Abstract
Airway remodeling is a structural alteration associated with chronic inflammatory and obstructive airway diseases, wherein fibroblasts are crucially involved. The present study investigates whether lung fibroblast proliferation is influenced by muscarinic mechanisms. For this purpose, expression of muscarinic receptors in MRC-5 human lung fibroblasts was characterized by semiquantitative RT-PCR, and the effects of muscarinic agonists and antagonists on (3H)-thymidine incorporation as a measure of proliferative activity were studied under different culture conditions. MRC-5 fibroblasts express mRNA encoding different subtypes of muscarinic receptors (M2 > M3 > M4, traces for M5 and no M1). Expression of M2 and M3 receptors was confirmed at the protein level by immunoblot analysis. Under different culture conditions, carbachol (up to 10 μM) or oxotremorine (10 μM) stimulated (3H)-thymidine incorporation, with maximum increases between about 40 and 100%. The stimulatory effect of 10 μM carbachol was prevented by pretreatment with pertussis toxin and antagonized in a concentration-dependent manner by the muscarinic receptor antagonists tiotropium, AQ-RA 741, AF-DX 384, 4-diphenylacetoxy-N-methylpiperidine methoiodide, himbacine, p-fluorohexahydrosiladifenidol, and pirenzepine, with concentrations producing 50% inhibition of 14 pM, 24, 64, 127, 187, 452 nM, and 1.5 μM, respectively. Primary human lung fibroblasts were also found to express mRNA for muscarinic receptors (M2 > M1 > M3, traces for M4 and no M5), and showed a pertussis toxin–sensitive proliferative response to muscarinic receptor stimulation. In conclusion, proliferation of human lung fibroblasts can be stimulated by activation of muscarinic receptors with a pharmacologic profile correlating best to M2 receptors.
Indeed, muscarinic agonists enhanced the proliferative response of human and bovine airway smooth muscle to epidermal growth factor and platelet-derived growth factor (PDGF), respectively (5, 6). Moreover, tiotropium was found to attenuate the increase in airway smooth muscle mass and myosin expression induced by repeated allergen challenges (7).
Apart from airway smooth muscle, almost every cell type in the airways expresses cholinoceptors (for review, see Refs. 8 and 9) and may, therefore, be a target for acetylcholine released from neuronal or nonneuronal sources (for review, see Refs. 8–10). In airway fibroblasts, expression of mRNA encoding different nicotinic receptor subunits (11) and M2 receptors (12) has been described, but a detailed study on expression of muscarinic receptor subtypes in lung and airway fibroblasts is still lacking. Activation of nicotinic receptors has been shown to stimulate collagen gene expression and fibronectin synthesis (13, 14), whereas the functional role of muscarinic receptors in airway fibroblasts remains to be determined.
The aims of this study were to characterize the expression pattern of muscarinic receptors in human lung fibroblasts and to investigate whether muscarinic receptors mediate effects on cell proliferation.
MCR-5 human lung fibroblasts (CCL-171; American Type Culture Collection, Manassas, VA) were grown in Eagle's minimum essential medium (MEM) with Earle's salts supplemented with 10% FCS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown in a humidified incubator at 37°C and 5% CO2 and passaged by trypsinization at near confluence. Early-passage cells were used in experiments.
Primary human lung fibroblasts were established from histologically normal areas of surgically resected lung tissue, which was obtained from lung cancer patients after thoracotomy. The protocol for obtaining human tissue was approved by the local ethics review board for human studies (Ethics Committee, Medical Faculty, University of Bonn, Bonn, Germany), and informed consent was obtained from the patient. Tissue was cut into small pieces, treated with pronase (1 mg/ml; Calbiochem Novabiochem, San Diego, CA) at 37°C for 30 min, placed in cell culture plates, and incubated in Eagle's MEM, supplemented as described previously here, with FCS increased to 15%. After 2 wk, fibroblasts had grown out from the tissue and were passaged by standard trypsinization. For experiments described here, cells of passages 1–10 were used.
