The Swedish CArdioPulmonary BioImage Study: objectives and design.

Cardiopulmonary diseases are major causes of death worldwide, but currently recommended strategies for diagnosis and prevention may be outdated because of recent changes in risk factor patterns. The Swedish CArdioPulmonarybioImage Study (SCAPIS) combines the use of new imaging technologies, advances in large-scale 'omics' and epidemiological analyses to extensively characterize a Swedish cohort of 30 000 men and women aged between 50 and 64 years. The information obtained will be used to improve risk prediction of cardiopulmonary diseases and optimize the ability to study disease mechanisms. A comprehensive pilot study in 1111 individuals, which was completed in 2012, demonstrated the feasibility and financial and ethical consequences of SCAPIS. Recruitment to the national, multicentre study has recently started.

Fibroblast growth factor 23 is an independent marker of COPD and is associated with impairment of pulmonary function and diffusing capacity.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone that in recent years has been reported to have significant effects on numerous tissues. Chronic obstructive pulmonary disease (COPD) is associated with hypophosphatemia but the evidence for elevated plasma levels of FGF23 in COPD subjects is ambiguous. Recently, FGF23 has even been shown to be involved in the inflammatory pathways activated in COPD, so FGF23 could be a novel biomarker for COPD and impairment of pulmonary function. The purpose was thus to explore the association of FGF23 with COPD and measures of pulmonary function. This was a cross sectional study of 450 subjects who underwent spirometry, body plethysmography, determination of diffusing capacity (DL,CO) and biomarker analysis of FGF23, interleukin (IL)-1 receptor antagonist, IL-6 and IL-8. Forty-four participants were excluded due to missing data or renal impairment (eGFR <45 mL/min/m2). Spirometry identified 123 subjects with COPD. FGF23 levels were elevated in COPD subjects compared to non COPD subjects, and this remained significant after adjustment for age, sex and smoking habits (OR = 1.6, p = 0.02). Linear regression showed significant relationships between FGF23 and FEV1 (ß = -0.15, p = 0.003), RV/TLC (ß = 0.09, p = 0.05) and DL, CO (ß = -0.24, p < 0.001). In conclusion we found that plasma levels of FGF23 are elevated in COPD subjects even when adjusting for traditional risk factors. Furthermore, FGF23 is associated with impairment in lung function as measured by FEV1 and DL,CO. Further studies are needed to establish whether FGF23 could serve as a novel biomarker of COPD and emphysema development.

Novel site-specific mast cell subpopulations in the human lung.

BACKGROUND: Lung mast cells are stereotypically divided into connective tissue (MC(TC)) and mucosal (MC(T)) mast cells. This study tests the hypothesis that each of these subtypes can be divided further into site-specific populations created by the microenvironment within each anatomical lung compartment. METHODS: Surgical resections and bronchial and transbronchial biopsies from non-smoking individuals were obtained to study mast cells under non-inflamed conditions. Morphometric and molecular characteristics of mast cell populations were investigated in multiple lung structures by immunohistochemistry and electron microscopy. RESULTS: MC(T) and MC(TC) coexisted in all compartments, with MC(T) being the prevailing type in bronchi, bronchioles and the alveolar parenchyma and MC(TC) being more abundant in pulmonary vessels and the pleura. Each of the MC(TC) and MC(T) phenotypes could be further differentiated into site-specific populations. MC(TC) were significantly larger in pulmonary vessels than in small airway walls, while the reverse was observed for MC(T). Within each MC(TC) and MC(T) population there were also distinct site-specific expression patterns of the IgE receptor, interleukin-9 receptor, renin, histidine decarboxylase, vascular endothelial growth factor, fibroblast growth factor, 5-lipoxygenase and leukotriene C4 synthase (eg, bronchial MC(T) consistently expressed more histidine decarboxylase than alveolar MC(T)). Renin content was high in vascular MC(TC) but markedly lower in MC(TC) in other compartments. For both MC(TC) and MC(T), the IgE receptor was highly expressed in conducting airways but virtually absent in alveolar parenchyma. CONCLUSIONS: These findings demonstrate novel site-specific subpopulations of lung MC(TC) and MC(T) at baseline conditions. This observation may have important implications in the future exploration of mast cells in a number of pulmonary diseases.

