 10–6 M) for 30 min before addition of pilocarpine (5 x 10–5 M), carbachol (10–4 M), or vehicle for 10 min.

 
EGF receptors in HSY cells are transactivated by pilocarpine, but not carbachol. Transactivation of EGF receptors has been shown to mediate muscarinic receptor-induced activation of ERK1/2 in a number of cell types (21, 22, 35). We tested whether EGF receptor transactivation might play a similar role in pilocarpine- or carbachol-induced activation of ERK1/2 in HSY cells. Western blot analysis revealed that pilocarpine treatment of HSY cells caused EGF receptor phosphorylation without affecting total EGF receptor protein levels (Fig. 2A). Pilocarpine-induced activation of EGF receptors exhibited similar kinetics and dose-response characteristics as observed for activation of ERKs (not shown). Phosphorylation of the EGF receptor at Y1068 after 10 min of treatment with pilocarpine (5 x 10–5 M) increased 2.0 ± 0.1 fold (n = 18, P < 0.0001) relative to control levels. In contrast to pilocarpine, carbachol (10–4 M) produced a slight, but significant, reduction (25%, P < 0.005) in EGF receptor phosphorylation at Y1068, despite its stimulatory effect on ERK1/2 (Fig. 2A). EGF receptor phosphorylation by pilocarpine was further confirmed by immunoprecipitation studies, in which treatment of HSY cells with pilocarpine for 10 min increased EGF receptor phosphorylation by 3.3 ± 1.0 fold (n = 6, P < 0.02) relative to untreated cells (Fig. 2B). In addition, pretreatment of HSY cells with the EGF receptor inhibitor tyrphostin AG-1478 reduced basal and pilocarpine-induced EGF receptor (Y1068) phosphorylation and abolished pilocarpine-stimulated ERK1/2 phosphorylation (Fig. 2C). Although pretreatment with AG-1478 significantly (P < 0.001) inhibited carbachol-induced ERK1/2 phosphorylation, the levels of phosphorylated ERKs in response to carbachol treatment remained elevated (P < 0.005) above control. Moreover, in AG-1478-pretreated cells, carbachol stimulated ERK phosphorylation by about sixfold relative to the basal level (i.e., in the presence of AG-1478 alone), which was equivalent to the fold stimulation by carbachol in the absence of AG-1478 (Fig. 2C). These results suggest that pilocarpine-induced ERK1/2 activation is mediated through EGF receptor transactivation. Pathways other than EGF receptor transactivation appear to be involved in mediating carbachol-induced ERK1/2 activation.


Figure 2
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  		Fig. 2. Involvement of epidermal growth factor (EGF) receptor transactivation in pilocarpine-induced activation of ERKs. A: transactivation of EGF receptor by pilocarpine. Cells were cultured and treated with pilocarpine (Pilo, 5 x 10–5 M) or carbachol (Carb, 10–4 M) for 10 min as described in Fig. 1 legend. Primary antibodies against EGF receptor phospho-Y1068 (pEGFR) and EGF receptor (EGFR) were used for Western blots. A representative blot and quantitative results (means ± SE, n = 17) of EGFR phosphorylation are shown. *P < 0.001; {dagger}P < 0.005 vs. CON. B: immunoprecipitation (IP) of EGF receptors. HSY cells were cultured to near confluence in DMEM and treated with pilocarpine (5 x 10–5 M) or vehicle (CON) for 10 min. Cell lysates were incubated with antibody to EGF receptor and precipitated with protein A-Sepharose. Immunocomplex was subjected to SDS-PAGE, and proteins were detected by immunoblot (IB) analysis using antibody against phosphotyrosine (pY) or EGF receptor (EGFR). Quantitation of band densities (means ± SE, n = 6) is also shown. *P < 0.01 vs. CON. C: effect of AG-1478 on pilocarpine- and carbachol-induced phosphorylation of EGF receptor and ERKs. HSY cells were pretreated with or without AG-1478 (5 x 10–6 M) for 30 min before addition of pilocarpine (5 x 10–5 M), carbachol (10–4 M), or vehicle (CON) for 10 min. Representative Western blots and quantitative results (means ± SE, n = 4–6) of ERK1/2 phosphorylation are shown. *P < 0.0001; {dagger}P < 0.005 vs. untreated.

