INTRODUCTION

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Angiotensin II is a potent vasoactive hormone that plays an important role in regulation of vasomotor tone and sodium and water homeostasis. Angiotensin II is formed from angiotensin I by the angiotensin-converting enzyme (ACE), which is heavily expressed in the lungs (1). It has been reported that activation of the renin-angiotensin system with elevation of plasma renin and angiotensin II levels is observed in asthmatic patients during acute severe attacks (2, 3). Angiotensin II causes bronchoconstriction in mildly asthmatic patients (4), and angiotensin II in subthreshold concentrations increases bronchial responsiveness to methacholine both in human bronchi in vitro and in mildly asthmatic patients in vivo (4). However, in contrast to its potentiation of the effect on methacholine-induced bronchoconstriction, angiotensin II has no effect on histamine-evoked bronchoconstriction in human bronchi in vitro or in vivo (5). Some animal studies have demonstrated that type 1 angiotensin II (AT1) receptors are involved in angiotensin II-induced bronchoconstriction in guinea pigs (6, 7), peptide leukotriene (LT) production in guinea pig airways (6), potentiating effect of angiotensin II on endothelin-1-induced contraction of bovine bronchial smooth muscle (8), and Cl secretion by canine tracheal epithelium (9). These observations suggest that angiotensin II could be a putative mediator in asthma. Indeed, both Northern (10) and Western (11) blot analyses have shown AT1 receptor expression in human lung tissues. In this study, we examined the effect of losartan on bronchial hyperresponsiveness of peripheral and central airways in patients with asthma, using FEV₁ and standardized partial expiratory flow at 40% of FVC (PEF₄₀) as sensitive indices of small airway bronchoconstriction.

	METHODS

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Patients

Eight asthmatic patients (7 male, 1 female, age 27 to 68 yr) with baseline FEV₁ values of 55 to 104% (80.9 ± 7.3%) of predicted values were studied (Table 1). None of the patients had ever smoked or experienced any occupational exposure, and each patient satisfied the American Thoracic Society (ATS) definition of asthma, with symptoms of episodic wheezing, cough, and shortness of breath responding to bronchodilators and reversible airflow obstruction (more than 15% reversibility in terms of FEV₁) documented on at least one pulmonary function study (12). None of the patients had a history of excessive mucus expectoration, and thin-slice chest computed tomography (CT) showed no low-attenuation area in any of the patients. None of the patients had taken theophylline, antihistamines, sodium cromoglycate, or oral corticosteroids for at least 2 mo before the study, and none experienced an upper respiratory tract infection in the preceding month or during the study. Permitted medication, which remained unchanged during the study, included inhaled ₂-agonists and inhaled corticosteroids. This study was carried out while the patients' symptoms were mild and stable. Informed consent was obtained from all patients. This study was approved by the ethics committee of our university hospital.

Study Protocol

The study was performed in a randomized, double-blind, placebo-controlled, two-period crossover fashion. The methacholine concentrations producing a 20% fall in FEV₁ (PC₂₀-FEV₁) and a 35% fall in standardized partial expiratory flow at 40% of FVC (PC₃₅-PEF₄₀) were measured on two occasions 2 wk apart. Losartan was given orally in a dose of 50 mg once a day, 30 min after breakfast, for 6 d and at 8:00 A.M. on the seventh day (test day). At the time of crossover from the first to the second treatment regimen, administration of the test drug was suspended for 1 wk. All medication except for pretreatment with losartan or placebo was stopped at 1:00 P.M. on the previous day to allow a washout time of at least 24 h. The bronchial responsiveness to inhaled methacholine was then determined at 1:00 P.M. after blood pressure was measured. Two weeks before the study (entry day), plasma angiotensin II concentration, baseline pulmonary function, and baseline PC₂₀-FEV₁ and PC₃₅-PEF₄₀ values were measured. The purpose of the challenges on entry day was to determine the repeatability of the parameters, such as FEV₁, PEF₄₀, PC₂₀-FEV₁, and PC₃₅-PEF₄₀.