Plasma markers of inflammation and incidence of hospitalisations for COPD: results from a population-based cohort study.

BACKGROUND: The relationship between plasma markers of inflammation and the incidence of chronic obstructive pulmonary disease (COPD) is still unclear. This population-based study explored whether raised levels of five inflammation-sensitive plasma proteins (ISPs) predicted hospital admissions for COPD during 25 years of follow-up. METHODS: Spirometric tests and measurements of five ISPs (fibrinogen, ceruloplasmin, alpha(1)-antitrypsin, haptoglobin, orosomucoid) were performed in 5247 apparently healthy men from the city of Malmö (mean age 46 years). The incidence of hospitalisations for COPD was studied in relation to the number of ISPs in the fourth quartile. RESULTS: During the follow-up period, 258 men were admitted to hospital with COPD, 211 of whom were smokers at baseline. The incidence of hospital admissions for COPD was significantly associated with the number of raised ISPs. Adjusted for risk factors, the hazards ratio (95% CI) was 1.00 (reference), 1.28 (0.9 to 1.9), 1.29 (0.8 to 2.0) and 2.30 (1.6 to 3.2), respectively, for men with 0, 1, 2 and >or=3 ISPs in the top quartile (p for trend <0.001). This relationship was consistent in men with high and low lung function at baseline. The relationship with the incidence of hospital admissions for COPD was largely the same for all individual ISPs. CONCLUSION: Raised plasma ISP levels are associated with an increased incidence of COPD requiring hospitalisation.

Matrix Metalloproteinases in COPD and atherosclerosis with emphasis on the effects of smoking.

BACKGROUND: Matrix metalloproteinases (MMP´s) are known biomarkers of atherosclerosis. MMP´s are also involved in the pathophysiological processes underlying chronic obstructive pulmonary disease (COPD). Cigarette smoking plays an important role in both disease states and is also known to affect the concentration and activity of MMP´s systemically. Unfortunately, the epidemiological data concerning the value of MMP´s as biomarkers of COPD and atherosclerosis with special regards to smoking habits are limited. METHODS: 450 middle-aged subjects with records of smoking habits and tobacco consumption were examined with comprehensive spirometry, carotid ultrasound examination and biomarker analysis of MMP-1, -3, -7, -10 and -12. Due to missing data 33 subjects were excluded. RESULTS: The remaining 417 participants were divided into 4 different groups. Group I (n = 157, no plaque and no COPD), group II (n = 136, plaque but no COPD), group III (n = 43, COPD but no plaque) and group IV (n = 81, plaque and COPD). Serum levels of MMP-1,-7,-10-12 were significantly influenced by smoking, and MMP-1, -3, -7 and-12 were elevated in subjects with COPD and carotid plaque. This remained statistically significant for MMP-1 and-12 after adjusting for traditional risk factors. CONCLUSION: COPD and concomitant plaque in the carotid artery were associated with elevated levels of MMP-1 and -MMP-12 even when adjusting for risk factors. Further studies are needed to elucidate if these two MMP´s could be useful as biomarkers in a clinical setting. Smoking was associated with increased serum levels of MMP´s (except for MMP-3) and should be taken into account when interpreting serum MMP results.

Obstructive airways diseases, smoking and use of inhaled corticosteroids in southern Sweden in 1992 and 2000.

OBJECTIVE: To examine the prevalence of obstructive pulmonary diseases, respiratory symptoms, smoking habits and pulmonary medication in an adult population, and to compare the results with a study performed in the same geographical area in 1992. DESIGN: In 2000, a postal questionnaire was sent to a randomly selected population of 5179 subjects aged 20-59 years living in southern Sweden. RESULTS: The participation rate was 71.3%. Self-reported asthma was reported by 8.5% of all respondents (vs. 5.5% in 1992, P < 0.001) and 14.5% of females aged 20-29 years. Self-reported chronic bronchitis and/or emphysema and/or chronic obstructive pulmonary disease (CBE/COPD) was reported by 3.6% (vs. 4.6% in 1992, non-significant) with the highest prevalence (5.7%) in the 50-59 year cohort. Smoking decreased from 33.3% in 1992 to 28.4% in 2000 (P < 0.05). About 46% of asthmatics reported nocturnal respiratory symptoms, and 69% reported having had asthma symptoms in the last 12 months. Use of inhaled steroids increased in subjects with asthma and CBE/COPD from 19.4% to 36.5% (P < 0.05) and from 8.6% to 30.0% (P < 0.05), respectively. CONCLUSIONS: Self-reported asthma increased significantly between 1992 and 2000, but the prevalence of CBE/COPD was unchanged. The high proportion of reported symptoms in asthmatics despite an increased use of steroids suggests that further efforts are needed to improve asthma treatment.

