INTRODUCTION

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Angiotensin-converting enzyme (ACE) inhibitors now play a significant role in the treatment of hypertension (1), congestive heart failure (2), and, more recently, acute myocardial infarction (3). Although these agents are well tolerated and have a low incidence of serious adverse reactions, a persistent, nonproductive cough is a well-documented side effect of this drug class (4). ACE inhibitor-induced cough is thought to result from inhibition of bradykinin degradation by ACE inhibitors, with accompanying effects of bradykinin on production of prostaglandins, leukotrienes, and substance P (5). On the other hand, the effect of ACE inhibitors on bronchial hyperresponsiveness has not been clearly established. Because potent bronchoconstrictors such as bradykinin and substance P are degraded by ACE and therefore may accumulate in the lungs of patients receiving ACE inhibitors, a theoretical basis exists for the induction of bronchospasm by ACE inhibitors. In animal experiments, it has been shown that inhibition of ACE potentiates airway obstruction and increases airway plasma leakage in response to bradykinin and substance P (6). In humans, bradykinin can induce bronchoconstriction in asthmatic subjects (7), and Lunde and coworkers reported several cases in which ACE inhibitors apparently caused or worsened airway obstruction and dyspnea in asthmatic patients (8). Losartan is the first angiotensin II type 1 (AT₁) receptor antagonist to become available for the treatment of hypertension. Because the mechanism of action of AT₁ receptor antagonists does not involve the inhibition of ACE, side effects induced by ACE inhibitors are not expected to occur with these agents. Losartan does not increase blood flow in the forearm after an infusion of bradykinin, because it would not be expected to increase bradykinin levels, unlike enalapril (9). If bradykinin causes ACE inhibitor-induced bronchoconstriction or angioedema, losartan should be nearly free of these side effects. However, recent reports have revealed several cases of AT₁ receptor antagonist-induced bronchoconstriction (10) and angioedema (11), suggesting that AT₁ receptor antagonists may not be entirely free of ACE inhibitor-related side effects. The aim of the present study was to examine the mechanism of AT₁ receptor antagonist-induced bronchoconstriction, considering in particular the involvement of endogenous nitric oxide (NO) released from airway epithelial cells in this process using anesthetized guinea pigs and cultured airway epithelial cells.

	METHODS

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Pulmonary Resistance (RL) Measurement

Hartly male guinea pigs (400 to 500 g) were used in this study. They were anesthetized using sodium pentobarbital (50 mg/kg, intraperitoneally; Abbott Laboratories, North Chicago, IL) and then ventilated artificially with a tracheal cannula using a constant-volume ventilator (Model 680; Harvard Apparatus Co., South Natick, MA) at a frequency of 60 breaths/min. The tidal volume was set at 6 ml/kg. Airflow was monitored continuously using a pneumotachograph (TV241T; Nihon Koden Co., Tokyo, Japan) connected to a differential pressure transducer (TP-602T; Nihon Koden Co.). The tidal volume was determined by electrical integration of airflow. A fluid-filled polyethylene catheter was introduced into the esophagus to measure esophageal pressure as an approximation of pleural pressure. Intratracheal pressure was measured using a polyethylene catheter inserted into a short tube connecting the tracheal cannula to the pneumotachograph. The transpulmonary pressure (defined as the pressure difference between the intratracheal and esophageal pressures) was measured with a differential pressure transducer. RL was calculated as previously described (12). Aerosols of test agents were generated with an ultrasonic nebulizer and delivered to the airways by the ventilator.

Effect of Ang II on Acetylcholine-induced Bronchoconstriction after Pretreatment with Ang II Receptor Antagonists or NO Synthase Inhibitor

We exposed guinea pigs to losartan (AT₁ receptor antagonist), PD-123319 (AT₂ receptor antagonist) (10⁷ M, 40 breaths), or L-N- nitroarginine-methylester (L-NAME; NO synthase inhibitor) or D-NAME (its inactive enantiomer) (5 × 10⁴ M, 40 breaths). Ten minutes after exposure to these agents, guinea pigs were exposed to Ang II (10⁷ M, 40 breaths) and then after another 5 min to acetylcholine (1 × 10³, 3 × 10³, or 5 × 10³ M, 40 breaths at each concentration). In another set of experiments, 5 min after exposure to L-NAME, guinea pigs were exposed to L-arginine (10³ M, 40 breaths at each concentration).

Measurement of Expired NO

Expired NO was measured on a chemiluminescence analyzer (CLM-500; Shimazu, Kyoto, Japan) sensitive to NO from 2 ppb, adapted for on-line recording of NO concentration. The inhaled gas was synthetic air (fraction of inspired oxygen [FI_O2] = 0.20, NO-free air < 1 ppb; Takachiho Chemicals, Kyoto, Japan). Exhaled gas was sampled from a reservoir tube placed immediately after the exhaust valve of the ventilator. A sample flow rate of 100 ml/min was controlled by a precision flow meter. Ten minutes after exposure to Ang II receptor antagonists (10⁷ M, 30 breaths), guinea pigs were exposed to Ang II (10⁷ M, 30 breaths) and then expired NO concentrations were recorded for each guinea pig.

