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J Appl Physiol 98: 1526-1533, 2005. First published December 3, 2004; doi:10.1152/japplphysiol.00948.2004
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TRANSLATIONAL PHYSIOLOGY

Increased relaxation of immature airways to {beta}2-adrenoceptor agonists is related to attenuated expression of postjunctional smooth muscle muscarinic M2 receptors

Michael Fayon,1,2 Eric Dumas De La Roque,1,2 Patrick Berger,1 Hugues Begueret,1 Olga Ousova,1 Mathieu Molimard,1 and Roger Marthan1

1Université Victor Segalen Bordeaux 2, Laboratoire de Physiologie Cellulaire Respiratoire, and Institut National de la Santé et de La Recherche Médicale E 356, Bordeaux, France; and 2CHU Bordeaux, Hôpital Pédiatrique, Unité de Pneumologie Pédiatrique et Centre de Recherche (CEDRE), Bordeaux, France

Submitted 30 August 2004 ; accepted in final form 24 November 2004

ABSTRACT

Spontaneous or agonist-induced contraction of airway smooth muscle can be observed very early in fetal life, thus explaining the possible occurrence of bronchospasm in very low birth weight infants within the first days of life. In an attempt to better manage such bronchospasms, the aim of the present study was to investigate the age-specific modifications in airway smooth muscle relaxation to {beta}2-agonists and muscarinic antagonists using a combination of functional and molecular techniques. In the rat, isometric relaxation to the {beta}2-agonist salbutamol was examined in tracheae; we also examined muscarinic receptor expression (M2R and M3R mRNA levels) in airway smooth muscle by immunochemistry, Western blotting, and real-time PCR. Compared with adults, salbutamol-induced relaxation was twofold greater in immature rat isolated tracheae preconstricted by carbachol. This effect was associated with a lower expression of M2R in the smooth muscle of immature animals (sixfold and almost twofold as assessed by immunochemistry and Western blotting, respectively). Real-time PCR data indicate that changes in M2R expression according to age occurred at a posttranscriptional level. In adult airways, there was a significantly greater functional efficacy of M2R blockade by methoctramine compared with that shown in immature rats. Because of the limited availability of human neonate lung tissue, only the molecular part of the study was performed, and we observed a qualitatively similar effect, i.e., a lower M2R expression in the neonatal airway smooth muscle, although this was quantitatively smaller. We conclude that {beta}2-agonist-induced relaxation is enhanced in immature compared with adult airways as a result of greater postjunctional M2R expression in adult airway smooth muscle. This finding may be of importance in the clinical management of bronchoconstriction in neonates.

acetylcholine; albuterol; muscarinic receptor subtypes; physiopathology; trachea

THE STUDY OF THE ONTOGENY of airway smooth muscle reactivity has revealed that spontaneous or agonist-induced contraction of airway smooth muscle can be observed very early in fetal life (22), thus explaining the possible occurrence of bronchospasm in very low birth weight infants within the first days of life (37). In humans, we have previously shown that most mechanisms controlling airway tone in adults are operative in the premature neonate (11). Whereas in vivo studies suggest that airway narrowing in human newborns may be less than that in adults (12), in vitro investigations in animals indicate that airway smooth muscle from neonatal or young generates greater isometric force compared with that from adult (27, 35). In this connection, it has been demonstrated that the force of airway smooth muscle contraction normalized to tissue weight, smooth muscle content, and/or tissue myosin content decreases systematically during maturation in the major resistance airways of swine (35).

Regarding airway smooth muscle relaxation, numerous in vivo studies indicate that bronchodilators may be efficacious in human neonates (6, 9, 26). The ontogeny of {beta}-adrenergic receptors in the developing lung has been previously studied in animals (21, 32); in rats for example, receptor density progressively increases as gestation advances and continues postnatally (32). Airway smooth muscle receptor density, as assessed by binding studies, increases 93% from day 1 to day 13, another 92% from day 13 to day 20, and remains unchanged thereafter (32). However, it has also been shown that {beta}-adrenoceptor responsiveness and sensitivity in guinea pig and rabbit airway smooth muscle decreases with age (5). Interestingly, the changes in functional responsiveness of tracheal tissue are not reflected by changes in the binding density or affinity of {beta}2-receptors or by changes in specific autoradiographic grain density over smooth muscle tissue (29). Evidence that ontogenic modification occurs at the receptor level has been provided by research showing that the effect of forskolin, a direct stimulator of adenylate cyclase, is not modified as a function of age in rats (13).