Predictors of lung function and its decline in mild to moderate COPD in association with gender: results from the Euroscop study.

BACKGROUND: There is increasing appreciation of gender differences in COPD but scant data whether risk factors for low lung function differ in men and women. We analysed data from 3 years follow-up in 178 women and 464 men with COPD, participants in the Euroscop Study who were smokers unexposed to inhaled corticosteroids. METHODS: Explanatory variables of gender, age, starting age and pack-years smoking, respiratory symptoms, FEV(1)%FVC and FEV(1)%IVC (clinically important measures of airway obstruction), body mass index (BMI), and change in smoking were included in multiple linear regression models with baseline and change in post-bronchodilator FEV(1) as dependent variables. RESULTS: Reduced baseline FEV(1) was associated with respiratory symptoms in men only. Annual decline in FEV(1) was not associated with respiratory symptoms in either men or women, and was 55 ml less in obese men (BMI 30 kg/m(2)) than men having normal BMI, an effect not seen in women. It was 32 ml faster in women with FEV(1)%FVC

Bronchial mucosal mast cells in asymptomatic smokers relation to structure, lung function and emphysema.

The pathologic mechanisms of chronic obstructive pulmonary disease (COPD) most certainly involves neutrophil granulocytes, cytotoxic T-cells, macophages and mast cells. The aim of this study was to investigate the relation between the number of mast cells in different compartments in bronchial biopsies of central proximal airways to structural changes, lung function tests and emphysema detected by high resolution computed tomography (HRCT). Twenty nine asymptomatic smoking and 16 never-smoking men from a population study were recruited. Central bronchial biopsies were stained to identify mast cells by immunohistochemistry. The number of mast cells in the epithelium, lamina propria and smooth muscle as well as epithelial integrity and thickness of the tenascin and laminin layer were determined. Smokers had increased numbers of mast cells in all compartments (P<0.001). Structural changes were correlated to mast cell numbers with the closest associations to mast cell numbers in the smooth muscle [epithelial integrity (R(S)=-0.48, P=0.008), laminin layer (R(S)=0.63, P=0.0002), tenascin layer (R(S)=0.40, P=0.03)]. Similar correlations between mast cells and lung function tests were seen [functional residual capacity (FRC) (R(S)=0.60, P=0.0006), total lung capacity (TLC) (R(S)=0.44, P=0.02) and residual volume (RV) (R(S)=0.41, P=0.03)]. No correlations could be detected between mast cells and FEV1 or to emphysema. Smoking is associated with an increase of mast cells in all compartments of the bronchial mucosa, including smooth muscle, and this is related to altered airway structure and function.

Pafenolol, a highly selective beta 1-adrenoceptor-antagonist, in asthmatic patients: interaction with terbutaline.

Pafenolol, a new beta 1-adrenoceptor antagonist, has been shown in animals to be more selective for beta 1-adrenoceptors than metoprolol. It was studied in asthmatic patients to evaluate whether it was more selective with respect to circulatory effects and especially whether this selectivity influenced bronchial muscle tone less than metoprolol, which has been shown to have beta 1-adrenoceptor selectivity similar to that of atenolol. Intravenous pafenolol (5 mg and 7.5 mg), metoprolol (15 mg), and saline were given double-blind at random and thereafter four increasing doses of terbutaline were given intravenously to seven asthmatic patients with reproducible reversibility of airways obstruction. After the terbutaline dose-response curve was determined, terbutaline was inhaled three times in increasing doses. A separately reported exercise study in the same patients showed that 5 mg pafenolol and 15 mg metoprolol were equipotent with respect to beta 1-adrenoceptor blockade, whereas 7.5 mg pafenolol tended to increase the blockade. The reflex tachycardia on terbutaline stimulation after pafenolol was greater than after metoprolol due to less blockade of beta 2-adrenoceptors in peripheral blood vessels. The bronchial effect of pafenolol was equal to that of saline, but there was a difference between the terbutaline dose-response curves after pafenolol and metoprolol that caused a rightward shift of the dose-response curve. Thus, pafenolol was shown to be more beta 1-selective than metoprolol.