Effect of Ang II Receptor Antagonists on Ang II-stimulated NO Production in Cultured Epithelial Cells

Tracheal epithelial cells were isolated by incubating the guinea pig trachea with a solution of 0.1% protease type XXIV in Krebs buffer solution for 20 min. Isolated cells were resuspended in a (DMEM/F₁₂) culture medium containing 10% fetal calf serum and seeded in 24-well tissue culture plates at a density of 2 × 10⁵ cells/cm². Cells in primary cultures were grown to confluence in a humidified atmosphere at 37° C in 95% O₂/5% CO₂ air. Epithelial cells generally formed confluent monolayers 4 to 6 d after seeding. Confluent cells were used in the following experiments. NO production from cultured epithelial cells was measured with an Iso-NO MARKII meter and sensor (World Precision Instruments, Mauer, Germany). Accurate NO measurements were taken over a range of NO concentrations from 1 nM to 20 µM in culture medium. The sensor probe housing was covered with a polymer membrane through which the NO diffuses. The electrode was filled with an electrolyte fluid for NO (13). Ten minutes after administration of Ang II receptor antagonists (10⁷ M), 10⁷ M Ang II was administered into the culture medium and the concentrations of NO release into the culture medium were then recorded.

Drugs

Losartan and PD123319 were gifted from DuPont Merck Pharmaceuticals Co. (Wilmington, DE) and Parke-Davis (Ann Arbor, MI), respectively. Acetylcholine, L-NAME, D-NAME, Ang II, L-arginine, D-arginine, and protease type XXIV were purchased from Sigma Chemical Co. (St. Louis, MO), and DMEM/F₁₂ culture medium was purchased from GIBCO BRL (UK).

Statistical Analysis

All values are expressed as the mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) followed by Scheffe's test. Values of p less than 0.05 were considered significant.

	RESULTS

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Effect of Ang II on Acetylcholine-induced Bronchoconstriction after Pretreatment with Ang II Receptor Antagonists or NO Synthase Inhibitor

Ang II (10⁸ ~ 10⁵ M), losartan (10⁷ ~ 10⁵ M), and PD123319 (10⁷ ~ 10⁵ M) each had no significant effect on baseline RL. Pretreatment with Ang II (10⁷ M) had no significant effect on acetylcholine-induced bronchoconstriction (Figure 1 [I]). Though losartan (10⁷ M) alone had no effect on acetylcholine-induced bronchoconstriction, pretreatment with losartan, but not with PD123319, markedly enhanced acetylcholine-induced bronchoconstriction at 5 min after exposure to Ang II (Figure 1 [II]). L-NAME and D-NAME (each 5 × 10⁴ M) each had no significant effect on baseline RL, and acetylcholine-and Ang II-induced bronchoconstriction. However, pretreatment with L-NAME, but not with D-NAME, potentiated acetylcholine-induced bronchoconstriction at 5 min after exposure to Ang II (Figure 2 [I]). L-Arginine (10³ M), but not D-arginine, reversed this action of L-NAME on the airway hyperresponsiveness to acetylcholine (Figure 2 [II]).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Effect of Ang II on acetylcholine-induced bronchoconstriction. Acetylcholine was administered 5 min after administration of saline or Ang II (107 M). Ang II receptor antagonists (losartan 107 M, PD123319 107 M) were administered 10 min before exposure to Ang II. Each point represents the mean ± SEM for six animals. There is a shift in the data points at each concentration of acetylcholine to avoid overlap of data points. **p < 0.01, *p < 0.05 versus Ang II alone.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Effect of L-NAME or D-NAME on Ang II-induced bronchial hyperreactivity to acetylcholine. Acetylcholine was administered 5 min after administration of Ang II (107 M). L-NAME (5 × 104 M) or D-NAME (5 × 104 M) was administered 10 min before exposure to Ang II. L-Arginine (L-Arg, 103 M) or D-arginine (D-Arg, 103 M) was administered 5 min after exposure to L-NAME. Each point represents the mean ± SEM for six animals. There is a shift in the data points at each concentration of acetylcholine to avoid overlap of data points. **p < 0.01, *p < 0.05 versus Ang II alone. §p < 0.05 versus Ang II + L-NAME.

Effect of Ang II Receptor Antagonists on Ang II-stimulated NO Release into Expired Air

Maximal NO concentration in expired air was 12.5 ± 1.5 ppb after saline administration (Figure 3). Administration of losartan (10⁷ M) and PD123319 (10⁷ M) alone did not change NO concentration in expired air. Ang II (10⁷ M) administration significantly increased NO concentration in expired air. After an initial peak (40 ± 5 ppb), expired NO concentration returned to pre-administration levels at 10 min. L-NAME significantly inhibited Ang II-stimulated NO release, and L-arginine, but not D-arginine, reversed the action of L-NAME on NO release. Losartan (10⁷ M), but not PD123319, also significantly inhibited Ang II-stimulated NO release from guinea pig airway (20 ± 3.5 ppb) (Table 1).

