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Journal List > Br J Pharmacol > v.154(8); Aug 2008

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Br J Pharmacol. 2008 August; 154(8): 1611–1618.
Published online 2008 June 2. doi: 10.1038/bjp.2008.208.
PMCID: PMC2518472
Copyright 2008, Nature Publishing Group
Isolated porcine bronchi provide a reliable model for development of bronchodilator anti-muscarinic agents for human use
G D'Agostino,1* A M Condino,1 L Gioglio,2 F Zonta,1 M Tonini,3 and A Barbieri1
1Department of Experimental and Applied Pharmacology, University of Pavia, Italy
2Department of Experimental Medicine, University of Pavia, Italy
3Department of Physiological and Pharmacological Sciences, University of Pavia, Italy
*Author for correspondence: Email: dagg/at/unipv.it
Received January 16, 2008; Revised April 7, 2008; Accepted April 18, 2008.
Abstract
Background and purpose: In human airways, muscarinic acetylcholine receptors (mAChRs) exert a predominant role in the control of airways resistance and anti-muscarinic agents are currently included in the pharmacological treatment of chronic obstructive pulmonary disease (COPD). However, the development of more effective mAChR antagonists is hampered by considerable species variability in the ultrastrucural and functional control of airway smooth muscle, making extrapolation of any particular animal model questionable. This study was designed to characterize the mAChRs in a bronchial preparation from pigs, animals considered to provide close models of human biology.
Experimental approach: Smooth muscle bronchial strips were examined by electron microscopy in order to compare their neuromuscular structure with that of human bronchi and used to study the affinity of a series of selective mAChR antagonists, estimated as pKis in competition binding assays with NMS and pA2, by Schild analysis, in contractile experiments.
Key results: Pharmacodynamic binding parameters and affinity profiles of a series of antagonists were consistent with the presence of a majority of M2 mAChRs along with a minor population of M3 mAChRs. Functionally, the highly significant correlation between postjunctional pA2 affinities and corresponding affinity constants at human recombinant M1–M5 subtypes indicated that smooth muscle contraction in porcine bronchi, as in human bronchi, was dependent on the M3 subtype.
Conclusion and implications: Based on the characterization of mAChRs, isolated porcine bronchi provide an additional experimental model for development of mAChR antagonists for the treatment of human airway dysfunctions.
Keywords: NMS binding, muscarinic antagonists, muscarinic receptor subtypes, pig airways, smooth muscle contraction
Introduction
The parasympathetic (cholinergic) nervous system represents the main excitatory neural pathway in the airways of mammals as well as in humans (Racké and Matthiesen, 2004). It plays a predominant role in the control of distal airway resistance although the density of parasympathetic innervation is greatest in proximal airways and diminishes peripherally (Barnes, 1986). ACh released from cholinergic nerve terminals regulates airway functions (smooth muscle tone and mucus secretion) through stimulation of muscarinic ACh receptors (mAChRs; Alexander et al., 2008). These receptors are members of the rhodopsin-like family of seven transmembrane receptors. Their predominant mode of signalling is mediated by the activation of GTP-binding proteins (G-proteins), although activation of other signalling molecules also occurs (Hall et al., 1999). Five (M1–M5) different subtypes of mAChRs have been identified by molecular biological techniques, but so far a convincing pharmacological and functional characterisation has been provided for four of them (M1–M4) only. Nonetheless, there is now emerging evidence that the M5 gene forms a functional M5 mAChR (Eglen and Nahorsky, 2000).
The distribution of mAChRs in airways has been mapped by receptor autoradiography and in situ hybridization (Mak and Barnes, 1990; Mak et al., 1992; Hislop et al., 1998), and binding and functional studies revealed that at least three mAChR subtypes, namely M1, M2 and M3, are expressed in airway smooth muscle and lung parenchyma of most mammals, including humans (Gies et al., 1989; Roffel et al., 1990; Patel et al., 1995; ten Berge et al., 1996). The presence of M1 receptor mRNA was described in human (Bloom et al., 1988; Mak and Barnes, 1989; Mak et al. 1992) and pig peripheral lung (Haddad et al., 1994; Hislop et al., 1998), whereas M2 and M3 subtypes were found in smooth muscle preparations from these (Gies et al., 1989; Roffel et al., 1993; Haddad et al., 1994; Watson et al., 1995) and other species, including rodents and ruminants (see Eglen et al., 1996 for review). Under basal conditions, the M3 receptor subtype is that involved in the bronchoconstriction action in all species. There is no evidence for expression of M4 or M5 receptors in human lung (Mak et al., 1992), although the presence of the M4 receptor subtype is supported by in situ hybridization (Dörje et al., 1991a; Mak et al., 1993; Yasuda et al., 1993) and binding experiments in the rabbit (Lazareno et al., 1990) and pig lung (Chelala et al., 1998), indicating important species difference in the distribution and subtype of expressed receptors (Barnes, 2004).