Total RNA was isolated by help of silica gel–based membranes, according to the manufacturer's instructions, including an additional DNase digestion protocol to avoid any contamination by genomic DNA (Qiagen, Hilden, Germany). First-strand cDNA was synthesised using Omniscript reverse transcriptase (Qiagen). Specific oligonucleotide primers were constructed based on human European Molecular Biology Laboratory (EMBL) sequences: β-actin, 5′-CACTCTTCCAGCCT TCCTTC-3′ and 5′-CTCGTCATACTCCTGCTTGC-3′; M1 receptor, 5′-CAGGCAACCTGCTGGTACTC-3′ and 5′-CGTGCTCGGTTCTC TGTCTC-3′; M2 receptor, 5′-CTCCTCTAACAATAGCCTGG-3′ and 5′-GGCTCCTTCTTGTCCTTCTT-3′; M3 receptor, 5′-GGACAGAG GCAGAGACAGAA-3′ and 5′-G AAGGACAGAGGTAGAGTGG-3′; M4 receptor, 5′-ATCGCTATGAGACGGTGGAA-3′ and 5′-GTT GGACAGGAACTGGATGA-3′; M5 receptor, 5′-ACCACAATG CAACCACCGTC-3′ and 5′-ACAGCGCAAGCAGGATCTGA-3′. PCR amplification was performed using Taq DNA polymerase in a programmable thermal reactor with initial heating for 3 min at 94°C, followed by 23 cycles (β-actin) or 35 cycles (M1–M5) of 45-s denaturation at 94°C, annealing at 53–60°C (30 s), extension at 72°C (1 min), and final extension for 10 min at 72°C. PCR products were separated by 1.2% agarose gel electrophoresis. Optical density of bands was quantified by RFLPscan 2.01 software (MWG, Ebersberg, Germany), corrected for β-actin, and referred to the respective amplification of genomic DNA to normalize for variations in PCR effectiveness.
Muscarinic receptor genes are intronless, and contamination of the RNA preparation by genomic DNA would cause false-positive PCR results; hence, the RNA preparation was treated with DNase before the RT reaction in order to remove traces of genomic DNA. In control experiments with cells not expressing muscarinic receptors, it was confirmed that false-positive results could be prevented by this pretreatment.
Cellular proteins were extracted in RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% sodium deoxycholat, 1% Nonidet P-40, 0.1% [wt/vol] SDS, 2 mM EDTA [pH 8.0]) containing the protease inhibitors PMSF (1 mM), pepstatin A (0.7 μg/ml), and leupeptin (0.5 μg/ml). Totals of 50–100 μg protein equivalents were mixed with reducing protein loading buffer (Roti-Load 1; Roth, Karlsruhe, Germany) and boiled for 3 min. Samples were separated on 10% acrylamide Tris-glycine precast gels (Mirador, Montreal, PQ, Canada) using the Laemmli buffer system (5% SDS, 125 mM Tris, 7.2% glycine) and transferred (20% methanol, 25 mM Tris, 14.4% glycine) to polyvinylidine difluoride membranes (Millipore, Billerica, MA). Blots were blocked in 5% dried milk protein Tris-buffered saline–Tween (150 mM NaCl, 50 mM Tris, 0.05% Tween 20), and proteins were detected by use of rabbit polyclonal antibodies anti-human mAChR M2 (H-170) and anti-human mAChR M3 (H-210) (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were visualized by peroxidase-conjugated secondary goat antibodies (Bio-Rad, Hercules, CA) employing Boehringer Mannheim (BM) chemoluminescence blotting substrate peroxidase (POD) (Roche, Mannheim, Germany), then blots were exposed to Hyperfilm enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Cells were trypsinized, harvested, and seeded into 12-well dishes at a density of 7.5 × 104 cells per well. Different protocols were tested in order to find conditions under which cholinergic effects might be particularly prominent. In one series of experiments, cells were first cultured for 24 h in the presence of 10% FCS, followed by an additional 48 h under FCS-free conditions. Test drugs were added either at the onset of the FCS-free period or 24 h later. (3H)-Thymidine (37 kBq) was present during the last 24 h of the 3-d culture period. In other series of experiments, fibroblasts were cultured for 30 or 48 h under FCS-free conditions from the onset. Test drugs were present from the beginning, and (3H)-thymidine during the last 24 h. In experiments of 48-h duration or longer, medium was renewed after 24 h to ensure the presence of active drugs during the whole culture period. At the end of incubation, cells were washed in ice-cold PBS and radioactivity incorporated into DNA was extracted, as described previously (15). Briefly, cells were denatured in trichloroacetic acid (10%) for 10 min, washed in ice-cold PBS, and DNA was extracted during incubation for 1 h in 0.1 mol/l NaOH at 37°C. Samples of supernatant solution (300-μl portions) were neutralized, combined with scintillation cocktail (Canberra Packard, Schwadorf, Austria), and radioactivity was determined by liquid scintillation spectrometry in a Packard 2100 TR liquid scintillation analyzer (Packard, Dreieich, Germany). External standardization was used to correct for counting efficiency.