Bronchial Responsiveness to Inhaled Methacholine

Methacholine was dissolved in physiological saline solution to produce concentrations of 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 mg/ml. Saline and each solution were inhaled from a DeVilbiss 646 nebulizer (DeVilbiss Co., Somerset, PA) operated by compressed air at 5 L/min. The nebulizer output was 0.14 ml/min. Saline was inhaled first for 2 min and partial and full flow-volume curves were measured on a dry wedge spirometer (Transfer test; P.K. Morgan Ltd., Chatham, Kent, UK). If the change in FEV₁ from the baseline value was 10% or less, inhalation of methacholine was started, and if the saline solution caused a change in FEV₁ > 10%, the test was stopped or postponed. Methacholine was inhaled for 2 min by tidal mouth breathing with the patients wearing a nose clip, and this was followed immediately by three measurements of partial and full flow-volume curves at 1-min intervals; the curve with the largest FVC was retained for analysis. Increasing concentrations of methacholine were inhaled until a decrease of 20% or more in FEV₁ occurred.

Partial expiratory flow-volume (PEFV) curves and full flow-volume curves (maximal expiratory flow-volume [MEFV] curves) were measured by the method of Fish and coworkers (13), using a dry wedge spirometer as mentioned previously, and recorded on an X-Y recorder (X-Y Recorder WX 441; Watanabe Co. Ltd., Tokyo, Japan). Flow-volume maneuvers were performed in the following manner: after a period of normal tidal breathing, patients momentarily held their breath at slightly above the end-tidal inspiration (approximately 60% of FVC), and then forcibly expired to residual volume (RV). After reaching RV, patients inspired to TLC as rapidly as possible and then immediately performed a full forced expiration. The expiratory flow at 40% (above RV) from the full FVC (MEF₄₀) was determined and used as a reference volume to compare the isovolume flow from the partial curve (PEF₄₀). The volume at 40% of the full curve was used as the reference volume for flow measurements on both curves because of discrepancies in RV obtained with the two maneuvers. To accurately define isovolume points of the partial and full curves, all intervening volume changes were continuously recorded during rebreathing from the spirometer. Small changes in volume caused by changes in respired gas composition were ignored.

Measurement of Plasma Angiotensin II

Plasma angiotensin II concentration was measured by radioimmunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The intra-assay coefficient of variation was 4.0% and interassay variation was 5.1%. The normal range was 9 to 47 pg/ml.

Data Analysis

PC₂₀-FEV₁ and PC₃₅-PEF₄₀ were determined by linear interpolation from the log dose-response curve. PC₂₀-FEV₁, PC₃₅-PEF₄₀, and plasma angiotensin II concentration values were logarithmically transformed for analysis and reported as the geometric mean (geometric standard error of the mean [GSEM]). All measurements except for PC₂₀-FEV₁, PC₃₅-PEF₄₀, and plasma angiotensin II concentration were expressed as mean ± SEM. The degree of change in methacholine responsiveness for FEV₁ (PC₂₀-FEV₁) and PEF₄₀ (PC₃₅-PEF₄₀) was calculated as the logarithmic value of each provocative concentration after losartan administration minus the corresponding logarithmic value of each provocative concentration after placebo. Change in baseline PEF₄₀ (PEF₄₀) and blood pressure between losartan day and placebo day was calculated as (baseline values after losartan minus baseline values after placebo)/baseline values after placebo. Statistical differences between two-period crossover examinations were determined by Student's paired t test. Correlations were obtained using Pearson's correlation coefficient. Repeatability between entry day (2 wk before the study) and placebo day was evaluated using the statistical method of Bland and Altman (14). A value of p < 0.05 was accepted as statistically significant.

