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1Department of Pediatrics, The Hospital for Sick Children, and 2Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada
Submitted 23 March 2006 ; accepted in final form 9 November 2006
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ABSTRACT |
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1- and 
1-subunits were significantly lower (P < 0.01) in the adult compared with the newborn bronchial tissue. Consistent with these results, the NO-stimulated sGC activity was significantly greater in the newborn compared with the adult (P < 0.01). In conclusion, the bronchial smooth muscle cGMP-, but not cAMP-dependent, relaxant response is developmentally regulated and significantly reduced in the adult rat. bronchodilation; lung; cAMP; cGMP; salbutamol
The main cyclic nucleotides involved in airway smooth muscle relaxation are the cAMP and cGMP. Commonly therapeutically used 2-adrenoceptor agonists act via cAMP to induce bronchodilation (12). The rat tracheal smooth muscle relaxant response to the adenylate cyclase stimulator forskolin does not change with age (11). Yet, the -adrenergic receptor density in the rat lung is developmentally regulated and progressively increases from fetal to postnatal life (22).
cGMP-mediated airway smooth muscle relaxation is present in several, if not all mammalian animal species, and nitric oxide (NO) is considered to be a weak bronchodilator based on data derived from adult animal and human studies (12). This contrasts with the important role of NO in the regulation of pulmonary vascular smooth muscle tone and the pathogenesis of pulmonary hypertension (23). The extent to which the limited NO-dependent bronchodilation relate to maturational differences in the protein expression and activity of soluble guanylate cyclase (sGC) merits further investigation.
Therefore, the purpose of the present study was to evaluate the newborn and adult bronchial cAMP- and cGMP-modulated relaxation response in the rat. A significant age-dependent difference for the latter, but not former, was documented that correlated with the higher expression of the two isoforms and greater activity of the sGC in the newborn rat bronchial tissue.
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METHODOLOGY |
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Functional studies. The airway rings (average diameter 70–100 µm, and length = 2 mm) were mounted on a wire myograph (Danish Myo Technology). Isometric changes were digitized and recorded online (Myodaq, Danish Myo Technology). Tissues were bathed in Krebs-Henseleit buffer (115 mM NaCl, 25 mM NaHCO3, 1.38 mM NaHPO4, 2.51 mM KCl, 2.46 mM MgSO4·7H2O, 1.91 mM CaCl2, and 5.56 mM dextrose), bubbled with air/6% CO2 and maintained at 37°C.
After 1 h of equilibration, the optimal resting tension of the tissue was determined by repeated stimulation with 128 mM KCl until maximum active tension was reached. All subsequent force measurements were obtained at optimal resting tension. Contractile responses were normalized to the tissue cross sectional area as (width x diameter) x 2, expressed as milli-Newtons per square millimeter, as previously described (3).
To study the relaxation response, the airways were precontracted with acetylcholine (EC75 for newborn and adult tissue = 10–5 M). This resulted in a stable contraction for up to 45 min. The relaxant agonist response was expressed as a percentage change from the initial precontracted force and a second order polynomial curve line fitted around the concentration-specific data. The NO-dependent relaxation was evaluated with the donor sodium nitroprusside. The sGC stimulation was induced with the BAY 41-2272 and YC-1 compounds. To test the cAMP-mediated relaxation, we used forskolin.
All drugs were dissolved in Krebs-Henseleit solution, except for the YC-1 in DMSO. All drugs were purchased from Sigma (Sigma, Oakvile, Ontario, Canada), except for YC-1 (Cayman Chemical).
Preparation of bronchi homogenates. The frozen bronchi were minced in liquid nitrogen and homogenized using a Teflon pestle of a Potter-Elvehjem homogenizer with 10 strokes in 4% wt/vol of tissue extraction buffer (50 mM Tris·HCl, pH 7.5, 500 mM NaCl, 10 mM MgCl2, 5 mM DTT). EDTA-free complete protease inhibitor tablets (Roche) were added to the tissue extraction buffer to prevent proteolysis. The homogenate was incubated for 1 h stirring on ice and centrifuged at 17,000 g for 10 min at 4°C. The protein concentrations were determined by the method of Bradford using bovine serum albumin as standard.
Western blot analysis.