IL-2 and IL-4 counteract budesonide inhibition of GM-CSF and IL-10, but not of IL-8, IL-12 or TNF-alpha production by human mononuclear blood cells.

1. The combination of interleukin-2 (IL-2) and IL-4 reduces the inhibitory effects of glucocorticoids on granulocyte-macrophage colony-stimulating factor (GM-CSF) production, in agreement with the hypothesis that this combination causes glucocorticoid resistance. Whether a general cytokine resistance to glucocorticoids is induced by IL-2 and IL-4 has not been reported. 2. Mononuclear blood cells from healthy individuals were pre-treated with IL-2, IL-4, or IL-2+ IL-4 (31.3-500 U ml(-1)) for 48 h, prior to lipopolysaccharide (LPS; 10 ng ml(-1); 20 h) and budesonide addition. Cytokine levels in the supernatants were analysed using specific immunoassays. DNA content was analysed to estimate cell numbers. 3. GM-CSF production was totally inhibited by budesonide at 10(-8) M in vehicle treated cultures, while IL-10 was inhibited to 33.4+/-4.3% of control. IL-2, IL-4, or IL-2 + IL-4 reduced the inhibitory effects of budesonide on GM-CSF to similar levels (23.7 6.7, 31.6+/-8.5 and 35.1+/-4.3% of control, respectively). IL-2, IL-4, or IL-2 + IL-4 also reduced the inhibitory effects of budesonide on IL-10 production (46.5+/-6.6, 55.9+/-7.3%, and 68.3+/-9.9% of control, respectively). In contrast, IL-8, IL-12 and TNF-alpha production did not become resistant to budesonide. 4. Thus, glucocorticoid resistance induced by IL-2 and IL-4 is not general at the cytokine production level. While the glucocorticoid sensitivity of GM-CSF and IL-10 production decreased, the sensitivity of IL-8, IL-12 or TNF-alpha production was unchanged. Also, the mixture of IL-2 and IL-4 is not crucial for induction of glucocorticoid resistance of GM-CSF production.

Non-adrenergic, non-cholinergic neural activation stabilizes smooth-muscle tone independently of eicosanoid factors in guinea-pig isolated airways.

1. We examined the effect of variations in resting smooth-muscle tone induced eicosanoid inhibitors on the direction and magnitude of the non-adrenergic, non-cholinergic (NANC) nervous response in guinea-pig isolated airways. 2. NANC responses (in the presence of 1 microM atropine, 10 microM guanethidine) to electrical field stimulation (1200 mA; 0.5 ms; 240 s; 3 Hz) were studied in guinea-pig isolated airway preparations (n = 4-7) taken from the proximal trachea, the distal trachea and the main bronchus. Tissues were treated with either the cyclo-oxygenase-blocker, indomethacin (10 microM), or the leukotriene receptor antagonist, FPL 55712 (11.5 microM), to modulate the resting tone. 3. Depending on the level of resting tone prior to electrical field stimulation, NANC activation induced either a contraction or a relaxation converging towards a similar level of tone, a 'tonus equilibrium'. This tonus equilibrium displayed an increasing level towards the periphery (3 +/- 3 (s.e.mean) % for the proximal trachea, 28 +/- 3% for the distal trachea and 54 +/- 4% for the main bronchus, in % of maximum active tension). After NANC activation, the tonus returned to a level similar to the resting tone. 4. We conclude that changes in resting smooth-muscle tone not only affect the magnitude but also the direction of the NANC response. It is suggested that the NANC nervous system is a stabilizing factor in the regulation of airway smooth-muscle tone, independent of eicosanoid factors. An increasing contractile component and a decreasing relaxant component of the NANC system towards the peripheral airways is indicated.

Non-adrenergic, non-cholinergic neural activation in guinea-pig bronchi: powerful and frequency-dependent stabilizing effect on tone.