Antimuscarinic agents are currently used in treating bronchoconstriction associated with chronic obstructive pulmonary disease and certain forms of asthma (Gosens et al., 2006), as an increase in cholinergic activity is a prominent pathophysiological mechanism in these conditions. Thus, mAChRs represent an attractive target for the development of novel antagonists that selectively block functional receptors in the effector cells; today, however, it is still a matter of controversy which animal species represents the best model for the search of bronchodilator anti-muscarinic agents for human use (Barnes, 2004).
The present study was designed to assess whether the distribution and the function of mAChRs in the airways of the pig, an excellent animal model for biomedical research, closely resemble those observed in humans. For this purpose, strips of porcine bronchi, denuded of mucosa to minimize the influence of airway epithelium (Stuart-Smith and Vanhoutte, 1988; D'Agostino et al., 1990) were used. The neuromuscular structure of the preparation was assessed by electron microscopical techniques. A characterization of mAChRs subtypes was carried out with a series of mAChRs antagonists, investigated in radioligand binding and functional experiments. The comparison of the results obtained here with published morphological and pharmacological findings revealed a similarity of porcine bronchi to human bronchi. Based on this evidence, porcine bronchi appear an appropriate animal model for studying novel mAChR antagonists targeted to a more efficacious therapeutic control of airway diseases.
Methods
Tissue preparation
Lungs of mature large white pigs (>5 months, carcass weight 180–200 kg) were obtained from a local abattoir and rapidly transported to the laboratory. The bronchial tree was dissected and rings (5–6 mm I.D.) of segmental bronchi (third order) were isolated. The mucosa was removed from the tissue by rubbing the luminal surface with a moistened pipe cleaner. Histological inspection by light microscopy indicated that such procedure did not damage the basal membrane (not shown; see Klapproth et al., 1997). The muscularis was carefully lifted away from underlying cartilage and connective tissue by using a surgical knife. Strips (4 mm width, 15–18 mm length and 40–60 mg wet weight) were obtained from transverse rings of airway wall.
Electron photomicrography
Specimens of bronchial smooth muscle were fixed for 4 h at 4 °C in 3.5% glutaraldehyde, dissolved in Millonig's phosphate buffer at pH 7, washed in Millonig's buffer, postfixed in a 1% osmium tetroxide solution in Millonig's buffer for 1 h at 4 °C and dehydrated in ascending concentrations of ethyl alcohol. Specimens were then treated with propylene oxide, embebbed in Epon 812, and cut into semithin and ultrathin sections using a Reichert OM 12 ultramicrotone. Semithin sections for light microscopy were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate, and then examined with a Zeiss 109 electron microscope.
Binding experiments
Bronchial strips, prepared as described above, were homogenized with an Ultraturrax at full speed for 30 s, followed by homogenization in a glass-on-teflon homogenizer in Na+/Mg++ HEPES buffer pH 7.4. (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES) (w/v final dilution 1:30). The homogenate was divided into 5 ml aliquots, and stored at −80 °C until use.
Displacement (competition) experiments were performed by incubating 980 μl of the homogenate (final tissue diluition 1:140) at 30 °C for 45 min in the presence of 0.3 nM [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and increasing concentrations of unlabeled compounds (mAChR antagonists) dissolved in the assay buffer (Na+/Mg++ HEPES buffer pH 7.4). Incubation volume was 1 ml. The reaction was terminated by rapid filtration and washing (3 × 2 ml) of ice-cold buffer using an IH 110 Inotech cell harvester (type G-7 IH 201 glass filters, Inotech Biotechnologies, Basel, Switzerland). The filters were the placed in plastic vials containing 4 ml of Filter Count (PerkinElmer Life and Analytical Sciences, Milan, Italy) and counted for radioactivity by liquid scintillation analyzer (PerkinElmer Tri-Carb 2800 TR).