All values are means ± SEM of n experiments. Statistical significance of differences was evaluated by ANOVA, followed by Dunnett's or Bonferroni's test using GraphPad InStat (GraphPad Software, San Diego, CA); a P value of < 0.05 was accepted as significant. Concentrations producing 50% inhibition (IC50) values and correlation coefficients were calculated by the use of computer programs GraphPad Prism (GraphPad Software and see Ref. 16).
AF-DX384 (5,11-dihydro-11-{[(2-{2-[(dipropylamin)methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6H-pyrido(2,3-β) (1, 4)benzodiazepine-6-one methanesulfonate), AQ-RA741 (11-({4-[4-(diethylamino)butyl]-1-piperidinyl}acetyl)-5,11-dihydro-6H-pyrido(2,3-β) (1, 4)benzodiazepine-6-one hydrochloride), pirenzepine, and tiotropium bromide were gifts from Boehringer Ingelheim (Ingelheim, Germany). Atropine sulphate, carbachol (carbamylcholine chloride), 4-diphenylacetoxy-N-methylpiperidine methoiodide (4-DAMP), himbacine, parafluorohexahydrosiladifenidol (p-FHHSiD), oxotremorine sesquifumerate, pertussis toxin (PTX), PDGF, desoxynucleotide mixture, penicillin–streptomycin solution, and trypsin were purchased from Sigma (Deisenhofen, Germany); Eagle's MEM with Earle's salts and L-glutamine, nonessential amino acids, were purchased from PAA (Coelbe, Germany); FCS was purchased from Biochrom (Berlin, Germany); Taq DNA-polymerase was purchased from Invitrogen (Karlsruhe, Germany); Omniscript reverse transcriptase, RNeasy Mini kit, and RNase-free DNase set were purchased from Qiagen. Oligodesoxynucleotides for RT-PCR were obtained from MWG Biotech (Ebersberg, Germany).
As shown in Figure 1, MRC-5 fibroblasts express mRNA encoding different muscarinic receptor subtypes. The highest expression was found for mRNA encoding M2 receptor, followed by M3 receptor transcripts. Lower but still detectable expression was observed for M4 receptor, whereas only traces of mRNA encoding M5 receptor were detected, and no transcript for M1 receptor was detected. The described pattern of muscarinic receptor mRNA expression appears to be stable under the culture conditions, as a similar pattern was found in different passages (passages 1–13).
Figure 1. (A) Samples of RT-PCRs of human muscarinic receptors (M1–M5) on RNA isolated from MRC-5 human lung fibroblasts. Cells were grown in 55-mm culture dishes to confluence, total RNA was isolated, treated with DNase, and used for RT-PCR with primers specific for the human muscarinic receptors or β-actin. To control effectiveness of primer pairs, PCRs were also performed on genomic DNA (gDNA) isolated from MRC-5 cells. One PCR lacking template DNA was regularly performed to account for any contamination (negative control, − Ctr.). PCR products were separated on a 1.2% agarose gel. (B) Densitometric evaluation of a series of experiments. Given are mean values (+ SEM) of three experiments performed with cells of passages 1–13. Values were normalized first for β-actin to correct for quality of the cDNA preparation and second for the respective amplification products obtained on gDNA to correct for effectiveness of the respective primer pairs.
[More] [Minimize]The expression of M2 and M3 receptors could also be confirmed at the protein level by Western blot analysis using specific, commercially available polyclonal antibodies (Figure 2). The immunoblot for M2 receptor showed a single band, somewhat larger than 80 kDa. A band of similar size was also observed for the M3 receptor, but here two additional products, with a lower molecular weight, were also detected. Most likely, they reflect cellular degradation products, which are still recognized by the antibody. For comparison, Figure 2 also shows immunoblots performed with proteins extracted from rat lung tissue.