 
ERK activation by carbachol, but not pilocarpine, is PKC dependent in HSY cells. Stimulation of muscarinic receptors causes Gq-mediated activation of PLCβ, leading to generation of DAG and IP3; DAG in turn activates PKC, whereas IP3 increases [Ca2+]i mobilization (3, 30). Activation of PKC has been linked to muscarinic receptor-induced ERK1/2 activation in several cell types (20, 23). To determine whether PKC plays a role in pilocarpine- and carbachol-induced ERK1/2 activation in HSY cells, we measured the effects of the two agonists on ERK phosphorylation under conditions of PKC depletion, i.e., after prolonged (20 h) pretreatment with a high concentration (10–6 M) of PMA (25). Prolonged PMA treatment decreased carbachol-induced ERK1/2 phosphorylation by 60% but had no effect on pilocarpine responsiveness (Fig. 3A). In control experiments, acute treatment of HSY cells with PMA (10–7 M, 15 min) induced ERK phosphorylation, and this effect of PMA was attenuated by the PKC inhibitor GF-109203X (Fig. 3B). Thus, in HSY cells, ERKs are PKC responsive, and ERK activation by carbachol, but not pilocarpine, appears to be PKC dependent.


Figure 3
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  		Fig. 3. Involvement of PKC in carbachol-induced ERK activation. A: pilocarpine- and carbachol-induced ERK phosphorylation: effect of PKC downregulation by prolonged pretreatment with PMA. HSY cells were pretreated with or without PMA (10–6 M) for 20 h before addition of pilocarpine (5 x 10–5 M), carbachol (10–4 M), or vehicle for 10 min. Representative Western blots and quantitative results (means ± SE, n = 3–6) are shown. *P < 0.001 vs. untreated. {dagger}P < 0.0001 vs. carbachol. B: PMA-induced ERK phosphorylation: attenuation by the PKC inhibitor GF-109203X. HSY cells were pretreated with GF-109203X (GFX, 10–6 M) or vehicle for 30 min before addition of PMA (10–7 M) for 15 min. Representative Western blot and quantitative results (means ± SE, n = 3) of ERK1/2 phosphorylation are shown. *P < 0.005 vs. untreated. {dagger}P < 0.005 vs. PMA.

 
Additional experiments were performed to investigate the role of [Ca2+]i signaling in muscarinic receptor-mediated ERK1/2 phosphorylation. We previously showed that carbachol stimulates [Ca2+]i mobilization in HSY cells (47). Consistent with this earlier finding, in the present study, carbachol (10–4 M)-responsive [Ca2+]i mobilization was evident, whereas no [Ca2+]i response to pilocarpine (10–4 M) was detected (Fig. 4A). On depletion of intra- and extracellular Ca2+ pools with EGTA (5 x 10–3 M, 2 h pretreatment), carbachol-induced [Ca2+]i mobilization was completely abolished in HSY cells (Fig. 4B). However, EGTA pretreatment had no effect on carbachol- or pilocarpine-induced ERK1/2 phosphorylation in HSY cells (Fig. 4C). Carbachol and pilocarpine responses were also unaffected by pretreatment of cells with BAPTA-AM, another [Ca2+]i chelator (data not shown). Thus, in contrast to PKC, [Ca2+]i signaling appears to play no role in ERK activation by pilocarpine or carbachol.


Figure 4
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  		Fig. 4. Lack of involvement of intracellular Ca2+ ([Ca2+]i) mobilization in pilocarpine- and carbachol-induced ERK activation. A: [Ca2+]i mobilization by carbachol but not pilocarpine. Fura 2-AM-loaded HSY cells were treated with pilocarpine (10–4 M) followed by carbachol (10–4 M), and [Ca2+]i responses were determined. [Ca2+]i trace represents results from 3 independent experiments. Arrows indicate addition of agonist. B: blockade of carbachol-induced [Ca2+]i mobilization by EGTA. HSY cells were pretreated with the Ca2+-chelating agent EGTA (5 x 10–3 M) for 2 h, and carbachol-induced [Ca2+]i responsiveness was measured as described in A. A representative trace is shown. C: carbachol- and pilocarpine-induced ERK phosphorylation in EGTA-treated HSY cells. Cells were incubated with EGTA (5 x 10–3 M) for 2 h before addition of carbachol (10–4 M), pilocarpine (5 x 10–5 M), or vehicle for 10 min, and cell lysates were subjected to Western blot analysis. Western blot represents results from 3 independent experiments.