1. We examined non-adrenergic, non-cholinergic (NANC) stimulation for its stabilizing effect on bronchial smooth-muscle tone with respect to its regulatory power and the effect of variations in neural impulse frequency. 2. The guinea-pig isolated main bronchus (n = 4-12) was pretreated with indomethacin (10 microM) and incubated with atropine (1 microM) and guanethidine (10 microM). Electrical field stimulation (EFS: 1200 mA, 0.5 ms, 240 s) was applied at various levels of tone prior to EFS: first without tone, then at a moderate tone induced by histamine (0.3 microM) and, finally, at a high tone induced by histamine (6 microM). Three different stimulation frequencies (1, 3 or 10 Hz) were used in order to produce moderate to near-maximum contractile and relaxant NANC neural responses. Both the contractile and the relaxant NANC responses were tetrodotoxin-sensitive in the guinea-pig isolated main bronchus (3 Hz). 3. Without tone prior to EFS, NANC activation (1, 3 or 10 Hz) induced a pronounced contractile response. At a moderate level of tone prior to EFS, NANC activation induced a less pronounced contractile response. At the highest level of tone prior to EFS, NANC activation induced a relaxant response. All these NANC responses adjusted the tone towards a similar level and this 'stabilization level' was 56(6)% at 1 Hz, 65(3)% at 3 Hz and 56(5)% at 10 Hz, expressed as a percentage of the maximum histamine-induced (0.1 mM) tone in each airway preparation. 4. There was a difference of approximately 90% of maximum between the highest and the lowest tone level prior to NANC activation. This difference was reduced by the converging contractile and relaxantNANC responses and the magnitude of this 'convergence effect' was 40(8)% at 1 Hz, 72(4)% at 3 Hz and 90(2)% at 10 Hz.5. These findings indicate that NANC neural activation stabilizes bronchial smooth-muscle tone via a contraction when the tone is low prior to activation and via a relaxation when the tone is high prior to activation. The NANC stabilizing effect on tone appears to be powerful and its magnitude can be controlled by the neural impulse frequency. The level of tone towards which the NANC responses converge does not appear to be markedly altered by variations in the impulse frequency. Our findings are consistent with a regulatory role for NANC responses in the control of bronchial smooth-muscle tone.

The non-adrenergic, non-cholinergic response counteracts changes in guinea-pig airway tone with and without sympathetic activation.

1. We examined whether the non-adrenergic, non-cholinergic (NANC) neural response can counteract changes in smooth-muscle tone with and without simultaneous sympathetic activation in guinea-pig airways. 2. Isolated airway preparations were pretreated with indomethacin (10 microM) and incubated with either atropine (1 microM) and guanethidine (10 microM) or atropine (1 microM) alone. The response to electrical field stimulation (EFS: 1200 mA, 0.5 ms, 3 Hz for 240 s) was studied at various levels of tone prior to EFS: first without induced tone, then at a moderate tone induced by histamine (0.3 microM) and finally at a high tone induced by histamine (6 microM). 3. The response to EFS was a contraction when the tone prior to EFS was low and a relaxation when the tone prior to EFS was high. These responses converged towards a similar level of tone, in the distal trachea and in the main bronchus, with and without guanethidine. 4. The mean (s.e. mean) level of tone towards which the responses to EFS converged was lower after incubation with atropine alone compared with incubation with atropine and guanethidine, both in the distal trachea [8 (1)% compared with 30 (6)% of maximum tone] and in the main bronchus [28 (4)% compared with 57 (2)% of maximum tone]. In separate experiments, the guanethidine-induced effect on the responses to EFS was imitated by propranolol (1 microM) but not by prazosin (0.3 microM) and yohimbine (1 microM). 5. These findings indicate that the NANC neural response can counteract changes in airway smooth-muscle tone via a contraction or via a relaxation, depending on the tone prior to activation.This stabilizing effect on tone does not appear to depend upon adrenergic activation per se. The level of tone towards which the NANC responses converge can, however, be reduced by beta-adrenoceptor activation, thus suggesting an interaction which provides protection from severe airway smooth-muscle contraction.

