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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 257-262, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0128OC

Muscarinic M3-Receptors Mediate Cholinergic Synergism of Mitogenesis in Airway Smooth Muscle

Reinoud Gosens, S. Adriaan Nelemans, Mechteld M. Grootte Bromhaar, Sue McKay, Johan Zaagsma and Herman Meurs

Department of Molecular Pharmacology, University Centre for Pharmacy, Groningen, The Netherlands

Address correspondence to: Reinoud Gosens, Department of Molecular Pharmacology, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail: R.Gosens{at}farm.rug.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [3H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M3-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 µM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1–10 µM), but only in cells having functional muscarinic M3-receptors. This synergism could be suppressed significantly by the selective M3-receptor antagonists DAU 5884 (0.1 µM) and 4-DAMP (10 nM), but not at all by the M2-subtype selective antagonist gallamine (10 µM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M3-receptors in bovine tracheal smooth muscle cells.

Abbreviations: airway smooth muscle, ASM • bovine tracheal smooth muscle, BTSM • Chinese Hamster Ovary, CHO • Dulbecco's modified Eagle's medium, DMEM • extracellular signal regulated kinase, ERK • fetal bovine serum, FBS • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • platelet-derived growth factor, PDGF • protein kinase C, PKC • pertussis toxin, PTX


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Airway smooth muscle (ASM) expresses muscarinic receptors of the M2- and the M3-subtype. The M3-subtype is responsible for contraction, largely through Gq-mediated activation of phosphoinositide turnover and subsequent Ca2+ mobilization (1). The role of the majority of M2-receptors in ASM is still unclear, however. In bovine tracheal smooth muscle (BTSM), a role for the M2-receptor in functional antagonism of ß-adrenergic responses through inhibition of adenylyl cyclase has been suggested. This was observed only after substantial 4-DAMP mustard-induced alkylation of the M3-receptor population (2); under normal conditions, M2-receptors are not interfering with the isoprenaline-induced relaxation of cholinergic tone, as demonstrated by the lack of effect of selective M2-receptor blockade (3). Recently, however, a role for M2-receptors in Ca2+ sensitization and cytoskeletal reorganization has been proposed (4–6). Furthermore, M2-receptors may stimulate nonselective cation channels through Gi/G(o) proteins, resulting in a rise in [Ca2+]i (7).

Muscarinic receptor agonists have been reported to be mitogenic for human ASM cells, though at most modestly, and to respond synergistically in combination with growth factors (8, 9). Although carbachol-induced mitogenesis has been reported to be pertussis toxin (PTX)-sensitive (8, 10), suggesting a role for the Gi-protein–coupled muscarinic M2-receptor, measurements were performed using human ASM cells in culture, which are known to have a relatively small functional M3-receptor population compared with noncultured cells. This loss in receptor function is far less profound for the M2-receptor subtype (11). A role for the M3-subtype in proliferation can therefore not be ruled out, nor can the putative relevance of the M2-subtype in ASM proliferation be properly estimated because of the diminished presence of muscarinic M3-receptors. Moreover, although PTX was found to decrease the carbachol-induced mitogenic response, proliferation to all applied stimuli was reduced to a similar extent by treatment with PTX (8). Therefore, it may not be appropriate to conclude that muscarinic receptor stimulation–induced mitogenic responses are M2-receptor–mediated.

Theoretically, both M2- and M3-receptors could account for the mitogenic contribution of muscarinic receptor stimulation. The mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 (MAPK/ERK) pathway is generally associated with proliferation and is known to be involved in the proliferative responses to various mitogens in BTSM (12–15). Muscarinic M3-receptor stimulation induces a considerable rise in [Ca2+]i, which may lead to the activation of MAPK/ERK through Pyk2- and Ras-dependent mechanisms (16). Moreover, the M3-receptor subtype activates protein kinase C (PKC), which may lead to MAPK/ERK activity through PKC{alpha}-mediated phosphorylation of Raf-1 (17). In support of this hypothesis, Chinese Hamster Ovary (CHO) cells transfected with the wild-type M3-receptor subtype, demonstrated activation of the MAPK/ERK pathway induced by carbachol (18). This M3-receptor–mediated MAPK/ERK activity was inhibited almost completely using the PKC inhibitor Ro-318220. Moreover, stimulation with the PKC activator PDBu could partially mimic this response, suggestive of a significant role for PKC in the response (18, 19).