 
Involvement of M1 and M3 muscarinic receptor subtypes in pilocarpine- and carbachol-induced ERK activation in HSY cells. We examined whether pilocarpine and carbachol activate ERKs in HSY cells via signaling through muscarinic receptor subtypes predominating in salivary cells, i.e., M1 and M3. Pretreatment of HSY cells with the nonselective muscarinic receptor antagonist atropine (10–6 M, 30 min) reduced the stimulatory effects of pilocarpine and carbachol on ERK activation (Fig. 5A). HSY cells apparently possess M1 and M3 muscarinic receptors, insofar as carbachol-induced [Ca2+]i mobilization in these cells is attenuated by selective inhibition of M1 and M3 receptors with pirenzepine and 4-DAMP, respectively (data not shown). Exposure of HSY cells to the M3 receptor inhibitor 4-DAMP blocked ERK responses to both pilocarpine and carbachol (Fig. 5B). Pretreatment of cells with the M1 inhibitor pirenzepine attenuated carbachol-induced ERK1/2 phosphorylation by 52% (P < 0.005) but had no apparent effect on pilocarpine responsiveness (Fig. 5C). Interestingly, 4-DAMP and pirenzepine each caused an elevation of basal phosphorylated ERK1/2 (~2.0-fold relative to untreated cells). Recent studies indicate that a single agent may act as receptor antagonist and partial agonist, depending on receptor interactions with downstream signaling effectors (2, 10). In this respect, 4-DAMP and pirenzepine may be partial agonists for receptor-mediated signals leading to basal ERK activation.


Figure 5
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  		Fig. 5. Effects of M1 and M3 muscarinic receptor antagonists on pilocarpine- and carbachol-induced ERK activation. HSY cells were preincubated with or without nonselective muscarinic receptor antagonist atropine (10–6 M, A), M3 muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate (4-DAMP, 10–6 M, B), or M1 muscarinic receptor antagonist pirenzepine (10–6 M, C) for 30 min before addition of pilocarpine (5 x 10–5 M), carbachol (10–4 M), or vehicle for 10 min. Representative Western blots of pERK1/2 and ERK1/2 and quantitative results (means ± SE) of ERK1/2 phosphorylation are shown. In A (n = 4–6), *P < 0.0001 vs. untreated; {dagger}P < 0.001 vs. pilocarpine; {ddagger}P < 0.0001 vs. carbachol. In B (n = 3–7), *P < 0.0001 vs. untreated; {dagger}P < 0.003 vs. pilocarpine; {ddagger}P < 0.0001 vs. carbachol. In C (n = 3), *P < 0.006 vs. untreated; {dagger}P < 0.005 vs. carbachol.

 
To confirm the role of M1 and M3 receptors in ERK activation, we performed experiments in which HSY cells were transfected with M1 or M3 siRNA before stimulation with agonists. In transfected cells, immunoreactive M1 and M3 receptor protein levels were reduced by 46% (n = 2) and 41% (n = 2), respectively, compared with controls (Fig. 6A). Similar to our results with muscarinic receptor-specific inhibitors, ERK1/2 activation by pilocarpine was inhibited by transfection with M3 siRNA but was unaffected by M1 siRNA; in control experiments, transfection of irrelevant siRNA (GAPDH) also did not affect the pilocarpine response (Fig. 6, B and C). M1 and M3 siRNA transfection had no apparent effect on carbachol-induced ERK activation (Fig. 6D). Taken together, our results suggest that pilocarpine-induced activation of ERK1/2 is mediated primarily via M3 muscarinic receptors. Blockade of the carbachol response by M1- and M3-specific antagonists implicates carbachol actions at both muscarinic receptor subtypes, in which case the lack of appreciable inhibition by either siRNA probably reflects incomplete knockdown of the two receptors.