Specific binding of [3H]NMS was defined as the binding displaceable by 1 μM QNB (3-quinuclidinylbenzilate, racemic mixture). Protein content was determined with Bio-Rad Protein Assay kit (Bio-Rad, Mississauga, ON, Canada) using bovine serum albumin as standard.
Contractile experiments
Porcine bronchi strips (4 mm wide and 15 mm length) were suspended isometrically and equilibrated for 60 min under a resting tension of 10 mN in a 10 ml organ bath containing Krebs–Henseleit solution (composition in mM: NaCl 118, KCl 5.6, CaCl2.2H2O 2.5, MgSO4.7H2O 1.19, NaH2PO4 1.3, NaHCO3 25 and glucose 10), oxygenated (95% O2 and 5% CO2) and thermoregulated at 37 °C. Indomethacin (3 μM) was added to avoid the possible influence of prostanoids on contractile response (D'Agostino et al., 1990; Catalli et al., 2002). In each preparation, a cumulative concentration–response curve was determined twice, 60 min apart, for any mAChR receptor agonist: McN-A 343 (3–300 μM), carbachol (0.01–30 μM), bethanecol (0.1–300 μM), methacholine (0.01–100 μM), muscarine (0.01–30 μM), oxotremorine (0.01–30 μM) and muscarone (0.01–1 μM).
The antagonist properties of the mAChR blockers were evaluated on concentration–response curves produced by muscarone. After construction of the first muscarone concentration–response curve, a 30 min washout period was allowed to return the basal tone (that is, 10 mN). The preparation was then exposed for 30 min to one of the following antagonists: atropine (1–30 nM), pirenzepine (1–10 μM), AF-DX 250 (3–30 μM), methoctramine (1–10 μM), tripitramine (1–10 μM), HHSiD (0.03–1 μM), 4-DAMP (10–100 nM), DAU 5884 (10–100 nM), pF-HHSiD (0.1–10 μM) and AQ-RA 741 (0.3–3 μM). A second muscarone concentration–response curve was then carried out in the presence of the antagonist under test. Only one concentration of antagonist was tested on each preparation and at least three increasing concentrations were assayed in separate experiments. The pA2 values were determined according to Arunlakshana and Schild (1959). Time-matched control experiments were carried out in the absence of antagonists.
Calculation and statistical analysis
The maximal binding site (Bmax) and the equilibrium dissociation constant (KD) of the ligand [3H]NMS was determined by Scatchard analysis from the saturation curve. Competition binding data from ligand displacement curves were analysed using Prism programme (GraphPAD software version 5, San Diego, CA, USA) to provide IC50 s values and Hill coefficients (nH). The best fit to one- or two-sites-binding model were determined as an F-test (P<0.01) on the residual variance. Significance testing of the Hill coefficient (nH) was carried out with an unpaired Student's t-test. Values were considered significantly different when P<0.05. The affinity (Ki) values of competing compounds were calculated from IC50 values according to the Cheng–Prusoff equation (Ki=IC50 [1+[D]KD−1; Cheng and Prusoff, 1973) and converted to pKi (−logKi).
In functional experiments, agonists' potency values were expressed as −logEC50, where EC50 indicates molar agonist concentration inducing 50% of the maximum effect of the curve evaluated by nonlinear curve fitting. Intrinsic activity (α) of an agonist was determined as percentage of muscarone maximal response. Antagonists affinity (pA2) values were determined by the Schild regression analysis, using agonist concentration ratios determined at EC50 levels on concentration–response curves in absence and in presence of antagonist (Arunlakshana and Schild, 1959). Means±s.e.m. and confidence limits at 95% probability were evaluated by using a computer program (PHARM/PCS, version 5).
Drugs and chemicals
Carbachol, bethanecol, methacholine, oxotremorine, 4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethylammonium chloride (McN-A343), atropine, pirenzepine and indomethacin were purchased from Sigma Chemical Company (St Louis, MO, USA); 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one (AQ-RA 741), muscarine, para-fluorohexahydrosiladifenidol (pF-HHSiD), HHSiD and methoctramine were purchased from Research Biochemicals, Natick, MA, USA); mamba toxin-3 (MT3) from Peptide International (San Diego CA, USA).
Quinuclidinyl benzilate (QNB) was synthesized at Dr Karl Thomae GmbH (Biberach, Germany); 8-methyl-8-azabicyclo-3-endooct-3-yl-1,4-dihydro-2-oxo-3(2H)-quinazoline carboxylic acid ester (DAU 5884) and dextrorotatory 11-(−1-piperdinyl]acetyl)-5,11-dihydro-6H-pyridobenzodiazepine-6-one (AF-DX 250) were provided by Boehringer Ingelheim Italy (Milan, Italy).