Figure 2. Immunoblot analysis of protein extracts of MRC-5 cells for comparison of rat lung tissue using commercially available antibodies against M2 and M3 receptor protein. Given are representative samples of SDS-PAGE.
[More] [Minimize]In order to test whether putative muscarinic effects on fibroblast proliferation depend on culture conditions and exposure time, various experimental conditions were explored. In a first set of experiments, cells were first cultured for 24 h in the presence of 10% FCS, followed by 24-h culture in FCS-free medium. Thereafter, cells were cultured for a further 24 h under FCS-free conditions in the presence of (3H)-thymidine and in the absence or presence of increasing concentrations of carbachol. Under control conditions (i.e., absence of drugs), (3H)-thymidine incorporation amounted to 7,281 ± 548 d.p.m. (disintegration per minute) (n = 52). As shown in Figure 3A, carbachol caused an increase in (3H)-thymidine incorporation (by 38 ± 18% at 10 μM) in a concentration-dependent manner. This effect was abolished in the presence of 1 μM atropine (Figure 3), which alone had no effect on (3H)-thymidine incorporation. Although the stimulatory effect of carbachol was clearly significant, the magnitude of the effect showed considerable variation.
Figure 3. Effects of muscarinic agonists and antagonists on (3H)-thymidine incorporation in MRC-5 human lung fibroblasts. A total of 7.5 × 104 cells were seeded in 12-well dishes in the absence or presence of 10% FCS, as indicated. In each series, (3H)-thymidine (37 kBq) was present for the last 24 h. (A) Effects of carbachol, at concentrations indicated by the abscissa, in the absence (open symbols) or presence (filled symbols) of 1 μM atropine. Test drugs were added simultaneously to (3H)-thymidine. (B and C) Carbachol (C, 10 μM), oxotremorine (O, 10 μM) and/or atropine (A, 1 μM) and/or tiotropium (T, 0.1 μM) were present 24 h before addition of (3H)-thymidine through the end of the protocol. Data are means (± SEM) of n = 3–12 (mostly n ⩾ 6). ** P < 0.01 and *** P < 0.001 vs. controls; + P < 0.05, ++ P < 0.001, and +++ P < 0.001 vs. the respective value in the absence of antagonists.
In a second protocol, cells were again cultured for 24 h in the presence of 10% FCS, followed by 48 h FCS-free conditions in the absence or presence of carbachol (10 μM). Under these conditions, carbachol enhanced (3H)-thymidine incorporation by 52 ± 10% (Figure 3B). Again the effect of carbachol was prevented by atropine and also by the long-acting muscarinic antagonist, tiotropium (Figure 3B). Furthermore, oxotremorine caused a similar increase (by 61%), an effect also inhibited by atropine (Figure 3B). For comparison, when FCS (1 or 10%) was present instead of a muscarinic agonist during the last 48 h, (3H)-thymidine incorporation was enhanced by 69 ± 17% (n = 6) and 216 ± 30% (n = 9), respectively, compared to FCS-free conditions.
In a further set of experiments, cells were FCS-deprived and drugs were applied from the onset of the test culture period. Under these conditions, 10 μM carbachol or oxotremorine increased (3H)-thymidine incorporation by 64 ± 8% and 100 ± 26%, respectively, when present 24 h before (3H)-thymidine, for a total of 48 h. These effects were again shown to be atropine-sensitive (Figure 3C). When carbachol was applied 6 h before (3H)-thymidine, for a total of 30 h, (3H)-thymidine incorporation was increased by 76 ± 4% (n = 80). These conditions were held for the subsequent interaction experiments with subtype-preferring muscarinic receptor antagonists.
As summarized in Figure 4, all muscarinic receptor antagonists tested here inhibited the stimulatory effect of carbachol on (3H)-thymidine incorporation in a concentration-dependent manner. Tiotropium was by far the most potent drug, with an IC50 value of 14 pM. The M2/M4 receptor–preferring antagonists, AQ-RA 741, AF-DX 384, and himbacine, showed IC50 values of 24, 64, and 187 nM, respectively, whereas, for the M3/M1 receptor–preferring antagonists, 4-DAMP and p-FHHSiD, IC50 values of 127 and 452 nM were determined. The IC50 values for the M1/M4 receptor–preferring antagonist pirenzepine was 1.5 μM. None of the antagonists at any of the concentrations studied significantly affected (3H)-thymidine incorporation on its own (data not shown, each n = 6–12). When the potencies of these antagonists as determined here were correlated to their reported affinity constants to cloned human muscarinic receptors, the best and highly significant correlation was obtained for the M2 receptor. Figures 4B–4D present only the correlations for M1, M2, and M3 receptors, as affinity data of tiotropium for cloned M4 and M5 receptors have not yet been published. For these two receptors, the respective correlations calculated on the basis of the remaining six antagonists failed to be statistically significant, with r = 0.60 and 0.27 for M4 and M5 receptors, respectively.