Gi proteins, however, activated by muscarinic M2-receptors, may also activate the MAPK/ERK pathway through either Gi{alpha}- (20) or Giß{gamma}-dependent (21) mechanisms. In canine colonic smooth muscle, it has been demonstrated that M2- rather than M3-receptors are responsible for MAPK/ERK activation (22). Moreover, it has been demonstrated that M2-receptors activate the nonreceptor tyrosine kinase Src in the same tissue (23), which acts as a key intermediate in tyrosine kinase signaling. In addition, in CHO cells transfected with the wild-type M2-receptor, methacholine-induced MAPK/ERK activation has been reported (24).

Proliferative responses following selective M2- or M3-muscarinic receptor stimulation in ASM have not yet been described. In the present study, we investigated their putative involvement in BTSM cells. Because muscarinic M3-receptors have been reported to lose their function rapidly in culture (11), we used both passaged and unpassaged BTSM cells. To gain insight into the receptor subtype(s) involved in methacholine-induced mitogenic responses, we studied the effects of subtype-selective receptor blockade. It was demonstrated that mitogenic responses to muscarinic receptor stimulation alone were absent. However, the mitogenic responses to platelet-derived growth factor (PDGF) were augmented by methacholine, which was solely mediated by the M3-receptor subtype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Dulbecco's modification of Eagle's Medium (DMEM) and methacholine hydrochloride were obtained from ICN Biomedicals (Costa Mesa, CA). Fetal bovine serum (FBS), NaHCO3 solution (7.5%), HEPES solution (1 M), sodium pyruvate solution (100 mM), nonessential amino acid mixture, gentamycin solution (10 mg/ml), penicillin/streptomycin solution (5,000 U/ml/5,000 µg/ml), amphotericin B solution (250 µg/ml) (Fungizone), and trypsin were obtained from Gibco BRL Life Technologies (Paisley, UK). PDGF AB (human recombinant), insulin (from bovine pancreas), apotransferrin (human), soybean trypsin inhibitor, and gallamine triethiodide were obtained from Sigma Chemical Co. (St. Louis, MO). DAU 5884 was a kind gift of Dr. H. N. Doods (Dr. Karl Thomae GmbH, Biberach, Germany), and 4-DAMP methobromide was kindly provided by Dr. R. B. Barlow, Bristol, UK. [Methyl-3H]-thymidine (specific activity 25 Ci/mmol) was obtained from Amersham (Buckinghamshire, UK). [3H]myo-inositol (specific activity 59.9 Ci/mmol) was obtained from NEN Life Sciences Products (Boston, MA). Papain and collagenase P were from Roche Diagnostics (Mannheim, Germany). All other chemicals were of analytical grade.

Isolation of Bovine Tracheal Smooth Muscle Cells
Bovine tracheae were obtained from local slaughterhouses and transported to the laboratory in Krebs-Henseleit buffer of the following composition (mM): NaCl 117.5, KCl 5.60, MgSO4 1.18, CaCl2 2.50, NaH2PO4 1.28, NaHCO3 25.00, and glucose 5.50, pregassed with 5% CO2 and 95% O2; pH 7.4. After dissection of the smooth muscle layer and removal of mucosa and connective tissue, tracheal smooth muscle was chopped using a McIlwain tissue chopper, three times at a setting of 300 µm and three times at a setting of 100 µm. Tissue particles were washed two times with DMEM, supplemented with NaHCO3 (7 mM), HEPES (10 mM), sodium pyruvate (1 mM), nonessential amino acid mixture (1:100), gentamicin (45 µg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (1.5 µg/ml), and 0.5% FBS. Enzymatic digestion was performed using the same medium, supplemented with collagenase P (0.75 mg/ml), papain (1 mg/ml), and Soybean trypsin inhibitor (1 mg/ml). During digestion, the suspension was incubated in an incubator shaker (Innova 4000; New Brunswick Scientific, Edison, NJ) at 37°C, 55 rpm for 20 min, followed by a 10-min period of shaking at 70 rpm. After filtration of the obtained suspension over 50 µm gauze, cells were washed three times in DMEM supplemented as above and containing 10% FBS.