There is cross-talk between muscarinic receptors and {beta}2-adrenoceptors (30, 31). Whereas M3 receptors (M3R) have been associated with smooth muscle contraction, the prejunctional M2-receptor subtype (M2R) modulates ACh release from postganglionic cholinergic nerve endings (31). Other postjunctional M2Rs localized to the airway smooth muscle have been implicated in G{alpha}i protein-coupled inhibition of adenylate cyclase (1, 30, 33). A number of studies have implicated these postjunctional M2Rs in the functional antagonism between airway smooth muscle contraction and relaxation by muscarinic agonists and the relaxation elicited by {beta}-adrenoceptor agonists in canine and guinea pig trachea (31, 33).

It was our hypothesis that developmental aspects of postjunctional M2R may play a role in the differential effects of {beta}-adrenoceptor agonists between neonatal and adult airway smooth muscle. Because selective muscarinic antagonists are becoming available, this issue may be of importance for the clinical management of bronchoconstriction in neonates. The aim of the present study was thus to investigate the age-specific modifications in airway smooth muscle relaxation to {beta}2-agonists and muscarinic antagonists with a combination of functional and molecular techniques, including immunochemistry, immunoblotting, and real-time PCR.

MATERIALS AND METHODS

Animal Studies

Our laboratory has the approval of the Animal Ethics Committee of our Institution to house rats and conduct these experiments. Adult as well as pregnant pathogen-free Wistar rats of known gestational age were obtained from Dépré, France, and allowed food and water ad libitum. After parturition in the laboratory, the pups were housed with their nursing mother during the entire experimental time. The immature and adult rats underwent euthanasia at the age of 12 days and 15 wk, respectively.

Isometric relaxation measurements.   Isometric relaxation measurements were done according to a previously described method (8). After rings were set at optimal resting tension, carbachol (Sigma, Saint-Quentin-Fallavier, France) was used to preconstrict the rat airway tissue. Unlike in steady-state cumulative concentration experiments, a single 10–6 and 3.10–7 M concentration of carbachol was used in adult and immature rats, respectively. This corresponded to EC70, i.e., the log concentration of carbachol inducing 70% of the maximal response. Once equilibrium tension, i.e., stable isometric tension after preconstriction, was achieved, salbutamol (salbutamol hemisulfate salt; Sigma) or atropine (Sigma) was added to each organ bath, and isometric tension was recorded. Cumulative concentration-response curves to salbutamol (10–9 to 10–4 M) and atropine (10–8 to 10–6 M) were then generated in a cumulative manner. Maximal relaxation (Emax) was elicited by the terminal administration of bamyphylline (3.10–3 M).

All solutions were freshly prepared on the day of the experiment. For each ring, relaxation to salbutamol was expressed as active force, stress, i.e., the active force divided by muscle cross-sectional area (g/mm2) (see Morphological study below), and percentage of Emax induced by bamyphylline.

Further experiments were done to assess the functional role of M2R, as well as the cyclooxygenase and nitric oxide synthase pathways. In a separate set of experiments, immature and adult tracheae were preincubated with 0.3 or 1 µM methoctramine (30 min) (31), 10–5 M indomethacin (1 h) (24), and 10–4 M nitro-L-arginine methyl ester (L-NAME; 1 h) (24) before tone was built up, and relaxation to salbutamol was again studied. All of these drugs were purchased from Sigma.

Morphological study.   Morphological study was done according to a previously described method in glycolmethacrylate-embedded tracheae (8). The mean within-observer coefficient of variation for smooth muscle surface area measurement was 5.4% (range 3.9–6.6%). The mean coefficient of variation for smooth muscle surface area of rings taken from the same trachea was 12.2% (range 1.1–35.9). Using x40 magnification, we measured the circumference of the tracheal lumen (epithelium) in immature and adult rats.