Figure 4. (A) Inhibitory effects of different muscarinic receptor antagonists on carbachol-induced stimulation of (3H)-thymidine incorporation in MRC-5 human lung fibroblasts. A total of 7.5 × 104 cells were seeded in 12-well dishes under FCS-free conditions. Carbachol (10 μM) was added from the onset of incubation for a total of 30 h, of which (3H)-thymidine (37 kBq) was present for the last 24 h. In the absence of antagonists, carbachol enhanced (3H)-thymidine incorporation by 98 ± 10% (n = 53). Antagonists were applied immediately before addition of carbachol at the concentrations given by the abscissa. Ordinate: stimulatory effect of carbachol in the presence of antagonists, expressed as percent of mean stimulatory effect of carbachol in the absence of antagonists, as observed in the respective series of experiment. Data are means of n = 3–12 (mostly n ⩾ 6); SEM, 6–12%. (B–D) Correlation between the potencies of the antagonists, as determined in the present experiments (ordinate), and the reported affinity constants, as determined on cloned human muscarinic receptors (abscissa) (23, 24). Open squares, 4-DAMP; closed triangles, AQ-RA741; open diamonds, AF-DX384; closed diamonds, p-FHHSiD; closed circles, tiotropium; open circles, himbacine; open inverted triangles, pirenzipene.
[More] [Minimize]Pretreatment of the fibroblasts with PTX resulted in a slight reduction in (3H)-thymidine incorporation, and abolished the stimulatory effect of carbachol (Figure 5). In order to test the specificity of PTX, the effects of this toxin were also studied on the proliferative action of PDGF, which is known to act via a different class of receptors. Pretreatment of the cells with PTX, as described in Figure 5, did not inhibit the PDGF-induced stimulation of (3H)-thymidine incorporation (increase by 219 ± 40% [n = 6] and 283 ± 40% [n = 3] in the absence and presence of PTX, respectively).
Figure 5. Effects of carbachol and/or PTX on (3H)-thymidine incorporation in MRC-5 human lung fibroblasts. A total of 7.5 × 104 cells were seeded in 12-well dishes and cultured under FCS-free conditions. PTX (0.1 μg/ml) was present from the onset of incubation. Carbachol (Carb., 10 μM) was applied 3 h after PTX. Finally, (3H)-thymidine (37 kBq) was added 6 h later and was present for 24 h. Data are means (± SEM) of n = 6–12. * P < 0.01 and ** P < 0.001 vs. controls (Ctr.); *,+ P < 0.01 vs. Ctr. and P < 0.001 vs. Carb. alone.
[More] [Minimize]When 1% FCS was present from the onset of the test culture period (6 h before (3H)-thymidine, for a total of 30 h), (3H)-thymidine incorporation was increased by 450% (Figure 6). Additional presence of 10 μM carbachol or oxotremorine induced a further significant increase in (3H)-thymidine incorporation, by about 300 and 240%, respectively, relative to the FCS-free controls (Figure 6). This corresponds to an increase of 54 and 43%, respectively, when expressed in relation to FCS-containing control experiments.
Figure 6. Comparison of the effects of carbachol and oxotremorine on (3H)-thymidine incorporation in MRC-5 human lung fibroblasts in the absence and presence of FCS. A total of 7.5 × 104 cells were seeded in 12-well dishes and cultured under FCS-free conditions or in the presence of 1% FCS. Carbachol (Carb, 10 μM) or oxotremorine (Oxo, 10 μM) was added from the onset of incubation and was present for a total of 30 h; (3H)-thymidine (37 kBq) was present for the last 24 h. Data are means (± SEM) of n = 3–12. *** P < 0.001 vs. controls (Ctr); + P < 0.01 vs. the respective value in the presence of FCS alone.