Bradykinin-induced airflow obstruction and airway plasma exudation: effects of drugs that inhibit acetylcholine, thromboxane A2 or leukotrienes.

1. The mechanisms behind bradykinin-induced effects in the airways are considered to be largely indirect. The role of cholinergic nerves and eicosanoids, and their relationship in these mechanisms were investigated in guinea-pigs. 2. The role of cholinergic nerves was studied in animals given atropine (1 mg kg-1, i.v.), hexamethonium (2 mg kg-1, i.v.), or vagotomized. To study the role of eicosanoids, animals were pretreated with a thromboxane A2 (TxA2) receptor antagonist (ICI 192,605; 10(-6) mol kg-1, i.v.) or with a leukotriene (LT) receptor C4/D4/E4 antagonist (ICI 198,615; 10(-6) mol kg-1, i.v.). 3. After pretreatment with a drug, bradykinin (150 nmol) was instilled into the tracheal lumen. We measured both airway insufflation pressure (Pi), to assess airway narrowing, and the content of Evans blue dye in airway tissue, to assess plasma exudation. 4. Bradykinin instillation into the trachea caused an increase in Pi and extravasation of Evans blue dye. The increase in Pi was significantly attenuated by atropine or the TxA2 receptor antagonist, but not by hexamethonium, vagotomy or the LT receptor antagonist. 5. The bradykinin-induced exudation of Evans blue dye was significantly attenuated in the intrapulmonary airways by the TxA2 receptor antagonist, but not by atropine, hexamethonium, cervical vagotomy or the LT receptor antagonist. 6. A thromboxane-mimetic U-46619 (20 nmol kg-1, i.v. or 10 nmol intratracheally), caused both an increase in Pi and extravasation of Evans blue dye at all airway levels. Atropine pretreatment slightly attenuated the peak Pi after the intratracheal administration of U-46619, but not after i.v. administration. 7. We conclude that peripheral cholinergic nerves are involved in bradykinin-induced airflow obstruction but not plasma exudation, and that TxA2 is involved in both airflow obstruction and airway plasma exudation induced by bradykinin given via the airway route. TxA2-induced airflow obstruction is mediated only to a minor degree, via the release of acetylcholine in the airways.

Leukotriene D4- and prostaglandin F2 alpha-induced airflow obstruction and airway plasma exudation in guinea-pig: role of thromboxane and its receptor.

1. We studied the effects of a thromboxane A2 receptor (TP receptor) antagonist, ICI-192,605 (0.5 mg kg-1, i.v.) and a selective thromboxane (Tx) synthetase inhibitor, OKY-046 (30 mg kg-1, i.v.), on airway responses induced by leukotriene D4 (LTD4; 0.2 nmol) or prostaglandin F2 alpha (PGF2 alpha; 20 nmol) instilled via the airways route to anaesthetized guinea-pigs. For a comparison, airway responses to a TxA2-mimetic, U-46619 (0.02 nmol) were also studied. We measured both lung resistance (RL) to monitor airflow obstruction, and extravasation of Evans Blue dye to quantify airway plasma exudation. 2. Instilled LTD4 into the tracheal lumen induced an immediate peak and subsequently persistent increase in RL and produced a large amount of extravasation of Evans Blue dye at all airway levels. Both ICI-192,605 and OKY-046 significantly attenuated the persistent increase in RL following the immediate response and reduced LTD4-induced extravasation of Evans Blue dye in the trachea and proximal intrapulmonary airway. Instilled LTD4 produced significant increases in immunoreactive TxB2 in bronchoalveolar lavage fluid obtained 1.5 min after instillation of LTD4. 3. Instilled PGF2 alpha into the tracheal lumen induced an immediate increase in RL which peaked at approximately 15 s. We also observed a delayed sustained increase in RL, reaching a second peak at approximately 4 min. PGF2 alpha produced small but significant increases in the amount of Evans Blue dye at all airway levels. As with PGF2 alpha, instillation of U-46619 produced a biphasic increase in RL and extravasation of Evans Blue dye. The potency of PGF2a, in inducing these airway responses was about 1000 times less than U-46619. ICI-192,605 abolished both the immediate and the delayed increase in RL after PGF2a, and also blocked PGF2a,-induced extravasation of Evans Blue dye. However, OKY-046 had no inhibitory effects on these responses.4. We conclude that airflow obstruction and airway plasma exudation induced by instilled LTD4 is, in part, mediated via TxA2 generation and subsequent activation of TP-receptors. On the other hand,instilled PGF2a, while inducing similar responses, does so primarily by direct activation of TP receptors,rather than via TxA2 generation.