Immunohistochemistry.   Tracheae (n = 5 per age group) were fixed in 10% formalin and embedded in paraffin. Serial sections, 3 µm thick, were cut. For immunophenotypic characterization, we performed immunohistochemical studies on paraffin-embedded contiguous sections using the biotin-streptavidin-peroxidase LSAB kit (DAKO) with goat polyclonal primary antibodies (Santa Cruz Biotechnology) directed against muscarinic M3Rs (clone C-20 at 1/50) and M2Rs (clone C-18 at 1/50), as well as heterotrimeric G protein {alpha}-subunits (G{alpha}i/o/t/z, clone D-15 at 1/50) and biotinylated swine secondary antibody (Multilink, DAKO; at 1/200). Protease digestion (trypsin, Prolabo, Créteil, France) was used at a dilution of 0.1% during 10 min at 37°C for the enhancement of the immunoreactivity. Control slides were treated similarly, by using an unrelated antibody. The sections were counterstained with Mayer's hematoxylin. The staining process was done with the peroxidase substrate 3-amino-9-ethylcarbazole (Sigma Aldrich Chimie SARL, Lyon, France). All staining was performed at the same time. Relative staining density in the smooth muscle was determined with Quancoul software (Quant'Image) by measuring the area stained vs. the total area of the smooth muscle.

Immunoblotting for M2R.   Experiments were performed in smooth muscle extracts. Immediately after euthanasia, the tracheal pars membrana was removed and the surrounding tissue was manually teased away and scraped with microforceps and scissors; this procedure was done via a Nikon SMZ 800 microscope (optical microscope examination of embedded dissected muscle showed minimal residual surrounding tissue). The tissue was frozen in liquid N2 and maintained at –80°C until use. Smooth muscle extracts were prepared from a pool of eight and five immature and adult rats, respectively. All samples were branded in liquid N2 and homogenized in RIPA buffer (1% NP-40, 0.5% deoxycholate sodium, 0.1% SDS, 10 µg/ml aprotinin, 100 µg/ml leupeptin, 1 mM AEBSF, 4-(2 aminoethyl)benzenesulfonylfluoride in PBS) with a Polytron (Bioblock Scientific, Illkirch, France) and then centrifuged. After centrifugation, the protein content of the supernatant was assayed with the use of an improved Lowry assay (Bio-Rad DC protein assay) and verified by Coomassie staining (Bio-Rad). A portion was reduced with {beta}-mercaptoethanol and subjected to electrophoresis on a 10% acrylamide reducing gel. Ten micrograms of each sample were loaded in 10% resolving acrylamide gel and transferred to Immobilon-P PVDF membranes (Millipore). The immunoblots were then developed with goat polyclonal anti-muscarinic ACh M2R (Santa Cruz Biotechnology clone C-18 at 1/1,000) or mouse monoclonal anti-{beta}-actin (Santa Cruz Biotechnology clone C-2 at 1/500). A biotinylated swine antibody reacting with all immunoglobulin classes of goat, mouse, and rabbit (Multilink, Dako, at 1/2,500) was used as secondary antibody. A streptavidin-biotinylated horseradish peroxidase kit (Dako, at 1/50) was used for amplification, and the immunoblots were revealed by enhanced chemiluminescence (Uptima, Interchim, Montluçon, France). For quantification, we used BioCaptMW software (Fischer Bioblock Scientific). The experiments were repeated three times with the same protein isolation, so as to assess their reproducibility.

Real-time PCR.   We extracted total RNA from rat tracheal smooth muscle cells using 400 and 600 µl of Trizol per immature and adult trachea, respectively, according to the manufacturer's instructions (GIBCO, Invitrogen, Cergy Pontoise, France). RNA integrity (the integrity of the 28S and 18S fractions of rRNA) was verified by gel electrophoresis. The concentration of total RNA was measured by GeneQuant RNA/DNA calculator (Amersham Pharmacia). The reverse transcription reaction was carried out as mentioned previously (2). Real-time PCR was performed with a Rotor-Gene 2000 (Corbett Research; Morlake, Sidney, Australia) as described earlier (2). All specific primers were designed by using oligo primer analysis software (Oligo 6.6, Molecular Biology Insights, Cascade, CO) and ordered from Sigma-Genosys (Sigma-Genosys, Cambridge, UK). The primer pair sequences are indicated in Table 1. The amplified cDNA products for M2, M3, GAPDH, acidic ribosomal protein PO (PRLPO), {beta}-actin, and YWHAZ are 148, 172, 153, 208, 231 and 291 bp, respectively. The efficiency of all of the PCR reactions was more than 90%. Of note, actins are highly conserved proteins. Therefore, the {beta}-actin primer constructed may also have amplified {alpha}-actin due to the high homology of nucleotide sequences. The specificity of the amplified products was examined in 2% agarose gel containing ethidium bromide. The accuracy of the normalization of real-time PCR data was verified by geometric averaging of four internal control genes (see above), according to a previously described method [Genorm (38)].