Laemmli sample buffer was added to 15 µg of protein samples, and the proteins were resolved on 10% SDS polyacrylamide electrophoresis gels. After the transfer of proteins to nitrocellulose membranes, the membrane was reversibly stained with Ponceau S, and the nonspecific protein-binding sites were blocked for 1 h in TBST buffer (10 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk. The following antibodies (Sigma, Oakvile, Ontario, Canada) against the housekeeping protein actin, 1, and 1 sGC subunits were used for detection: actin (1:500), 1 (1:5,000), and 1 (1:4,000); and these were incubated for 1.5 h in TBST buffer at room temperature. The membranes were washed three times for 10 min with TBST and subsequently incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000; Cell Signaling). After three washes with TBST buffer the membranes were processed with the enhanced chemiluminescence Western blotting detection system according to the manufacturer's recommendations (Perkin Elmer).
Guanylate cyclase activity assay.
Guanylate cyclase activity was measured as described by Koglin et al. (16). The activity of the obtained protein samples (10–100 µg/assay tube) was determined by incubation for 10 min at 37°C in the presence of 50 mM triethanolamine-HCl buffer, pH 7.4, containing 3 mM dithiothreitol, 1 mM 3-isobutyl-1-methylxanthine, 1 mM cGMP, 5 mM creatine phosphate, 10 units per tube creatine phosphokinase, 0.5 mM [-32P]GTP (
1 mCi), and 3 mM MgCl2 with or without 100 µM 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO) in a total volume of 0.1 ml. The incubation was stopped by the addition of 500 µl of NaHCO3 (120 mM) and 500 ml zinc acetate (125 mM). The radiolabeled cGMP was isolated according to the method of Jakobs et al. (13) using aluminum oxide columns.
Statistical analysis. Data of the functional studies were evaluated by one- or two-way analysis of variance (ANOVA) and the Tukey-Kramer test was used for multiple comparisons. The guanylate cyclase activity data were analyzed by the Student's unpaired t-test. All data are reported as means ± SE, and P < 0.05 was considered statistically significant.
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RESULTS |
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To evaluate the maturational changes in the cyclic nucleotide-dependent bronchial relaxation, we first tested the cAMP pathway. Forskolin, a cAMP-mediated agonist, induced a similar bronchial muscle relaxation of newborn, compared with the adult bronchial smooth muscle (Fig. 1).
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1- and 1-subunits of sGC in bronchi was significantly (P < 0.01) reduced in the adult tissue (Fig. 4). The newborn and adult bronchial tissue basal sGC activity was similar (Fig. 5). Yet, in keeping with the maturational differences in NO-mediated relaxation, the bronchial tissue sGC activity under NO-stimulated condition was significantly (P < 0.03) reduced in the adult when compared with the newborn values (Fig. 5).
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DISCUSSION |
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To offset the tendency for bronchoconstriction and to minimize the airway resistance under physiological conditions, the bronchial smooth muscle is maintained in a relaxed state via distinct mechanisms. The most important are the cGMP- and cAMP-mediated responses and K+ channel openers, although the latter play only a minor role in humans (1).
Limited data are available on the maturational changes in bronchial smooth muscle relaxation. Chitano et al. (6) have recently shown that airway smooth muscle relaxation potential increases with age, and these maturational-dependent changes in airway smooth muscle relaxation may have important implications in the pathogenesis of asthma (7).
Salbutamol, a 2-agonist that relaxes airway smooth muscle via cAMP stimulation, has been shown to have a greater response in the newborn, compared with the adult rat trachea (10). Maturational differences in cAMP-mediated bronchial muscle relaxation do not account for the age-dependent salbutamol effect since the forskolin-induced response was similar for the newborn and adult rat. This apparent discrepancy between the salbutamol and forskolin age-related relaxation response has been shown by Fayon et al. (10) to be secondary to the greater muscarinic type 2 receptor expression in the adult airway resulting in functional antagonism between the cAMP-induced relaxation and muscle contraction.