Cell Culture
After isolation, BTSM cells were either used directly for experiments (unpassaged cells) or seeded in 25 cm2 culture flasks at a density of 1 x 106 cells/ml for further culturing. Cultured cells were kept viable in medium containing 10% FBS at 37°C in a humidified 5% CO2 incubator. Medium was refreshed every 48–72 h. Cell cultures were allowed to grow and, upon confluence, were passaged further at a 1:2 split ratio by means of trypsinization. Cultured cells were used for experiments in passage 2.

[3H]Thymidine Incorporation
BTSM cells were plated in 24-well cluster plates at a density of 30,000 cells per well, and were allowed to attach overnight in 10% FBS containing medium at 37°C in a humidified 5% CO2 incubator. Cells were washed two times with sterile phosphate-buffered saline (PBS, composition [mM] NaCl, 140.0; KCl, 2.6; KH2PO4, 1.4; Na2HPO4.2H2O, 8.1; pH 7.4) and made quiescent by incubation in FBS-free medium, supplemented with apo-transferrin (5 µg/ml), ascorbate (100 µM), and insulin (1 µM) for 72 h. Cells were then washed with PBS and stimulated with mitogens in FBS-free medium for 28 h, the last 24 h in the presence of [3H]thymidine (0.25 µCi/ml), followed by two washes with PBS at room temperature and one with ice-cold 5% trichloroacetic acid. Cells were treated with this trichloroacetic acid solution on ice for 30 min; subsequently, the acid-insoluble fraction was dissolved in 1 ml NaOH (1 M). Incorporated [3H]thymidine was quantified by liquid-scintillation counting.

Accumulation of [3H]inositol Phosphates
BTSM cells were plated after isolation or after passage 2 in six-well cluster plates at a density of 1 x 106 cells/well. After attachment overnight in medium containing 10% FBS at 37°C in a humidified 5% CO2 incubator, cells were washed twice in sterile PBS and treated for 72 h with serum-free medium containing apo-transferrin (5 µg/ml), ascorbate (100 µM), insulin (1 µM), and [3H]inositol (2 µCi/ml). Next, cells were washed twice with Ringer buffer containing (in mM) NaCl 125.0, KCl 6.0, MgCl2 2.5, CaCl2 1.2, NaH2PO4 1.2, HEPES 25.0, and glucose 11.0, pH 7.4. After a 15-min incubation period in the same buffer, supplemented with 5 mM LiCl, cells were stimulated with methacholine in varying concentrations for another 30 min. Reactions were terminated by replacing the Ringer buffer for 1 ml of a methanol:0.12 mM HCl mixture (1:1 vol/vol), which had been previously kept at -20°C, and cells were allowed to lyse for another 30 min at -20°C. A quantity of 800 µl of the lysate was neutralized to pH = 7 using 3 ml buffer (composition 25 mM Tris / 0.5 M NaOH/H2O 10:1:30 vol/vol/vol), and [3H]inositol phosphates were finally separated form free [3H]inositol using Dowex-AG 1X8 anion exchange chromatography as described by Hoiting and coworkers (25).

Data Analysis
All data represent means ± SEM from n separate experiments. The statistical significance of differences between data was determined by the Student's t test for paired observations or one-way ANOVA where appropriate. Differences were considered to be statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Methacholine-Induced Accumulation of Inositol Phosphates in Unpassaged and Passage 2 BTSM Cells
BTSM cells treated for 1 d with 10% FBS, followed by 3 d with serum-deprived medium (unpassaged BTSM cells), responded to methacholine with an increase in the accumulation of inositol phosphates (maximal effect [Emax] = 348 ± 43% of basal, sensitivity [pEC50] = 5.61 ± 0.21). In contrast, in cells cultured up to passage 2 no dose-dependent effects of methacholine could be determined. In this condition, basal formation of inositol phosphates remained unchanged for all concentrations of methacholine applied (average 98 ± 6%, Figure 1) .