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  		Table 1. Primer pairs used for rat airway smooth muscle gene expression analysis by real-time reverse transcription PCR

 
Human Studies

The study was conducted in accordance with the institutional ethics policies and with the written, informed consent of the parents. Human lung was obtained at autopsy from premature and term neonates and infants who had died from extrapulmonary causes and at thoracotomy in adults undergoing resection for pulmonary carcinoma, as previously described (2, 11). Adult patients were nonatopic and did not report any clinical history of asthma. Obtained tissues (trachea in neonates and third to fifth generation airways in adults) were used for cell cultures.

Immunohistochemistry.   Blocks of bronchial tissue obtained from a tissue bank in the pathology department [n = 5 per age group in adults and infants (<1 yr old)] were processed according to the method described above.

Immunoblotting for M2R.   Immunoblotting for M2R experiments were performed in airway smooth muscle cell culture (trachea in neonates, 3rd to 5th generation bronchi in adults, n = 2 separate individuals per age group). Cleaned airway smooth muscle was dissected with the aid of a microscope, and the cells were cultured in uncoated plastic culture plates in DMEM supplemented with 10% fetal bovine serum (GIBCO BRL), 1% nonessential amino acids (Sigma), 2 mM L-glutamine (GIBCO BRL), 1 mM sodium pyruvate (Sigma), 0.5 U/ml penicillin, and 0.5 mg/ml streptomycin and 0.25 µg/ml amphotericin B. Cells were grown at 37°C in a humidified incubator under 5% CO2. For passage of cultures at confluence, cells were lifted using 0.05% trypsin and 0.5 mM EDTA and reseeded into new culture plates. Cells from passage 1 or 2 were used in the present study. Extraction of proteins from cell culture was done without serum deprivation, since it has previously been shown that M2R protein decreased with serum deprivation in cultured canine tracheal myocytes (decrease to 25% of day 0 values after 2 days) (25). The cells were washed with PBS, and then total protein homogenates were prepared in extraction buffer (20 mM Tris, pH 7.5, 2 mM sodium orthovanadate, 1 mM EDTA, 20 µg/ml aprotinine, 5 mM MgCl2, 1 mM 1,4-dithio-DL-threitol, 1 mM AEBSF, 100 µM leupeptin, and 1% Triton X-100). Proteins from whole cell lysates were size fractionated by SDS-PAGE and then electroblotted, as described above.

Statistical Analysis

Reported values are means ± SE. Statistical analysis was performed with the software package NCSS 6.0.21 (Kaysville, UT). Differences between groups of animals regarding smooth muscle surface area, real-time PCR, and Emax were compared with unpaired t-tests. The effects of age and pharmacological agent concentration on smooth muscle relaxation were analyzed by a two-way multivariate analysis of variance. A P value of <0.05 was considered significant. Bronchodilator apparent EC50 (i.e., the log concentration of salbutamol that induces 50% of the maximal response) was determined with Boltzman's nonlinear curve fit, considering that Emax was that induced by the maximal concentration of bronchodilator used.