Maturational changes in NO-mediated airway muscle relaxation have been reported by others. In the rat, Mhanna et al. (19) showed that substance P-induced, NO-mediated relaxation decreases with age. In this study, we have also documented a greater relaxation in the newborn, compared with the adult rat bronchial muscle, in response to the NO donor sodium nitroprusside. In addition, we further confirmed this age difference to be sGC-related, given the similar response pattern to the enzyme stimulants BAY 41-2272 and YC-1. In fact, YC-1 in high doses induced airway smooth muscle contraction in the adult but not newborn for reasons that are not presently clear. The YC-1 site of action on sGC is not known (17), and possibly the contraction responses are unrelated to their effect on this enzyme.
Based on our previous observation of greater sGC protein expression in the newborn, compared with the adult lung (2), we speculated and further demonstrated in this study developmental changes in the expression and activity of this enzyme in the rat. sGC is a key enzyme responsible for the NO-triggered cGMP-dependent smooth muscle relaxation. This heterodimeric heme-containing enzyme consists of four subunits named 1, 2, 1, and 2. The 1- and 1-subunits are present in the lung (9, 14, 15), whereas the 2- and 2-subunits are expressed in other organs (5). In the Sprague-Dawley (4) and Wistar (2) rat, lung tissue sGC activity is highest in neonates and decreases with age.
The lung is composed of vascular, airway, and parenchymal compartments, and each populated by different cells expressing sGC. Therefore, in the present study, we measured sGC expression and activity in the same generation airway tissue used for the functional studies. We documented that the sGC 1- and 1-subunits protein expression and enzyme activity are markedly reduced in the adult rat bronchi, accounting for the lesser cGMP-dependent relaxation later in life.
Although the present data was obtained from rat airways, the clinical relevance of NO-mediated airway smooth muscle relaxation needs to be revisited with attention to the developmental changes in sGC expression. Most of the published studies comparing the airway muscle relaxation potential of different agonists were carried out in animal tissue. Based on these mostly adult tissue data, NO is considered to have a potency intermediate between the -adrenergic agonist and the phosphodiesterase inhibitor theophylline (12). In contrast, the data from this study indicate that NO, the -adrenergic agonist salbutamol, and the adenylate cyclase activator forskolin have similar bronchial smooth muscle relaxation potential in the newborn rat.
In addition, it has been recently reported that asthma-like disease in adult mice is associated with reduction in sGC activity, thus further limiting the potential therapeutic benefit of cGMP-mediated bronchodilation in adults (20). Taken together and to the extent that extrapolations can be made to humans, the evidence from ours and other studies suggest that cAMP-mediated bronchodilation should be preferentially sought in adults, whereas for the newborn airway, muscle relaxation is equally effective when either cyclic nucleotide is stimulated.
In summary, we documented a significant reduction in the 1- and 1-subunit expression and NO-stimulated sGC activity in adult rat bronchial tissue. This finding was associated with a significantly reduced cGMP-mediated relaxation in the adult rat airway muscle. Further comparative maturational studies of the adenylate and guanyl cyclase airway expression and activity are warranted to better address the pharmacological selection of bronchodilators to treat airway reactivity in humans.
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FOOTNOTES |
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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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REFERENCES |
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2-adrenoceptor agonists is related to attenuated expression of postjunctional smooth muscle muscarinic M2 receptors. J Appl Physiol 98: 1526–1533, 2005.This article has been cited by other articles:
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  W. J. Pearce, J. M. Williams, C. R. White, and T. M. Lincoln Effects of chronic hypoxia on soluble guanylate cyclase activity in fetal and adult ovine cerebral arteries J Appl Physiol, July 1, 2009; 107(1): 192 - 199. [Abstract] [Full Text] [PDF]
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 R. B. Sopi, M. A. Haxhiu, R. J. Martin, I. A. Dreshaj, S. Kamath, and S. I. A. Zaidi Disruption of NO-cGMP signaling by neonatal hyperoxia impairs relaxation of lung parenchyma Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L1029 - L1036. [Abstract] [Full Text] [PDF]
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 C. E. Teixeira, F. B. M. Priviero, and R. C. Webb Effects of 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) on Smooth Muscle Tone, Soluble Guanylyl Cyclase Activity, and NADPH Oxidase Activity/Expression in Corpus Cavernosum from Wild-Type, Neuronal, and Endothelial Nitric-Oxide Synthase Null Mice J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1093 - 1102. [Abstract] [Full Text] [PDF]
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