Different effects of salmeterol, formoterol and salbutamol on cholinergic responses in the ferret trachea.

1. In the present study, the inhibitory effects of the selective beta 2-adrenoceptor agonists, salmeterol, formoterol and salbutamol, have been investigated on contractions of ferret trachea induced both by endogenous and exogenous acetylcholine. The aim of the study was to evaluate quantitative and/or qualitative differences in response which may indicate both pre- and post-junctional sites of action. The non-selective beta-antagonist, sotalol, was used to estimate beta-adrenoceptor involvement. 2. Isometric tension was measured in ferret isolated tracheal strips. The inhibitory effects of the drugs were studied on tonic contractions induced by pre-junctional activation with electrical field stimulation (EFS) (2 Hz, 700 mA) or post-junctional activation with exogenous acetylcholine (ACh) (0.5 microM, about EC80), giving a similar degree of smooth muscle response. 3. Concentration-response experiments were performed with formoterol (0.3 nM-0.3 microM) and salmeterol and salbutamol (10 nM-10 microM). The experiments ended with the addition of sotalol (10 microM). 4. All three beta-agonists inhibited the contractions in a concentration-dependent manner. Salbutamol, formoterol and salmeterol inhibited the EFS-induced contractions by 66(8)%, 105(5)% and 103(8)% (mean(s.e. mean)) respectively. ACh-induced contractions were inhibited by 37(6)%, 72(11)% and 33(8)%. Theophylline (10 nM-3 mM) inhibited the contractions to the same degree. 5. beta-Adrenoceptor blockade by sotalol significantly antagonized the inhibitory effects of salbutamol and formoterol on both EFS- and ACh-induced contractions. The effect of salmeterol on ACh-induced contraction was also significantly antagonized, whereas the inhibition of EFS-induced contraction was virtually unaffected. 6. In conclusion, salbutamol, salmeterol and formoterol produced greater inhibitory effects in preparations contracted by EFS than in preparations contracted by exogenously-added ACh. In the case of formoterol and salbutamol, the effects on both levels are most probably due to beta-adrenoceptor stimulation, whereas for salmeterol the dominant pre-junctional effect is probably not mediated via beta-adrenoceptors. This non-beta-mediated effect could represent an additional relaxant mechanism for salmeterol.

Extent of salmeterol-mediated reassertion of relaxation in guinea-pig trachea pretreated with aliphatic side chain structural analogues.

1. Salmeterol is a potent, selective and long acting beta 2-adrenoceptor agonist. In vitro, salmeterol exerts 'reassertion' relaxation of airways smooth muscle. Reassertion relaxation refers to the capacity of salmeterol to cause repeated functional antagonism of induced contraction when airway smooth muscle is intermittently exposed to, then washed free from, beta-adrenoceptor antagonists such as sotalol. The mechanism(s) underlying reassertion relaxation are unknown but may relate to high affinity binding of the long aliphatic side chain of salmeterol to an accessory site, distinct from the agonist recognition site, in or near the beta 2-adrenoceptor (exosite binding hypothesis). 2. In order to test the exosite hypothesis, three pure analogues of salmeterol, each exactly preserving the molecular structure of the aliphatic side chain but with zero or low efficacy at the beta 2-adrenoceptor were synthesized. The effect of pre-incubating guinea-pig tracheal smooth muscle with these analogues on salmeterol-induced reassertion relaxation was determined. 3. Computer Assisted Molecular Modelling of these molecules revealed that each of them exactly preserved the low energy linear conformation of the aliphatic side chain of salmeterol. Measurement of lipophilicity (octanol:water partition coefficient; log P) and direct partition into synthetic membranes (membrane partition coefficient; Kpmem) showed that all compounds had high affinity for lipids and membranes. In particular the biophysical properties of CGP 59162 (log P 1.89, Kpmem 16500) were very similar to salmeterol (log P 1.73, Kpmem 16800). 4. Two of the analogues, CGP 54103 and D 2543 (1 microM), which are structural mimics of the side chain of salmeterol, differing slightly in their length, did not prevent either the initial relaxation induced by salmeterol (0.1 microM) or the reassertion relaxation; however, it was not possible to determine whether either of these molecules occupied the beta 2-adrenoceptor. 5. The third analogue, CGP 59162, which has the substituents on the active saligenin head group of salmeterol in transposed positions, itself exerted a weak beta 2-adrenoceptor-mediated relaxation antagonized by ICI 118551 (beta 2-selective antagonist) but not CGP 20712 (beta 1-selective antagonist) and, at higher concentrations CGP 59162 caused reassertion relaxation suggesting that it may occupy and activate the beta 2-adrenoceptor in a manner analogous to salmeterol. 6. CGP 59162, at concentrations up to ten fold molar excess, did not prevent or reduce salmeterol-induced reassertion relaxation. 7. In conclusion these data are not consistent with the existence of a distinct 'exosite' recognising the aliphatic side chain of salmeterol mediating reassertion.