	RESULTS

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There were no significant differences between treatment periods in FEV₁ (placebo: 1.88 ± 0.31 L, losartan: 1.80 ± 0.29 L, p = 0.428) or PEF₄₀ (placebo: 0.86 ± 0.22 L/s, losartan: 0.89 ± 0.21 L/s, p = 0.671) values at baseline. Although the PC₂₀-FEV₁ values after placebo (2.037 [GSEM, 0.210] mg/ml) and losartan (2.098 [GSEM, 0.239] mg/ml) were identical (p = 0.840) (Figure 1), the geometric mean PC₃₅-PEF₄₀ values significantly (p = 0.034) increased from 0.258 (GSEM, 0.156) mg/ ml with placebo to 0.456 (GSEM, 0.186) mg/ml with losartan. There was no correlation between losartan and placebo days in individual changes in PC₃₅PEF₄₀ (PC₃₅-PEF₄₀) and baseline PEF₄₀ (PEF₄₀) (r = 0.182, p = 0.874). Repeatability of the two methacholine inhalations between entry day (2 wk before the study) and placebo day was assessed using the method described by Bland and Altman (14) to establish a repeatability coefficient, by comparing the standard deviations of the differences in PC₂₀-FEV₁ and PC₃₅-PEF₄₀ for each asthmatic patient for the two methacholine inhalations. This gave a repeatability coefficient (2 × SD of the differences) of 0.620 log mg/ml and 0.570 log mg/ml for PC₂₀-FEV₁ and PC₃₅-PEF₄₀, respectively, with all values lying within this range, suggesting good repeatability for both PC₂₀-FEV₁ and PC₃₅-PEF₄₀ (Figure 2). No significant correlation was observed between the plasma angiotensin II concentration and the change in methacholine responsiveness (PC₂₀-FEV₁ [r = 0.244, p = 0.782] and PC₃₅-PEF₄₀ [r = 0.312, p = 0.664]) (Figure 3). Losartan lowered the systolic (placebo: 139.5 ± 6.9 mm Hg, losartan: 126.9 ± 8.3 mm Hg, p = 0.007) and diastolic (placebo: 88.0 ± 3.1 mm Hg, losartan: 80.1 ± 4.7 mm Hg, p = 0.027) blood pressure without any effects on the heart rate (placebo: 87.4 ± 4.8/min, losartan: 81.3 ± 4.1/min, p = 0.160). There was no correlation between the change in blood pressure and the change in methacholine responsiveness (PC₂₀-FEV₁ [systole: r = 0.037, p = 0.994; diastole: r = 0.499, p = 0.318] and PC₃₅-PEF₄₀ [systole: r = 0.117, p = 0.946; diastole: r = 0.327, p = 0.637]) (Figure 4). No patient complained of adverse effects.

View larger version (15K): [in this window] [in a new window]   Figure 1.   The effect of losartan on bronchial hyperresponsiveness to methacholine in asthmatic patients.  : geometric mean; *p = 0.034.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Repeatability of measurement of PC20-FEV1 and PC35-PEF40. Averages (x-axis) of and differences (y-axis) in log values for PC20-FEV1 and PC35-PEF40 on two separate days (entry day and placebo day) are plotted.

View larger version (12K): [in this window] [in a new window]   Figure 3.   Relationship between plasma angiotensin II concentration and change in PC20-FEV1 (PC20-FEV1) and PC35-PEF40 (PC35-PEF40). No significant correlation was observed between them.

View larger version (11K): [in this window] [in a new window]   Figure 4.   Relationship between change in blood pressure and PC35-PEF40 (PC35-PEF40). No significant correlation was observed between them.

	DISCUSSION

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In this study, we investigated the effect of losartan on bronchial hyperresponsiveness to methacholine in eight patients with stable mild asthma. The results showed that bronchial hyperresponsiveness to methacholine, in terms of PC₃₅-PEF₄₀, but not PC₂₀-FEV₁, was attenuated by losartan. The effect of losartan on PC₃₅-PEF₄₀ appears to be specific, because the drug itself did not affect baseline PEF₄₀ values as shown in this study. Therefore, the effect of losartan on PC₃₅-PEF₄₀ cannot be attributed to bronchodilatory effects of the compound.