Figure 6
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  		Fig. 6. Effects of M1 and M3 small interfering RNAs (siRNAs) on pilocarpine- and carbachol-induced ERK activation. HSY cells were transfected with M1 or M3 siRNA for 72 h and stimulated with pilocarpine (5 x 10–5 M) or carbachol (10–4 M) for 10 min. A: Western blot analysis of M3 and M1 receptors. ERKs were used as loading controls. CON, untransfected control; Lipo, Lipofectamine treatment; siM3 and siM1, transfection with siRNA against M3 and M1 receptor, respectively. B: cells were transfected with irrelevant siRNA (GAPDH) and then stimulated by pilocarpine. Western blot analysis of pilocarpine-induced ERK activation is shown. C: Western blot analyses of pilocarpine-responsive ERK phosphorylation in cells transfected with M3 (left) or M1 (right) siRNA. D: carbachol responses after transfection with M3 (left) or M1 (right) siRNA. Quantitative results from 3–4 experiments are shown as fold increase in pERK-to-ERK ratio relative to values from cells treated with Lipofectamine alone. *P < 0.05.

 
Involvement of Src in pilocarpine-induced ERK activation, but not carbachol responsiveness. In a variety of cell types, GPCR-induced stimulation of ERKs by transactivation of EGF receptors is dependent on activation of nonreceptor protein kinases of the Src family (32, 46). To determine whether Src mediates pilocarpine- or carbachol-induced ERK activation in HSY cells, we pretreated cells with the selective Src family inhibitor PP2 before addition of muscarinic agonist. Incubation with PP2 significantly attenuated pilocarpine-induced ERK1/2 phosphorylation with negligible effect on carbachol responsiveness (Fig. 7A). Pretreatment of cells with PP3, an inactive PP2 analog, had no inhibitory effect on ERK activation by either agent (Fig. 7B). PP2 also blocked pilocarpine-induced EGF receptor phosphorylation (Fig. 7C). These results indicate that pilocarpine-induced ERK activation involves Src at a signaling step proximal to EGF receptor transactivation. In contrast, carbachol-induced ERK activation appears to be Src independent.


Figure 7
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  		Fig. 7. Effect of Src inhibition on pilocarpine- and carbachol-induced ERK activation. A: pilocarpine- and carbachol-induced ERK1/2 phosphorylation in the absence and presence of the Src inhibitor PP2. Incubation of HSY cells with or without PP2 (5 x 10–6 M) for 2 h was followed by addition of pilocarpine (5 x 10–5 M), carbachol (10–4 M), or vehicle for 10 min. Representative Western blots of pERK1/2 and ERK1/2 and quantitative results (means ± SE, n = 3–6) of ERK phosphorylation are shown. *P < 0.0001 vs. untreated. {dagger}P < 0.004 vs. pilocarpine. B: no effect of the inactive PP2 analog PP3 on pilocarpine- and carbachol-induced ERK activation. Cells were treated with PP3 (5 x 10–6 M) for 2 h and pilocarpine, carbachol, or vehicle was added as described in A. A representative Western blot is shown. C: effect of PP2 on pilocarpine-induced EGF receptor phosphorylation. Western blot analysis of cell lysates for phosphorylated EGF receptor [pEGFR(Y1068)] and total EGF receptor and quantitative results (means ± SE, n = 3–6) of EGFR phosphorylation are shown. *P < 0.0001 vs. untreated. {dagger}P < 0.0001 vs. pilocarpine.

 
Activation of ERK by pilocarpine and carbachol in rat parotid acinar cells. To determine whether stimulation of muscarinic receptors also induces activation of ERKs in normal salivary cells, we prepared rat parotid acinar cells for studies examining ERK phosphorylation by pilocarpine and carbachol. As in HSY cells, pilocarpine and carbachol each activated ERK1/2 in normal parotid cells (Fig. 8A). Furthermore, [Ca2+]i mobilization in parotid cells was elicited by carbachol, with negligible response to pilocarpine (Fig. 8B). These results indicate that the actions of pilocarpine and carbachol in HSY cells also extend to native salivary tissue.