[N-methyl-3H]-scopolamine (2.94 TBq mmol−1 specific activity) was obtained from New England Nuclear (Boston, MA, USA). Tripitramine sesquifumarate and muscarone were a generous gift of Professor C Melchiorre (University of Bologna, Italy) and Professor C De Micheli (University of Milan, Italy), respectively. All drugs were dissolved in distilled water, with the following exceptions: indomethacin, AF-DX 250, HHSiD and pF-HHSiD were prepared in ethanol (stock solutions) and diluted further in distilled water.
Results
Electron microscopy study
The ultrastructural morphology of the bronchial smooth muscle strips, devoid of epithelium, was investigated by transmission electronic microscopy. The smooth muscle cells were arranged in bundles and separated by space containing an amorphous matrix. No ganglia were evident. Nerve fasciculi related to smooth muscle ran parallel to muscle bundles (Figure 1aFigure 1) and close contacts with smooth muscle membrane were more reliably detected when muscle bundles were cut in cross-section (Figure 1bFigure 1). Micrographic analysis also revealed that in porcine bronchi most nerve fibres presented axonal varicosities containing predominantly (along with few large granular vesicles) or exclusively small agranular vesicles. Their profile resembles the profile of cholinergic nerves in primates and humans (El-Bermani and Grant, 1975; Daniel et al., 1986).
Figure 1
Figure 1
Figure 1
(a) TEM photomicrograph illustrating a nerve fibre (N) located between bronchial muscle cells (SM). Scale bar, 1 μm. Magnification, × 20000. (b) TEM photomicrograph showing nerve fibre (N) containing predominantly small agranular (more ...)
Binding experiments
In crude membrane homogenates prepared from porcine bronchi in Na+/Mg++ HEPES buffer, binding of 0.3 nM [3H]NMS (888 KBq) was inhibited by 85±5% by 1 μM QNB. Therefore, specific binding amounted to about 85% of total binding at a concentration of the radioligand near its KD value (0.61 nM). The binding of [3H]NMS was specific and saturable (data not shown). Scatchard analysis of the saturation curve indicated that the non-selective antagonist bound to an apparently homogeneous population of mAChRs with a total density (Bmax) of high-affinity binding sites of 425±65 fmol (mg protein)−1.
Pirenzepine (M1 selective), DAU 5884 (M1/M3 selective), methoctramine (M2/M4 selective), tripitramine (M2 selective) and MT3 (M4 selective) were studied in competition experiments, testing the concentration-dependent inhibition of [3H]NMS binding by increasing concentrations of the putative selective antagonists.
According to Scatchard and Hill plot analyses, the pirenzepine displacement-curve fitted a single-site model (nH=0.89±0.04; pKi=6.43). At variance, nonlinear least squares regression analysis of occupancy–concentration curves of DAU 5884, methoctramine and tripitramine differed from a one-binding-site model. Hill coefficients significantly less than unit (nH=0.75±0.03, 0.73±0.06 and 0.78±0.05, respectively) could reflect the presence of a heterogeneous population of mAChRs.
DAU 5884 showed a best fit to a two-site model, with high affinity (pKi=8.29) and low affinity (pKi=7.05) for 23% and 77% of receptor sites, respectively. According to the two-binding-site model, methoctramine recognized with high affinity (pKi=7.87) 86% of binding sites and displayed low affinity (pKi=7.06) for the remaining 14%. A similar pattern was observed for tripitramine, the recognized proportion being equivalent to 78% (pKi=8.91) and 22% (pKi=7.10) of total bindings sites. As regards MT3, the peptide from green mamba venom, this peptide was completely inactive in the range of concentrations used (10–1000 nM).
The binding parameters of the mAChR antagonists used here are summarized in Table 1.
Table 1
Table 1
Binding parameters of mAChR ligands derived from displacement (competition) experiments against 0.3 nM [3H]NMS in pig bronchi
Functional experiments
A series of mAChR agonists induced concentration-dependent contraction of the smooth muscle bronchial strips. All the compounds behaved as full agonists, compared to muscarone, except oxotremorine and McN-A 343, which showed a maximal contractile response (Emax) of 80.04±1.1 and 21.8±6%, respectively. The rank order of potencies (−logEC50) was muscarone (6.9±0.06; n=6)>oxotremorine (6.82±0.09; n=4)>carbachol (6.38±0.09; n=5)>muscarine (6.33±0.10; n=6)>methacholine (6.07±0.08; n=5)>bethanecol (5.21±0.05; n=6) [dbl greater-than sign]McN-A 343 (4.67±0.6; n=4).