[More] [Minimize]As Figure 7 shows, mRNA encoding muscarinic receptors was also detected in primary human lung fibroblasts obtained from patients with lung cancer. At different passages, it was observed that expression of M2 receptor transcripts was most prominent, followed by mRNA encoding M1 and M3 receptors, whereas only traces of mRNA for M4, and none for M5, were found.
Figure 7. (A) Samples of RT-PCRs of the five human muscarinic receptors (M1–M5) on RNA isolated from primary human lung fibroblasts. For further details, see Figure 1. (B) Densitometric evaluation of a series of experiments. Data are means (+ SEM) of three experiments performed on cells of passages 1, 5, and 10. For further details, see Figure 1.
[More] [Minimize]In coherence to MRC-5 cells also in these primary lung fibroblasts, 10 μM carbachol or oxotremorine stimulated the incorporation of (3H)-thymidine, and the presence of tiotropium or atropine, as well as pretreatment with PTX, abolished the stimulatory effect of carbachol (Figure 8). Also, in primary human lung fibroblasts, PTX pretreatment failed to inhibit the proliferative effect of 100 ng/ml PDGF (increase by 403 ± 49% [n = 12] and 487 ± 80% [n = 3] in the absence and presence of PTX, respectively).
Figure 8. Effects of carbachol and/or PTX, tiotropium or atropine on (3H)-thymidine incorporation in primary human lung fibroblasts. A total of 7.5 × 104 cells were seeded in 12-well dishes and cultured under FCS-free conditions. Cells were incubated for 30 h with carbachol (Carb, 10 μM) and/or tiotropium (T, 0.1 μM), atropine (A, 1 μM), or oxotremorine (Oxo, 10 μM), as indicated. (3H)-thymidine (37 kBq) was added 6 h after onset of exposure to these drugs. In the respective experiments, PTX (0.1 μg/ml) was already present 3 h before carbachol. Data are means (± SEM) of n = 3–12. ** P < 0.001 vs. controls; + P < 0.01 vs. carbachol alone.
[More] [Minimize]Almost every cell type in the lung and airways expresses cholinoceptors, but expression of muscarinic receptors in lung fibroblasts and their functional role have not yet been characterized. The present experiments demonstrate that human lung fibroblasts express muscarinic receptors, which mediate a stimulatory effect on cell proliferation.
Using semiquantitative RT-PCR, several muscarinic receptors were found to be expressed in MRC-5, as well as in primary lung fibroblasts. In both, the cell line and primary cells, the strongest mRNA expression was observed for M2 receptor, whereas some gradual differences were found with regard to the other muscarinic receptors. In MRC-5 cells, the levels of mRNA encoding M3 receptor appeared to be higher than in primary cells, which, to the contrary, seemed to express a clear message for M1 receptor. As mentioned previously here, the genes of muscarinic receptors are intronless. Therefore, the RNA preparations were treated with DNase before the RT reaction to prevent contamination by genomic DNA. The differential expression pattern observed in the present study—in particular, the lack of PCR products accounting for M1 and M5 receptors in MRC-5 and primary lung fibroblasts, respectively—argues against an artifact. Moreover, in MRC-5 cells, expression of M2 and M3 receptors could be confirmed at the protein level by Western blot analysis.
Proliferation of lung fibroblasts in vitro largely depends on the presence of FCS, which obviously contains a cocktail of diverse factors promoting fibroblast growth. Therefore, the effects of muscarinic agonists were first tested in FCS-deprived conditions (i.e., in absence of other mitogenic stimuli). Under these conditions, carbachol and oxotremorine caused an increase in (3H)-thymidine incorporation in both MRC-5 and primary human lung fibroblasts. In both cell types, the proliferative effects of carbachol or oxotremorine were blocked by the muscarinic antagonist, atropine, and the long-acting muscarinic antagonist, tiotropium, at concentrations at least 10- and 100-fold lower, respectively, than those of the agonists, proving the involvement of specific muscarinic receptors. The antagonists had no effect on their own, which excludes significant effects of nonneuronal acetylcholine on fibroblast proliferation under the present culture conditions.