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  		Figure 1. Methacholine-induced inositol phosphates (IPx)-accumulation in unpassaged (open circles) and passage 2 (closed circles) BTSM cells. Data represent means ± SEM from three experiments, each performed in triplicate.

 
Effects of Methacholine, Alone and in Combination with PDGF on [3H]thymidine Incorporation in Unpassaged and Passage 2 BTSM Cells
Stimulation of passage 2 BTSM cells with PDGF (10 ng/ml) induced an increase in [3H]thymidine incorporation to 255 ± 46% of basal. Methacholine (10 µM) did not induce any proliferative response (91 ± 14% of basal), nor did it potentiate the proliferative response to PDGF (295 ± 59%, Figure 2A) .



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  		Figure 2. [3H]thymidine incorporation induced by 10 µM methacholine (MCh), 10 ng/ml PDGF, and the combination in both passaged (passage 2) BTSM cells (A) and unpassaged BTSM cells (B). Data represent means ± SEM from four (passaged) and six (unpassaged) experiments, each performed in triplicate. *P < 0.05 compared with basal; {dagger}P < 0.05 compared with PDGF.

 
In unpassaged BTSM cells, methacholine (10 µM) was unable to produce a proliferative response by itself (114 ± 14%). Interestingly, the PDGF-induced increase in incorporated thymidine was significantly potentiated in these cells (246 ± 33% and 371 ± 59% for PDGF and PDGF + methacholine-treated cells, respectively; P < 0.05) Note that passage number did not influence the PDGF response (Figure 2B).

The effects of methacholine appeared to be concentration-dependent. Although at concentrations of 0.1–10 µM methacholine was unable to induce a proliferative response by itself, the agonist raised the Emax and reduced the EC50 of PDGF in a concentration-dependent fashion, which was most pronounced at 10 µM methacholine (Emax = 326 ± 45% and 421 ± 46%, P < 0.001; and EC50 = 4.8 ± 0.8 and 3.0 ± 0.5 ng/ml, P < 0.05 for PDGF and PDGF + 10 µM methacholine, respectively; Figure 3) .



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  		Figure 3. Dose-dependent [3H]thymidine incorporation in response to PDGF in unpassaged BTSM cells in the absence (open circles) or presence of 0.1 µM (filled circles, A), 1 µM (filled squares, B), and 10 µM methacholine (filled triangles, C). Data represent means ± SEM from 6 (0.1 and 1 µM methacholine) and 12 (10 µM methacholine) experiments, each performed in triplicate. *P < 0.05; ***P < 0.001 compared with control.

 
Effects of Subtype-Selective Antagonists on Methacholine and PDGF-Induced Synergism in [3H]thymidine Incorporation
To establish the muscarinic receptor subtype(s) involved in the mitogenic responses to methacholine, we measured the inhibitory effects of selective receptor antagonists (DAU 5884, 4-DAMP, and gallamine) on the methacholine-induced potentiation of the proliferative response to PDGF. Synergism was calculated as the methacholine-induced increase in the PDGF response and expressed relative to a control response (10 ng/ml PDGF). As shown in Figure 4A , synergism was dependent on the dose of methacholine applied. Maximal methacholine-induced synergism averaged 8.9 ± 10.6, 36.0 ± 15.9, and 55.3 ± 11.9% for 0.1, 1, and 10 µM methacholine, respectively.



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  		Figure 4. (A) Synergism in [3H]thymidine incorporation in unpassaged BTSM cells, induced by 0.1 µM (triangles), 1 µM (inverted triangles), and 10 µM (open circles) methacholine in combination with PDGF. Data represent means ± SEM from 6–12 experiments, each performed in triplicate. (B) Synergism in [3H]thymidine incorporation in unpassaged BTSM cells, induced by 10 µM methacholine in combination with PDGF in the presence of 0.1 µM DAU 5884 (diamonds), 10 nM 4-DAMP (filled circles), or 10 µM gallamine (squares). Data represent means ± SEM from four to six experiments, each performed in triplicate. *P < 0.05 compared with absence of antagonist.