Peptidase modulation of noncholinergic vagal bronchoconstriction and airway microvascular leakage.

We investigated whether inhibition of neutral endopeptidase 24.11 (NEP) and/or angiotensin-converting enzyme (ACE) modifies vagally induced nonadrenergic noncholinergic (NANC) airflow obstruction and airway microvascular leakage as measured by extravasation of Evans blue dye (intravenous) in anesthetized guinea pigs. We gave phosphoramidon to inhibit NEP and enalapril maleate or captopril to inhibit ACE. Animals pretreated with inhaled phosphoramidon (7.5 or 75 nmol), enalapril maleate (87 or 870 nmol), or captopril (350 nmol) reached higher peak lung resistance (RL) values (14.3 +/- 2.7, 15.7 +/- 3.8, 16.7 +/- 3.8, 11.4 +/- 1.6, and 24.6 +/- 3.5 cmH2O.ml-1.s, respectively) than saline-treated animals (5.9 +/- 1.1; P less than 0.05) after bilateral vagus nerve stimulation (5 Hz, 10 V, 10 ms, 150 s). Intravenous phosphoramidon (1 mg/kg), but not intravenous captopril (6 mg/kg), potentiated peak RL (22.9 +/- 6.9 and 7.1 +/- 1.5 cmH2O.ml-1.s, respectively). Vagal nerve stimulation (1 and 5 Hz) increased the extravasation of Evans blue dye in tracheobronchial tissues compared with sham-stimulated animals, but this was not potentiated by inhaled enzyme inhibitors or intravenous captopril. However, intravenous phosphoramidon significantly augmented the extravasation of Evans blue dye in main bronchi and intrapulmonary airways. We conclude that degradative enzymes regulate both NANC-induced airflow obstruction and airway microvascular leakage.

Effect of nedocromil sodium on non-adrenergic, non-cholinergic neural responses in guinea pig bronchi in vitro.

OBJECTIVE: Nedocromil sodium (nedocromil) improves the clinical condition of asthmatic subjects but its mechanism of action is not fully understood. This study aimed to determine whether nedocromil alters the ability of contractile and relaxant non-adrenergic, non-cholinergic neural (NANC) responses to stabilise tone by inhibiting or potentiating these responses in bronchial smooth muscle and, if so, whether the action is on a pre- or postjunctional level. RESULTS: Nedocromil attenuated contractile but not relaxant NANC responses (elicited by electric field stimulation) significantly (P < 0.05) in guinea pig main bronchi in vitro. However, the ability of NANC responses to stabilise tone (convergence effect) was not significantly impaired by nedocromil. Furthermore, nedocromil did not significantly shift the concentration response curve (-log EC50) to neurokinin A (NKA), the dominating contractile NANC transmitter, or alter the maximum response to NKA (P > 0.05). Submaximum or maximum contractile responses to histamine were not markedly affected by nedocromil (P > 0.05). CONCLUSIONS: Nedocromil exerts selective neural inhibition of the contractile but not of the relaxant NANC responses on a pre-junctional level in bronchial smooth muscle. Nedocromil does not, however, markedly impair the ability of NANC response to stabilise bronchial smooth muscle tone.