RESULTS

Animal Studies

Isometric relaxation measurements and morphometry.   Relaxation to salbutamol in immature airways was significantly greater than in mature airways at most of the concentrations (Fig. 1). When expressed as percent of Emax to 10–3 M bamyphylline (Fig. 1A), salbutamol produced similar Emax in immature compared with mature rats. However, Emax was significantly enhanced in immature rats when the response was expressed as force per surface area of smooth muscle (stress) (Fig. 1B). Absolute values of maximal contraction and relaxation were twofold greater in adults compared with immature rats, but percent maximal contraction-to-Emax ratio was similar in both age groups (Table 2). Salbutamol potency was also significantly greater (P < 0.001) in immature (EC50 of 2.73 x 10–8 M; 95% confidence interval of 6.29 x 10–9 to 1.19 x 10–7 M) vs. adult rats (EC50 of 5.67 x 10–6 M; 95% confidence interval of 1.75 x 10–6 to 1.83 x 10–5 M).



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  		Fig. 1. Mean concentration response curves for salbutamol-induced relaxation of rat isolated tracheae precontracted with carbachol at a concentration inducing 70% of the maximal response (EC70). A: relaxation expressed as percent of maximal relaxation induced by bamyphylline. B: relaxation expressed as stress (normalization of force to smooth muscle surface area). Enhanced relaxation and greater sensitivity are present in immature compared with adult rats. Values are means ± SE; n = 13 for adults ({blacktriangleup}) and 9 for immature rats ({circ}). *P < 0.05, adult vs. immature, for the same concentration of salbutamol.

 

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  		Table 2. Functional and morphometric data in immature and adult rats

 
Atropine induced rapid relaxation in both groups (–52.9% and –72.5% of Emax to bamyphylline at 10–8 M in adults and immature rats, respectively) with no difference in either maximal response or potency according to age.

Immunohistochemistry.   M3R, M2R, and G{alpha} protein labeling was present in the epithelium, smooth muscle, and glandular tissues. There was no difference between immature and adult animals regarding M3R and G{alpha} labeling density (data not shown). M2R density in the smooth muscle was consistently six times less in immature rats compared with adults [7.6 ± 2.1 vs. 47.0 ± 3.0% (P < 0.01), respectively] (Fig. 2).



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  		Fig. 2. M2R staining. A: representative photographs of antimuscarinic type 2 receptor (M2R) density in tracheal smooth muscle in adult (a and c) and immature (b and d) rats or adult and immature human bronchi (e and f) (x20 magnification). a and b: Control experiments using an unrelated antibody. *Smooth muscle. Note the decreased 3-amino-9-ethylcarbazole (red) staining in immature rat smooth muscle and sparse staining in humans. Scale bar = 100 µm. B: percent of M2R staining normalized to smooth muscle surface area in rats (n = 5 immature and adult human bronchi and n = 5 immature and n = 5 adult rat tracheae). *P < 0.05.

 
M2R protein expression.   Figure 3A shows bands that had migrated at 65 kDa, compatible with M2R, similar to the findings of others (23) in the airways. The density of the M2R bands was 67% greater in adult vs. immature rats.



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  		Fig. 3. M2R density. A: representative experiments of anti-M2R density in pooled, cultured rat and human tracheal smooth muscle by Western blotting. B: results expressed as relative (adult/immature) density (mean ± SE; n = 3 experiments using the same pooled sample for the assessment of the quantitative reliability of repetitive immunoblots) of M2R ratio. Note that, in both rats and humans, there was a higher adult-to-immature M2R ratio.

 
Real-time PCR.   Using Genorm software (38), we found the most stable housekeeping genes for the rat airway smooth muscle to be, in rank order of stability, {beta}-actin > YWHAZ > GAPDH > PRLPO, with an average expression stability value (M) of 0.45, 0.48, 0.58, and 0.59, respectively (the lowest M values have the most stable expression). The real-time PCR expression of the target gene was normalized to a combination of the first three endogenous references (GAPDH, {beta}-actin, and YWHAZ) because this was deemed adequate by Genorm analysis. After corrections by the respective normalization factors were made, the relative quantities of M2 mRNA (compared with the lowest value) in adult and immature rats were 0.82 ± 0.14 and 1.46 ± 0.41, respectively [not significant (NS)]. M3 mRNA relative quantities were 4.44 ± 0.75 and 4.65 ± 0.89, respectively (NS). The greater protein expression in rats was thus not linked to a significant increase in mRNA levels. This was also confirmed by the 2–{Delta}{Delta}CT method (the delta-delta cycle threshold method for comparing relative expression results between treatments in real-time PCR; Perkin Elmer, Forster City, CA) (28), for both M2R and M3R (2{Delta}{Delta}CT of 1.6 and 1.3, respectively).