The first and the second concentration–response curves of muscarone were superimposable. The muscarone concentration–response curve was shifted to the right in a parallel manner and without depression of the maximal response by atropine or subtype-preferring mAChR blockers, thus, indicating competitive antagonism as shown by Schild plot analysis (Figure 2Figure 2). Postjunctional pA2 values are listed in Table 2 and compared with affinity estimates for the same antagonists at human M1–M5 subtypes stably expressed in CHO (Chinese hamster ovary) cells. Their correlation analysis is shown in Figure 3Figure 3. In Table 3, the postjunctional pA2 values in pig bronchi are compared with corresponding pA2/pKB values obtained in human bronchi.
Figure 2
Figure 2
Figure 2
Schild plots for atropine and M1/M3 mAChR antagonists (a) and for M2/M4 mAChR antagonists (b). Each point represents the mean±s.e.m. of 4–9 individual observations. The pA2 values with slopes ±s.e.m. for the regression lines are (more ...)
Table 2
Table 2
Comparison of affinity (pKi/pA2) estimates of selective antagonists at human mAChR subtypes (pKi) expressed in CHO cells and at porcine mAChRs (pA2) in pig bronchi
Figure 3
Figure 3
Figure 3
Correlation of postjunctional affinity (pA2) values at mAChRs in pig bronchi with corresponding affinity constants (pKi) at human cloned muscarinic receptor subtypes (hM1-hM5) expressed in CHO cells. r=correlation coefficient of Pearson; SSQ=sum of squares (more ...)
Table 3
Table 3
Comparison of postjunctional pA2 estimates of mAChR antagonists in pig isolated bronchus with pA2/pKB values obtained in human bronchus
Discussion and conclusions
Acetylcholine exerts its effects on smooth muscle, nerves and secretory cells through activation of mAChRs (Barnes, 1987). These receptors were described on different target cells by means of different techniques, but the narrow ‘selectivity window' of mAChR ligands used in the past generated some uncertainty about the expression of the mAChRs subtypes involved in the control of airway functions (see Racké and Matthiesen, 2004, for review). In this study, we used isolated bronchi of the pig, an excellent animal model for biomedical research, as a large body of evidence supports the notion that pigs and humans are similar in many aspects of both infant and adult anatomy, physiology, biochemistry, pathology and pharmacology (Hawarth and Hislop, 1981; Eglen et al., 1996). In this respect, transmission electronic microscopy photomicrographs of sections of pig bronchial strips, offering a fine anatomical view of the neuromuscular structure, support the similarity of porcine with human bronchi (Daniel et al., 1986; van Koppen et al., 1988). The morphological analysis showed a preponderance of small agranular vesicles which are characteristic of cholinesterase-positive cholinergic nerve terminals, innervating the bronchial smooth muscle of large mammals and primates (El-Bermani and Grant, 1975; van Koppen et al., 1987).
Based on this evidence, our strategy was to obtain an appropriate characterization of mAChRs in this bronchial preparation by means of a series of subtype-preferring mAChR antagonists. Their pharmacodynamic parameters, assessed in radioligand binding and functional experiments, are discussed in comparison with corresponding values reported in human recombinant mAChRs.
In binding assays, the non-selective ligand [3H]NMS binds a homogeneous population of mAChRs yielding values in agreement with other non-selective mAChR antagonists tested in pig (Haddad et al., 1994) and human airways (van Koppen et al., 1985, 1988). With regard to the characterization of the mAChRs, the [3H]NMS displacement curve for the M1 antagonist pirenzepine was best fitted to a one-binding-site model with a pKi of 6.43, a value close to that found for human cloned M2 receptors (6.65; see Table 2, for comparison), thus excluding the participation of the M1 receptor subtype. Conversely, a Hill coefficient significantly less than unity for DAU 5884 (M1/M3 selective), methoctramine (M2/M4 selective) and tripitramine (M2 selective), suggests the presence of a heterogeneous expression of mAChRs in porcine bronchi (Table 1). The high affinity values of methoctramine and tripitramine and the low affinity value of DAU 5884 are in agreement with the pharmacological profile of an M2 receptor subtype, whereas the low affinity values of methoctramine and tripitramine and the high affinity value of DAU 5884 are consistent with the presence of a minor population of M3/M5 receptors (Table 2).