 
The potentiation induced by 10 µM methacholine was almost completely suppressed by DAU 5884 (0.1 µM) (Figure 4B). The concentration of DAU 5884 applied results in an almost complete blockade of the M3-receptor, with minor effects on the M2-receptor (Table 1). Another M3-receptor selective antagonist, 4-DAMP (10 nM), with a lower fractional M3-receptor occupancy (Table 1) also inhibited the methacholine-induced synergism, though not completely (Figure 4B). In contrast, the M2-receptor selective antagonist gallamine (10 µM; Table 1) did not alter the synergistic response (Figure 4B), which was virtually identical to control (compare with Figure 4A, open symbols).


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  		TABLE 1 Fractional occupancy of muscarine M2- and M3-receptors by gallamine, DAU 5884, and 4-DAMP

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cultured ASM cells undergo a variety of changes in protein expression and function as a consequence of modulation toward a synthetic phenotype. This phenotypic modulation is known to diminish the contractile properties of the ASM cell; in contrast, their synthetic and proliferative capacities increase (26). Plasticity in receptor protein expression is one of the consequences of phenotypic modulation, which may lead both to downregulation (e.g., muscarinic M3-receptors in human ASM) and upregulation of receptor proteins (e.g., PDGF-ß receptors in porcine uterus) (27, 28). As regards muscarinic receptors in cultured ASM cells, the M3-subtype is known to be downregulated rapidly upon culturing, whereas the M2-receptor function is not markedly altered for up to passage 10 in human ASM cells (11). Therefore, it is important to characterize the functional presence of the M3-receptor under different cell culture conditions, particularly because our aim was to study the relative involvement of M2- and M3-receptors in mitogenic responses.

In the unpassaged condition, M3-receptor function, assessed by methacholine-induced increase in inositol phosphates formation, was comparable to earlier observations in freshly isolated BTSM cells (25). It is of importance to note that the M3-receptor is the only receptor mediating inositol phosphates formation in BTSM, as established using selective muscarinic receptor antagonists (29). In contrast to the unpassaged condition, the M3-receptor was no longer functional in cells that were passaged twice. It has been shown that passaged cultured canine ASM cells do not functionally express M3-receptors either, whereas subsequent prolonged deprivation of serum re-induces functional coupling of the M3-receptor selectively for a subset of elongated contractile cells (30). These elongated cells shorten by > 70% of their original length in response to acetylcholine and express M3-receptors on the outer membrane, whereas serum-fed cells demonstrate a perinuclear distribution of M3-receptors that are not functionally coupled to inositol phosphate production (31). This shows that cell surface–coupled M3-receptors are reversibly lost upon transition to the synthetic phenotype. Most likely, this plastic behavior in M3-expression is the basis behind the differences in functional M3-responses in the different cell culture stages. Recently, it was shown that activator protein (AP)-2{alpha} may act as an important transcriptional regulator in this process: the M3-receptor gene contains eight AP-2 consensus-binding motifs, and AP-2{alpha} is known to be upregulated upon serum withdrawal (32).

In passaged human ASM cells, it has been found that the inositol phosphate response to carbachol is still present, although decreased. The loss of functional M3-receptors in human ASM appeared to be largely determined by post-transcriptional regulation, not by decreased mRNA expression (11). This process might be even more active in BTSM cells in view of the total lack of response.

Interestingly, the same culture condition that maintained functional M3 expression ensured a synergistic mitogenic response for methacholine and PDGF. In passaged (passage 2) cells, however, this synergism in mitogenesis was absent, as was functional M3-receptor expression. These differences suggest a role for the M3-receptor in the proliferative potentiation induced by methacholine. Indeed, selective blockade of the M3-receptor by DAU 5884 or 4-DAMP resulted in suppression of this synergistic response. The suppression was not as profound for 4-DAMP as it was for DAU 5884. However, DAU 5884 was used in a concentration that results in 0.9% of the M3-receptor fraction available for stimulation by methacholine, whereas in the case of 4-DAMP, a higher fraction (4.6%) of M3-receptors remained unoccupied. Remarkably, selective blockade (98.8%) of the M2-receptor by gallamine was totally ineffective. Hence, the M3-receptor apparently is the only subtype involved in the regulation of the mitogenic responses by methacholine in unpassaged BTSM cells. Because M3-expression in cultured airway myocytes appears not to be homogeneous (30, 31), this could indicate that muscarinic agonist–induced growth synergy is mediated by a particular subset of these cells. In 1321N1 human astrocytoma cells, acetylcholine did also induce proliferation via M3-receptors, despite the presence of M2- and M5-subtypes (33).