Functional Studies

Percent relaxations to salbutamol of immature and mature tracheae after preincubation with methoctramine are shown in Fig. 4. This agent significantly enhanced relaxation (vs. controls) in both immature and adult airways. However, in adult airways, there was a significantly greater efficacy of M2R blockade; i.e., a greater increase in relaxation to salbutamol was observed in adult compared with immature airways, and this was more marked at a concentration of 0.3 µM methoctramine: 157.1 ± 7.8% (immature, n = 7) vs. 310.0 ± 47.0% (mature, n = 12) (P = 0.026). Percent relaxation vs. controls to salbutamol after pretreatment with 10–5 M indomethacin was 90.2 ± 11.1 and 81.4 ± 24.1% in immature (n = 7) and mature (n = 10) rats, respectively (NS). Similarly, there was no significant difference in salbutamol-induced relaxation in immature (n = 8) vs. mature (n = 10) rats, respectively (NS), after pretreatment with 10–4 M L-NAME (not shown)



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  		Fig. 4. Maximal functional efficacy of M2R blockade by methoctramine. Rat isolated tracheae were preincubated for 30 min with methoctramine before precontraction with carbachol (EC70) and addition of salbutamol. Values are means ± SE. Black bars and gray bars represent immature (n = 7) and adult (n = 12) rats, respectively. *P < 0.05, adult vs. immature, at 10–4 M salbutamol.

 
Human Studies

In humans, the difference in M2R density in the smooth muscle as measured by immunochemistry (Fig. 2A) was also significant, although of lower amplitude [3.2 ± 0.5 vs. 5.8 ± 0.7% (P = 0.01) in infants and adults, respectively]. The immunoblot analysis of M2R protein showed a 17% greater expression in adults compared with infants (Fig. 3B).

DISCUSSION

In the present study, we have shown that the {beta}2-agonist-induced relaxation in rat isolated tracheae preconstricted by carbachol is greater in immature animals. This effect is associated with a lower expression of M2R in the smooth muscle, as confirmed by immunoblotting and immunochemistry. Quantitative real-time PCR did not reveal significant changes in M2R mRNA according to age, suggesting a posttranscriptional mechanism. There was a significantly greater functional efficacy of M2R blockade by methoctramine in adult airways. Finally, similar qualitative data, although with a smaller quantitative amplitudes, were observed in human tissues. These findings therefore provide a mechanistic explanation for the greater effect of {beta}-agonists in newborns. Incidentally, from a technical point of view, this study also provides additional data for the optimal use of real-time PCR in ontogenetic studies in rat airway smooth muscle cells. The stability of a combination of {beta}-actin, YWHAZ, and GAPDH as housekeeping genes enables us to normalize accurately any other gene of interest in the airway smooth muscle in this animal species.

In rats, a comprehensive study combining physiological and molecular approaches including immunochemistry, immmunoblotting, and real-time PCR was performed in similar tracheal tissues. For the three latter determinations, as much care as possible was taken to obtain pure smooth muscle in which prejunctional M2Rs were considered to be absent as controlled by optic microscopy. Comparing M2R expression in two age groups by immunoblotting is hazardous, since the commonly used control protein, {beta}-actin, has not been validated for ontogenetic studies. We thus evaluated absolute values of M2R expression for the same protein load. However, taken altogether, the data consistently point to the greater role of postjunctional M2Rs in adult airway relaxation. Because of major difficulties in obtaining human neonatal airway tissue, only the molecular part of the study was performed in humans. It is better to avoid direct comparisons of results of immunoblotting in rats vs. humans since the origin of human airway smooth muscle varied (cultured cells from tracheae in neonates vs. intrapulmonary airways in adults and tissue bank for immunochemistry). Nonetheless, the fact that similar qualitative data, although quantitatively different, were observed in human and rat tissues suggests that the hypothesis of a change in postjunctional M2Rs playing a role in the differential effect of {beta}-adrenoceptor agonists between neonatal and adult airway smooth muscle may be clinically relevant.