In MRC-5 cells, muscarinic stimulation of proliferation was studied in more detail. The stimulatory effect of carbachol appeared to be somewhat more pronounced when the drug was added after dissemination isochronal to FCS-deprivation and when it was already present before the (3H)-thymidine exposure compared with conditions in which the agonist was applied simultaneously with (3H)-thymidine for the last phase of the incubation period. This may suggest that the muscarinic mitogenic stimulus is slow in developing, but is long lasting. A mitogenic effect of muscarinic agonists has also been demonstrated on airway smooth muscle (5, 6); however, in contrast to the present observations on fibroblasts, muscarinic agonists alone showed no proliferative effect in airway smooth muscle, but augmented the effect of other mitogenic stimuli, such as PDGF or epidermal growth factor. The present experiments, showing that the proliferative effects of carbachol and oxotremorine were basically the same in the absence or the presence of 1% FCS (Figure 6), indicate that, in human lung fibroblasts, the proliferative response induced by muscarinic receptor activation may be additive to proliferative effects caused by other mitogenic stimuli.
Both M2 and/or M3 receptors have been shown to mediate activation of the mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 pathway (17–19), which is known to mediate proliferative responses in various cell types (20–22). In order to characterize the muscarinic receptor subtype mediating the proliferative effect in MRC-5 fibroblasts, the M2/M4 receptor–preferring antagonists, AF-DX384, AQ-RA741, and himbacine, the M3/M1 receptor–preferring antagonists, 4-DAMP and p-FHHSiD, and the M1/M4 receptor–preferring antagonist, pirenzepine, were tested in addition to tiotropium. It should be emphasized that the subtype selectivity of the currently available “selective” muscarinic receptor antagonists is rather limited. Usually, they reveal a relatively high potency at least for two of the five muscarinic receptors, and the potency difference from the other muscarinic receptors is only about one order of magnitude. Nonetheless, comparing the potency of a series of subtype-preferring antagonists is an approach to deal with this problem (e.g., Ref. 23). When the antagonist potencies, as observed in the present experiments, were correlated to their reported potencies on cloned human muscarinic receptors (24, 25), the best correlation was obtained for the M2 receptor, supporting the assumption that this muscarinic receptor subtype may be essential in mediating the proliferative effects. This correlates well with the observation that, at the mRNA level, M2 receptors represented the major muscarinic receptor subtype in these cells. In line with this conclusion also lies the observation that PTX abolished the effect of carbachol, as PTX is known to inhibit Gi/o G proteins, to which only M2 and M4 receptors couple (26). An unspecific antiproliferative action of PTX may be excluded, as PTX did not inhibit the proliferative effect of PDGF, which is known to exert its effects via activation of a tyrosine kinase receptor, a class of receptors different from G protein–coupled receptors. Therefore, the PTX-induced reduction of baseline proliferation may reflect a background activation of Gi-mediated effects, possibly caused by paracrine mediators (as, for example, chemokines) released by the fibroblasts themselves.
The long-acting muscarinic antagonist, tiotropium, was by far the most potent antagonist. The affinity constants of tiotropium are similar for M1, M2, and M3 receptors, but the dissociation of the respective receptor complexes shows different kinetics, with the longest half-life for the M3 receptor (25). This kinetic subtype selectivity of tiotropium may not be of significance in the present experiments, because they were performed under equilibrium conditions. Whether the kinetical subtype selectivity of tiotropium is of clinical importance is still debatable (27). Because the half-life of the tiotropium–M2 receptor complex, although shorter than that of the M3 receptor complex, is still 3.6 h (25), it may be expected that tiotropium causes a significant blockade of M2 receptors in the lung under clinical conditions.
Although detailed pharmacologic experiments have only been performed on MRC-5 cells, the observations that PTX also abolished the proliferative effect of carbachol in primary human lung fibroblasts, and that M2 receptor mRNA represented the predominant muscarinic receptor transcript in these cells as well, suggest that M2 receptors might also mediate the proliferative stimulus in primary human lung fibroblasts.
In conclusion, human lung fibroblasts express muscarinic receptors, which obviously mediate a proliferative stimulus, an effect which might contribute to structural changes known to occur in chronic obstructive airway diseases. Prolonged blockade of these receptors may contribute to the long-term beneficial effects of anticholinergics, as observed, for example, for the long-acting muscarinic antagonist, tiotropium, in COPD (3, 4).
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