These findings raise the question why this synergistic response is absent in passaged BTSM cells, but present in passaged human ASM cells, as described previously (9). This difference may be the result of species differences in M3-receptor expression: although diminished, the presence of a functional M3-receptor population in cultured human ASM has been reported on several occasions, having a response of ~ 35% of that induced by histamine, as determined by the formation of inositol phosphates (11, 34). Furthermore, a small but not absent population of functional M3-receptors is consistent with the finding that carbachol is relatively weak in inducing proliferation synergy in human ASM cells when compared with other G protein–coupled agonists (8, 9).

Methacholine did not produce a proliferative response by itself, which is in line with observations by others, showing no (9) or a relatively small (8) increase in proliferation of ASM induced by muscarinic agonists. However, muscarinic receptor stimulation may play an important modulatory role. Muscarinic receptor stimulation was mitogenic only in combination with other mitogens, like growth factors. The combined response induced by methacholine (10 µM) and a concentration of PDGF (1 ng/ml), unable to induce proliferation by themselves, resulted in ~ 45% of the maximal PDGF-induced response under control conditions. This would imply a threshold either in the activation of transduction cascades or in the response of the cell to transductional activation.

The modulatory role may become of physiologic relevance, particularly in an environment in which growth factors are abundant, for instance due to secretion by inflammatory cells. Therefore, endogenous acetylcholine may contribute to the pathophysiology of inflammatory airway diseases, in which an increase in smooth muscle mass leads to airflow obstruction such as chronic asthma (35).

On a molecular level, M3-mediated mitogenic responses are in agreement with biochemical studies, showing that Gq-coupled muscarinic receptors may couple to pathways known to be involved in transcriptional regulation (36), such as the MAPK/ERK pathway (24), the PI-3 kinase/PKB pathway (37), and stress-activated members of the MAPK-superfamily, like the c-Jun N-terminal kinase (JNK) pathway (24) and the p38-MAPK pathway (38). In addition, coupling to G12/13-proteins may be responsible for the observed effects. Although less well studied, G{alpha}12/13 subunits are known to be involved in cellular growth and to potently activate JNK, whereas ERK activity may both be inhibited or stimulated, depending on the cell type (39).

The more challenging question is why M2-receptors do not seem to be relevant, because in theory, M2-receptors may couple to the same pathways (24, 40, 41). Moreover, other Gi–coupled stimuli have been shown to respond synergistically in combination with growth factors in human ASM cells (8). Perhaps other transductional pathways are involved in the muscarinic receptor–mediated mitogenic responses. However, differences in the kinetics of activation may also account for the unexpected observation. In CHO cells transfected with either human wild-type M2- or M3-receptors, it was found that both the M2- and the M3-transfected cells mediated MAPK/ERK and JNK activation; however, M2-receptor–mediated responses were transient, whereas M3-receptor responses were sustained (24). This difference may be of critical importance, because sustained MAPK/ERK activity determines the proliferative responses in human (42) and bovine ASM cells (14). Because little is known about the quantitative contribution of the pathways mentioned in regulating cross-talk between G-protein–coupled receptors and tyrosine kinase–coupled receptors, the absence of an M2-mediated mitogenic response might perhaps not be generalized.

In conclusion, in BTSM cells methacholine does not induce mitogenesis by itself, but potentiates PDGF-induced proliferation. This was dependent on the presence of functional M3-receptors as controlled by the cell culture conditions applied. This synergism could be abolished by selective M3-receptor antagonists, like DAU 5884 and 4-DAMP, but not by the M2-subtype selective antagonist gallamine. These results show that methacholine potentiates mitogenesis induced by PDGF through stimulation of M3-receptors in BTSM cells.


   Acknowledgments
 
This work was financially supported by the Netherlands Asthma Foundation, grant NAF 99.53.

Received in original form July 22, 2002

Received in final form September 16, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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