Immature airways are of small caliber with high resistance, and there might be some functional benefit to having greater airway smooth muscle relaxant properties. Preliminary data (not shown) as well as those from our previous studies (11) confirm greater sensitivity to {beta}2-agonists in immature human neonates compared with adults (as observed in rats in the present study), in agreement with the present molecular findings. It is unlikely that {beta}2-receptors account for the greater relaxation observed in immature animals. First, although the density of such receptors over rat bronchial smooth muscle increases threefold within the first 6 mo (32), we did not observe a greater relaxant effect to {beta}2-agonists in adult compared with juvenile rats. Second, the binding of {beta}2-agonists to only a fraction of the available receptors is sufficient to provoke a maximal effect, explaining why the changes in functional responsiveness of tracheal tissue are not reflected by changes in the binding density of {beta}2-receptors (5). We therefore reasoned that the difference between the two age groups may be due to a factor that is partially or completely independent of {beta}2-receptors, in particular, M2R as previously suggested (33), taking advantage of the possible use of molecular techniques to evidence this hypothesis. Our functional results add convincing evidence that such receptors are of lesser importance in immature vs. adult airways. Nevertheless, alternative mechanisms for enhanced airway relaxation in early life have also been proposed, such as the nitric oxide-prostaglandin pathway (4, 34). In the present study, neither of these pathways appears to contribute to the difference between the two age groups. Moreover, it should be kept in mind that the present experimental design also did not allow us to examine the kinetics of {beta}2-adrenoceptor agonist-induced relaxation, e.g., the mean half-relaxation time and the late phase of isotonic relaxation to submaximal concentrations of {beta}2-agonists (36). Therefore, information related to cross-bridge cycling rate could not be obtained. In light of the "plasticity theory" of which the most important feature is that active force is length independent, the eventual age-related differences in the above parameters warrant further investigation (36).

The mechanism underlying muscarinic inhibition of {beta}-adrenergic responsiveness is well established. Pharmacological criteria reveal four distinct receptor subtypes: M1 (parasympathetic ganglia), M2 (both postganglionic nerves and smooth muscle cells), M3 (smooth muscle cells), and M4 (autoinhibitory prejunctional receptors in guinea pigs). These subtypes have been identified in airway tissue by the use of selective antagonists (30). The most important muscarinic receptor mediating ACh-induced contraction of bronchial smooth muscle is of the M3 type (30). M1R and M3R are generally coupled to Gq/11 protein and phosphoinositide hydrolysis and thus to calcium signaling, whereas M2R and M4R are coupled to Gi proteins and adenylate cyclase inhibition and thus to cAMP signaling (30). When cAMP concentration is increased on {beta}2-adrenoceptor activation, M2R activation by carbachol inhibits cAMP formation (30). The M2R thus contributes to cholinergic functional antagonism and also causes indirect contraction of human bronchi by reversing sympathetically mediated relaxation. As an example, in intact isolated bronchi from adult humans (31), ACh-induced precontraction decreases isoproterenol potency and Emax (–log EC50 shift = –1.49 ± 0.16 and Emax inhibition for 100 µM ACh = 30%) more than histamine-induced equivalent contraction. In this connection, the M2R-selective antagonist methoctramine reduced this antagonism in ACh-contracted but not histamine-contracted tissues, suggesting participation of M2R.

Despite its clinical relevance, the ontogenesis of muscarinic receptors in airway smooth muscle has not been extensively studied, and the results are somewhat contradictory, possibly due to different experimental design and species specificity. Direct estimation by means of radioligand binding studies have shown considerable variation between species in the relative proportion of muscarinic receptor subtypes and lungs (17). Tracheae from rats show 79–90% of the total sites to be of the M2 subtype, whereas human lung apparently expresses no M2R (15), and there are few RNA transcripts for the M2R in human tracheal smooth muscle (20). In guinea pig airway smooth muscle, the majority of muscarinic receptors belong to the M3 subtype, with a very small population of the M2 subtype present (20). Two other reports have studied the ontogenesis of muscarinic receptors in whole lung. In the first report (39), muscarinic cholinergic receptors were characterized in crude membrane fractions of rat lung and trachea from day 17 of gestation to adulthood using (–)-[3H]quinuclidinyl benzilate. Muscarinic cholinergic receptor sites were identified as early as day 17 of gestation, decreasing progressively from days 17–18 to days 21–22 of gestation and remaining stable thereafter from birth to adulthood. A second report (18) in porcine peripheral whole lung membranes showed that maximal binding capacity of the nonselective muscarinic antagonist [N-methyl-3H]scopolamine and the affinity of the receptor significantly increase between birth and adulthood. Subtype affinity changed with age (greater affinity of M1R together with less M2R affinity), but the rank order was always M3 > M1 > M2. Finally, Northern blots showed that M2 mRNA was present at all ages and decreased with age. This decrease in M2R and mRNA with age is in contrast to the present study and functional data. This may be due to the different species studied. Moreover, the data in the reported literature were not specific to the airway smooth muscle itself.