Figure 8
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  		Fig. 8. Effects of pilocarpine and carbachol on ERK activation and [Ca2+]i mobilization in rat parotid acinar cells. A: acinar cell aggregates were treated with pilocarpine (5 x 10–5 M) or carbachol (10–4 M) for 10 min, and cell homogenates were subjected to immunoblot analysis using antibodies against pERK1/2 and ERK1/2. A representative Western blot and quantitative results (means ± SE, n = 3–4) of ERK phosphorylation are shown. *P < 0.05 vs. control. B: [Ca2+]i mobilization by carbachol and pilocarpine was monitored in acinar cell aggregates. A representative [Ca2+]i trace is shown. Arrows indicate addition of agonists.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The muscarinic agonist pilocarpine is commonly used to stimulate saliva secretion and, thereby, alleviate symptoms of xerostomia (dry mouth). The cellular signals mediating the action of pilocarpine on salivary gland tissues have not been clarified. In the present study, using the human salivary epithelial HSY cell line, we have demonstrated that pilocarpine activates the ERK cascade through muscarinic receptor-induced transactivation of EGF receptors, but not through classical muscarinic receptor-linked [Ca2+]i and PKC pathways. The signaling properties of pilocarpine leading to ERK activation in HSY cells differ from those of the muscarinic agonist carbachol. In contrast to carbachol, pilocarpine does not evoke [Ca2+]i mobilization. Although carbachol also activates ERK1/2, this action, in contrast to that of pilocarpine, is mediated through a PKC-dependent pathway. Thus the present study delineates the cellular actions of two muscarinic receptor agonists converging on the ERK cascade through two distinct signaling pathways.

Muscarinic receptor agonists activate ERKs in a variety of cell types (4, 14, 16, 20–22, 31, 32, 35, 39). In most systems studied, carbachol was employed as the agonist, with one exception, in which pilocarpine was used (4). In our experiments, activation of ERK1/2 in HSY cells by pilocarpine and carbachol was reduced by treatment with the MAPK kinase (MEK) inhibitor U-0126, indicating involvement of the classical Raf1-MEK-ERK1/2 cascade in the actions of both muscarinic agonists. Previous studies of carbachol responsiveness in other systems revealed at least two upstream pathways leading to muscarinic receptor-induced ERK1/2 activation: one involving the transactivation of EGF receptors and the other dependent on classical PLCβ-IP3/DAG-[Ca2+]i/PKC signaling.

Muscarinic receptor-induced transactivation of EGF receptors, with downstream activation of ERKs, has been demonstrated in several cell types (21, 22, 35, 38). In the present study, pilocarpine was found to increase EGF receptor phosphorylation in HSY cells (Fig. 2). However, in contrast to several other cell types in which carbachol elicits EGF receptor transactivation (21, 22, 35), in HSY cells no increase in EGF receptor phosphorylation following carbachol treatment was detectable using antibody against Y1068 (Fig. 2) or EGF receptor immunoprecipitation (data not shown). In HSY cells pretreated with the EGF receptor inhibitor AG-1478, pilocarpine-induced activation of ERK1/2 was completely abolished, whereas ERK activation by carbachol remained substantial (Fig. 2). Thus signaling pathways other than EGF receptor transactivation appear to be involved in carbachol-stimulated ERK1/2 phosphorylation in HSY cells. A decrease in carbachol-responsive ERK activation after AG-1478 pretreatment appears to reflect diminished basal levels of ERK activity (Fig. 2). The molecular mechanisms underlying Gq protein-coupled receptor-induced transactivation of EGF receptors are largely unknown but may involve Gq-mediated shedding of heparin-binding EGF (27). Similarly, the cellular signals linking pilocarpine stimulation to EGF receptor transactivation in HSY cells require further investigation (see also below).

PKC is thought to mediate muscarinic-responsive ERK activation in some systems (5, 20, 33, 44), but not others (19, 32). In the present study, rapid phosphorylation of ERKs by the PKC activator PMA, with attenuation of the PMA effect by the PKC inhibitor GF-109203X (Fig. 3B), indicates that PKC activation can induce ERK signaling in HSY cells. However, pilocarpine-responsive ERK activation in HSY cells does not appear to be mediated by PKC, insofar as PKC depletion by prolonged treatment with PMA did not affect pilocarpine-induced ERK1/2 phosphorylation (Fig. 3A). In contrast to the pilocarpine response, carbachol-induced ERK activation was markedly attenuated by PKC depletion (Fig. 3A). PKC is a family of serine/threonine kinases classified into three subgroups: Ca2+-dependent, or conventional, PKCs; novel, Ca2+-independent and DAG-dependent PKCs; and Ca2+/DAG-independent, or atypical, PKCs (17, 18). Carbachol stimulation of ERKs in some cell types has been linked to signaling via specific novel or atypical PKC subtypes (20, 44), although the role of specific PKC subtypes in the carbachol response of HSY cells remains to be determined.