Based on these data, the dominant receptor subtype representing approximately 80% of receptor sites (Table 1), is an M2 mAChR, a finding also corroborated by the high level of M2 mRNA transcript (Haddad et al., 1994).
The presence of a putative M4 mAChR subtype was excluded by the apparent inefficacy of MT3, used at concentrations that produce a complete blockade of the M4 mAChR subtype. The MT3 peptide used in this study is known to be effective against the M4 mAChR subtype as it showed activity in the human isolated detrusor (data not shown), a preparation which expresses these receptors (D'Agostino et al., 2000). In this respect, the absence of M4 mRNA in porcine bronchi (Haddad et al., 1994) should be noted.
The identification of the minor M3/M5 receptor population was achieved with the use of previous (pirenzepine, methoctramine, tripitramine and DAU 5844) and additional mAChR antagonists (atropine, AF-DX 250, AQ-RA 741, 4-DAMP, HHSiD and pF-HHSiD) in functional contractile smooth muscle experiments. Porcine bronchi contracted following exposure to the mAChR agonists muscarone, carbachol, muscarine, methacholine and bethanecol, demonstrating full agonist properties, whereas oxotremorine and Mc-NA 343 behaved as partial agonists. The concentration–response curve induced by the most potent agonist, muscarone, was shifted in a competitive fashion by all the mAChR blockers with a slope not significantly different from unity, probably indicating the involvement of a single muscular mAChR subtype. The high affinity values obtained for atropine, 4-DAMP and DAU 5884, the intermediate affinity values for HHSiD, pF-HHSiD, pirenzepine and tripitramine, as well as the low affinity estimates for methoctramine and AF-DX 250 (see Table 2) suggest a prevalent M3 mAChR pharmacological profile. Nevertheless, the participation of the M5 receptor subtype could not be excluded a priori, given the similarity in M3 and M5 profiles (difference <ten-times) of the antagonists used. The use of AQ-RA741, a ligand with preferential affinity (difference >10-fold) for the M3 over the M5 receptor in animal and human tissues (Eglen and Nahorsky, 2000), strongly suggested that M3 mAChRs mediate contractions of porcine bronchial smooth muscle. This conclusion is also supported by the finding of an excellent correlation between pA2 values obtained in pig bronchi and those obtained in CHO cells expressing the human M3 receptor (Figure 3cFigure 3). The data generated in pig bronchi are also largely consistent with those obtained in human tracheal and bronchial preparations (Roffel et al., 1990; Watson et al., 1995) suggesting that the same mAChR subtype (that is, the M3 subtype) is coupled to contraction. The difference in affinities observed with methoctramine, HHSiD and pF-HHSiD in human and pig bronchi could be explained in several ways. In human bronchi, methoctramine (3–300 μM) causes irregular rightward shifts of agonist concentration–effects curves accompanied by depression of the curve maximums, thereby, compromising estimation of its affinity. As far as HHSiD and pF-HHSiD are concerned, both compounds show some variability in pA2 values for the M3 receptor subtype, which are tissue and species-dependent. As such, the utility of these antagonists in muscarinic receptor classification is limited (Eglen et al., 1990).
In conclusion, isolated porcine bronchi seem to be a reliable model system to study mAChR function in peripheral airways and may represent an useful tool in the screening and development of novel bronchodilator agents for the therapy of airway diseases in humans.
Acknowledgments
This study was supported by funds from the University of Pavia (FAR-2006 and FAR-2007).
Abbreviations
AF-DX 250dextrorotatory 11-(-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyridobenzodiazepine-6-one
AQ-RA 74111-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one
4-DAMP4-diphenylacetoxy-N-methyl-piperidine
DAU 58848-methyl-8-azabicyclo-3-endooct-3-yl-1,4-dihydro-2-oxo-3(2 H)-quinazoline carboxylic acid ester
HHSiDhexahydrosiladifenidol
pF-HHSiDparafluorohexahydrosiladifenidol
mAChRmuscarinic acetylcholine receptor
McN-A 3434-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethyl-ammonium chloride
MT3mamba toxin-3
[3H]NMS[N-methyl-3H]scopolamine methyl chloride
QNBquinuclidinyl benzilate

Notes
Conflict of interest
The authors state no conflict of interest.
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