View larger version (13K): [in this window] [in a new window]   Figure 3.   Time course of Ang II-stimulated NO release into expired air. Each bar represents the mean ± SEM for seven animals.

Effect of Ang II Receptor Antagonists on Ang II-stimulated NO Release in Cultured Airway Epithelial Cells

The basal level of NO in the incubation media conditioned by guinea pig epithelial cells was 10 ± 4 nM (Table 2). Ang II (10⁷ M) resulted in a robust increase of NO production in the incubation medium (160 ± 25 nM). L-NAME, but not D-NAME, significantly inhibited NO production induced by Ang II. Losartan (10⁷ M) and PD123319 (10⁷ M) alone did not have an effect on NO production. However, the effect of this Ang II-induced NO release was significantly inhibited by 10-min pretreatment of epithelial cells with 10⁷ M losartan (25 ± 8 nM), but not that with PD123319.

	DISCUSSION

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In this study, we found that the bronchoconstriction induced by acetylcholine was not changed 5 min after administration of Ang II, and, in contrast, Ang II in the presence of losartan caused a significant increase in the acetylcholine responsiveness. Moreover, Ang II stimulated NO release in a rapid and short-lasting manner, and losartan inhibited Ang II-stimulated NO release in cultured airway epithelial cells and anesthetized guinea pigs. Nijkamp and coworkers demonstrated that aerosolized L-NAME induced an airway hyperresponsiveness for histamine in anesthetized guinea pigs (14). We also found that aerosolized L-NAME potentiated the bronchoconstriction induced by histamine, but not by acetylcholine. Moreover, pretreatment with L-NAME potentiated acetylcholine-induced bronchoconstriction 5 min after administration of Ang II, and L-arginine reversed this action of L-NAME on the acetylcholine responsiveness. These findings suggest that losartan-induced bronchoconstriction may have resulted from inhibition of endogenous NO release from airway epithelial cells. Airway epithelium appears to directly modulate the responsiveness of bronchial smooth muscle by releasing inhibitory factors, one of which is endogenous NO. Previous studies suggested that reduced NO synthesis in airway epithelial cells might be related to the development of airway hyperresponsiveness (15), and that exogenous and endogenous NO both have an inhibitory effect on bronchial obstruction (16, 17).

Recently, angiotensin receptors have been subcategorized into type 1 (AT₁) and type 2 (AT₂) based on functional characteristics and the use of specific antagonists (18). In rat lung, Ang II receptor sites are present, and specifically interact with AT₁ receptor antagonist (19). However, the effect of AT₁ receptor antagonist may not be entirely due to blockade of AT₁ receptor. When AT₁ receptor is blocked, Ang II may act via AT₂ receptor activation (20). We found that the combination of AT₁ and AT₂ receptor antagonists had no effects beyond those of AT₁ receptor antagonist alone on Ang II-stimulated NO release from cultured epithelial cells. This finding suggests that Ang II also stimulates NO release in epithelial cells via AT₁ receptor pathway. However, in rat kidney, Ang II stimulates NO production, and this action of Ang II is mediated at the AT₂ receptor (21). The physiological effects of Ang II at the AT₂ receptor have been difficult to elicit in guinea pig airway, at least in part because AT₂ receptors may be less expressed than AT₁ receptors, as in rat lung.

Ang II levels in plasma are elevated in patients with asthma (22). Airway inflammation such as asthma has been reported to be strictly associated with the development of bronchial hyperreactivity. It is possible that when alveolar capillary permeability is increased during airway inflammation, leakage of Ang II into the airspace is also increased, affecting airway function. Interestingly, all subjects in a previous study reporting that losartan-induced bronchospasm had a history of bronchial asthma (10). Under normal conditions, inhibition of endogenous NO release in the airway is only marginally effective in constricting airway smooth muscle. However, in asthmatic patients, stimulation of endogenous NO release by Ang II may counteract not only the effect of Ang II itself but those of various spasmogens as well. Therefore, administration of losartan can immediately induce bronchospasm in asthmatic patients. In this study, administration of NOS inhibitor did not reveal a bronchoconstrictor action of Ang II in the absence of acetylcholine. However, Ang II enhanced cholinergic bronchoconstriction in the presence of NOS inhibitor. It is possible that Ang II itself had very weak bronchoconstrictor action, but a priming effect which markedly enhanced cholinergic bronchoconstriction (23). NO production stimulated by Ang II might inhibit a priming effect of Ang II to the acetylcholine responsiveness. In fact, the dose of Ang II (10⁷ M) used in this study induced the release of NO into the airway via AT₁ receptor, and Ang II in the presence of losartan or L-NAME caused a significant increase in the acetylcholine responsiveness through inhibition of NO release. Whether the use of oral losartan at recommended doses results in significant bronchoconstriction remains to be determined as clinical experience with this drug increases.

In summary, losartan-induced bronchoconstriction may be due at least in part to inhibition of endogenous NO production by airway epithelial cells. Until more clinical experience with losartan has accumulated, we urge that this drug be used with caution in patients with a history of bronchial asthma.