Muscarinic receptor-induced [Ca2+]i mobilization has been reported to mediate activation of ERKs in some, but not all, systems studied (6, 14, 19–22, 41, 42, 44). In HSY cells, pilocarpine failed to increase [Ca2+]i mobilization (Fig. 4A), and depletion of intra- and extracellular Ca2+ with EGTA had no effect on pilocarpine-induced ERK activation (Fig. 4C). Although pilocarpine is widely considered a muscarinic receptor agonist, reports on the effects of pilocarpine on [Ca2+]i mobilization in salivary cells are limited; in these few instances, the [Ca2+]i response to pilocarpine was much less than that elicited by other muscarinic agonists, e.g., carbachol (36). The HSY cell line was originally derived from human parotid adenocarcinoma. Nevertheless, in experiments with rat parotid acinar aggregates (Fig. 8), we observed a profile of [Ca2+]i and ERK responsiveness to pilocarpine and carbachol equivalent to that demonstrated in HSY cells. Therefore, HSY cells appear to provide an adequate model for muscarinic signaling in normal salivary tissue. Notwithstanding a marked [Ca2+]i response to carbachol in HSY cells (Fig. 4A) (47), ERK activation by this agonist was unaffected by EGTA (Fig. 4C). Our results suggest that stimulation of ERKs by pilocarpine and carbachol in HSY cells occurs independently of [Ca2+]i mobilization and also provide evidence that any PKC(s) involved in the carbachol response is not Ca2+ dependent, i.e., is not of the conventional subtype.

In salivary gland cells, muscarinic receptors are predominantly of the M1 and M3 subtypes, although M2 and M4 subtypes have also been documented (9). Muscarinic receptor subtypes in HSY cells are not well characterized. In our studies, the [Ca2+]i response to carbachol was attenuated by pretreatment of cells with the M1-selective muscarinic antagonist pirenzepine or the M3 antagonist 4-DAMP, suggesting that HSY cells possess M1 and M3 receptors (data not shown). Pilocarpine- and carbachol-induced ERK activation was mediated through muscarinic receptors, since the nonselective muscarinic receptor antagonist atropine blocked the ERK response to either agent (Fig. 5A). 4-DAMP inhibited the stimulatory effects of pilocarpine and carbachol on ERK1/2 phosphorylation (Fig. 5B), and downregulation of M3 by siRNA attenuated pilocarpine-induced ERK activation (Fig. 6C). On the other hand, pirenzepine had no effect on pilocarpine-induced ERK activation but partially inhibited the carbachol response (Fig. 5C). On the basis of these results, we conclude that activation of ERKs by pilocarpine occurs mainly through the M3 receptor subtype, whereas M1 and M3 subtypes appear to contribute to carbachol-induced ERK activation. Our findings are consistent with the consensus view that the pharmacological action of pilocarpine on salivary secretion is mediated through muscarinic M3 receptors in salivary gland acinar cells (43). Whether the higher level of ERK activation induced in HSY cells by carbachol than by pilocarpine (Fig. 1) might reflect more receptor subtypes available to carbachol is not known. Whereas the majority of muscarinic receptors in rat parotid glands are of the M3 subtype (7), the muscarinic receptor subtypes in the major salivary glands of humans are less well characterized because of difficulties in obtaining clinical or autopsy tissue specimens. M1 receptors do exist in human minor salivary gland secretory cells, and the role of this receptor subtype in saliva secretion requires further clarification (34). Studies using transgenic M1-, M3-, and M1/M3-knockout mouse models have shown that the M1 subtype plays a role in saliva secretion especially under the condition of high-dose pilocarpine stimulation (29). Nerve-stimulated saliva secretion in sheep has also been found to be reduced after blockade of M1 receptors with specific antagonist (37). It remains unknown whether muscarinic receptor subtypes other than M1 and M3 (i.e., M2, M4, or M5) are expressed in HSY cells or play a role in mediating pilocarpine- and carbachol-induced ERK activation.

Src family protein tyrosine kinases are thought to act as early signaling intermediates in the pathway by which muscarinic agonists stimulate ERKs through transactivation of EGF receptors (1, 21, 22, 35). In contrast, muscarinic receptor-induced ERK activation mediated via PKC may be Src independent (5, 24, 39). In the present study, pretreatment of HSY cells with the Src inhibitor PP2 blocked pilocarpine-stimulated phosphorylation of EGF receptors and ERKs but had no apparent effect on carbachol-responsive ERK activation (Fig. 7). These findings support the notion that Src acts as an early signal mediating pilocarpine-induced EGF receptor transactivation and ERK activation yet plays no regulatory role in carbachol stimulation of ERKs via a PKC-dependent pathway.