In animals, the consequences of M2R ontogeny on airway relaxation have been studied in isolated trachea. For example, to determine whether the functional role of M2R varies with age, dose-dependent relaxation responses to isoproterenol were compared in 3-day-old and adult rabbits in the absence and presence of an M2R antagonist (33). In accordance with the present study, airway smooth muscle sensitivity to isoproterenol decreased with age and the expression of inhibitory G{alpha}i subunit proteins was similar in 3-day-old and adult airways. Furthermore, it was demonstrated that the magnitude of muscarinic functional antagonism of isoproterenol-mediated relaxation significantly increased with age. In contrast, however, with the present study, the inhibition of {beta}-agonist-induced relaxation via the M2R-Gi protein pathway appeared to be independent of age (33). In another study, the maximal relaxant effect of isoproterenol decreased from 4 to 11 wk in Sprague-Dawley rat airways (13) but not in the Wistar strain, suggesting that physiological responses to {beta}2-agonists may be strain dependent. In humans, to the best of our knowledge, there has been no report on the ontogeny of M2R and airway relaxation.

The relative difference between M2R in adult and immature tissue that we presently report supports the hypothesis of a change in postjunctional M2R playing a role in the differential effect of {beta}-adrenoceptor; this may be clinically relevant. In terms of translational physiology, the study of prejunctional and postjunctional M2R ontogeny is important for pathophysiogical, clinical, and pharmacological reasons. Experimental data have shown that double-stranded RNA, a product of viral replication, causes greater bronchoconstriction and bradycardia via increased release of ACh from the vagus nerves because of loss of M2R function on parasympathetic nerves in the guinea pig lung (3). It has also been shown that vitamin A deficiency promotes bronchial hyperreactivity in rats by altering muscarinic M2R function (23). Vitamin A deficiency remains an important health problem among children in developing countries; there, higher mortality from respiratory infections is found, which is likely related to suboptimal nutrition, including vitamin A deficiency. In the clinical setting, influenza virus and major basic protein from eosinophils may inactivate M2R (7, 10, 14, 19). This may account for greater cholinergic reflex bronchoconstriction during an exacerbation of asthma, due to either a viral infection or an allergen exposure in children. Finally, the use of newer and more specific cholinergic antagonists has recently been advocated (16). Nonselective muscarinic antagonists, by blocking the postjunctional M2R-mediated inhibition of relaxation, might be more efficient for treatment of bronchospasm than M3R-selective antagonists (16). In light of our findings, the relative clinical importance of the blockade of autoinhibitory M2R in children remains to be proven.

In conclusion, the present study adds evidence in support of greater Emax to {beta}2-agonists in immature compared with adult animals and provides data in favor of a mechanism related to attenuated muscarinic functional antagonism as a result of greater adult M2R expression in the airway smooth muscle observed in both rats and humans. Although the pathophysiological role of prejunctional M2R is better understood, the question regarding whether and how the airway smooth muscle M2R influences airway disease in children requires further clarification.

ACKNOWLEDGMENTS

The authors are grateful to Josette Arsaud, Renée Dalibart, Huguette Crevel and Béatrice Martinez for technical support with Western blotting and isometric contraction.

FOOTNOTES


Address for reprint requests and other correspondence: M. Fayon, Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E356, Université Bordeaux 2, 146 rue Léo Saignat, 33000 Bordeaux, France (E-mail: michael.fayon{at}chu-bordeaux.fr)

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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