It has been reported that there are two major receptor binding sites for angiotensin II that can be defined pharmacologically by losartan and PD123177 (15). A standard nomenclature for angiotensin receptor subtypes has been proposed and updated as AT1 and AT2 (16). The prototypical antagonist of the AT1 receptors is losartan and that of the AT2 receptors is PD123177 or CGP42112A. Losartan, as well as its active metabolite EXP3174, is over 10,000-fold more selective for the AT1 receptors than for the AT2 receptors, whereas PD123177 and CGP42114A are 3,000- to 4,000-fold more selective for the AT2 sites (17). All of the known physiological functions of angiotensin II, including vasoconstriction (18), aldosterone release (19), enhanced noradrenaline release (20), feedback control of renin release (21), and drinking behavior (19), are mediated by the AT1 receptor subtype.

Kanazawa and coworkers reported that inhaled angiotensin II caused bronchoconstriction via AT1 receptors, but not via AT2 receptors, in guinea pigs (7). Nally and coworkers demonstrated that angiotensin II potentiated contraction evoked by endothelin-1 via AT1 receptors in bovine bronchi (8). Although angiotensin II increases bronchial responsiveness to methacholine both in human bronchi in vitro and in mildly asthmatic patients in vivo (4), Dicpinigaitis and Dobkin (22) reported that losartan had no effect on PC₂₀-FEV₁ in stable mildly asthmatic patients. However, their study (22) had the following weak points: (1) cardiovascular effects, which are useful parameters for confirming the dose of losartan administered, were not assessed; and (2) lung function was evaluated only by FEV₁. In the present study, losartan was administered at doses sufficient to decrease blood pressure and the attenuating effect of losartan on methacholine responsiveness was detected only using PEFV curves. The deep inspiration to the level of TLC that is needed to perform a MEFV maneuver is well known to modify measurements of resting bronchomotor tone in normal and asthmatic subjects and to diminish the effects of bronchoconstrictors (23, 24). In addition, PEF₄₀ is predominantly influenced by changes in smaller airways compared with FEV₁. These observations suggest that an AT1 receptor-mediated mechanism plays an important role in bronchial hyperresponsiveness in peripheral asthmatic airways rather than central airways. Although the reason for this discrepancy is unclear, it is possible that the location of AT1 receptors or angiotensin II activity may be heterogeneous at different sites in the airways. However, it is unclear whether the location of AT1 receptors differs between central and peripheral airways.

It has been recognized that bronchodilator and bronchoprotective activities of antiasthmatic drugs are different in the case of the intensity and duration of action. In addition, angiotensin II in the subthreshold concentrations increases bronchial responsiveness to methacholine both in human bronchi in vitro and in mildly asthmatic patients in vivo (4). Taking together with the present results, it is speculated that subthreshold doses of angiotensin II, which do not cause bronchoconstriction per se, may increase bronchial responsiveness of asthma via AT1 receptors, especially in peripheral airways. However, its mechanisms are still unclear.

Pretreatment with losartan influenced blood pressure. This could potentially have an effect on airway responsiveness by a number of reflex mechanism such as a rise in circulating adrenaline resulting from the losartan-induced decrease in blood pressure. There was no correlation between the change in blood pressure and the change in PC₃₅-PEF₄₀, making it unlikely that the attenuating effect of losartan on PC₃₅-PEF₄₀ is indirect owing to a number of reflex mechanisms such as a rise in circulating adrenaline resulting from the losartan-induced decrease in blood pressure. However, it cannot be concluded that the effect observed is specific for the AT1 receptor antagonist. A control day using another antihypertensive compound needs to be included to prove the specificity to losartan.

In the present study, there was no correlation between plasma angiotensin level and the change in methacholine responsiveness. Plasma levels of angiotensin II are reported to be elevated in acute severe asthma (2), which suggests the need for further research on the role of an AT1 receptor-mediated mechanism in asthmatic patients during acute severe attacks.

At present, investigating the involvement of AT2 receptors in bronchial hyperresponsiveness in asthmatic patients is problematic, because there is no useful and safe method for determining the role of AT2 receptors in humans in vivo.

In summary, this study demonstrated that AT1 receptors are involved in bronchial hyperresponsiveness to methacholine in peripheral asthmatic airways. This is the first report demonstrating the involvement of AT1 receptors in bronchial asthma.