Muscarinic receptor-induced ERK activation has been associated not only with cell proliferation (20, 38, 44), but also with secretory function in several cell systems (21, 23, 31). Activation of ERKs is linked to muscarinic-responsive Na-HCO3 cotransport activity in kidney cells and mucin secretion in conjunctival goblet cells and may negatively regulate carbachol-stimulated Cl– secretion in colonic epithelial cells (21, 23, 31). Before the present study, pilocarpine was generally believed to stimulate salivary flow rates as an immediate consequence of muscarinic receptor-induced [Ca2+]i mobilization. Our results suggest that the secretory action of pilocarpine is mediated through signaling pathways involving stimulation of ERKs, but not [Ca2+]i mobilization. Alternatively, pilocarpine action on salivary secretion could be mediated indirectly through cholinergic receptors in the autonomic nervous system, which in turn trigger [Ca2+]i mobilization and secretion in salivary cells. Interestingly, in clinical studies, xerostomic patients experienced maximal therapeutic benefit from pilocarpine only after prolonged (>12 wk) treatment (11, 12). Whether mitogenic responses to pilocarpine-induced ERK activation might play a role in regeneration of salivary secretory tissue over the longer term remains an intriguing, albeit untested, possibility. Recent work from our laboratory (46) demonstrated that CD44, a cell adhesion molecule involved in growth and differentiation, is a downstream effector of ERK activation in HSY cells. However, the role of CD44 or other effectors in the action of pilocarpine on salivary cell growth and secretion is yet to be explored.

In summary, we have demonstrated that two muscarinic ligands induce ERK activation in HSY cells by distinct signaling pathways: 1) pilocarpine via muscarinic M3 receptors and Src-dependent transactivation of EGF receptors and 2) carbachol via M1/M3 receptors and activation of PKCs presumably of other than the conventional subtype (Fig. 9). To our knowledge, ERK signaling by distinct muscarinic ligand-dependent pathways intrinsic to a single cell type has not been reported previously. Our observations are in accord with earlier work indicating that activation of transfected M1 receptors by different ligands can stimulate distinct signaling pathways (15). Related studies have recently demonstrated ligand-dependent coupling of transfected β1- and β2-adrenergic receptors to different signal transduction effectors, i.e., adenylyl cyclase and ERKs (10). Ligand selectivity of receptor signaling may be attributable to ligand-dependent stabilization of different receptor conformations favoring distinct receptor affinities for G protein(s) and/or distal effectors (10, 15). Additional investigations into the molecular pharmacology of ligand-dependent muscarinic receptor functions in salivary cells will be critical to the development of effective drug therapies for salivary gland hypofunction and possibly for muscarinic-cholinergic-associated neurological disorders such as Alzheimer's disease and seizures.


Figure 9
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  		Fig. 9. Schematic illustration of distinct signaling pathways involved in pilocarpine- and carbachol-induced ERK1/2 activation in HSY cells. Pilocarpine binds to muscarinic receptors of the M3 subtype, causing Src-dependent EGF receptor (EGFR) transactivation and subsequent stimulation of the classical Ras/c-Raf1 cascade of ERK activation (right). In contrast, binding of carbachol to M1 and M3 receptor subtypes results in stimulation of PKC, which is known to activate Ras or c-Raf-1 (left). Signaling intermediates tested in the present study are shown in bold type. Sequence of individual signaling intermediates involved in the 2 pathways is adapted from recent consensus views of G protein-coupled receptor-mediated ERK activation (27). [Ca2+]i mobilization, which is stimulated by carbachol and linked to salivary fluid secretion, is not involved in either pathway of ERK activation.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institute of Dental and Craniofacial Research Grant R21 DE-15381 (to C.-K. Yeh) National Heart, Lung, and Blood Institute Grant R01-HL-075011 (to B.-X. Zhang), and Department of Veterans Affairs Merit Awards (to C.-K. Yeh and M. S. Katz).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C.-K. Yeh, Geriatric Research, Education and Clinical Center (182), Audie L. Murphy Division, South Texas Veterans Health Care System, 7400 Merton Minter Blvd., San Antonio, TX 78229-4404 (e-mail: yeh{at}uthscsa.edu)

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


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