INTRODUCTION

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The renin-angiotensin system is a metabolic pathway producing the octapeptide angiotensin II, which has vasoconstrictive effects and also is involved in regulation of water homeostasis and electrolyte balance. Angiotensin-converting enzyme (ACE) is highly expressed in the lungs (1) and plays a key role in the metabolism of angiotensin II and bradykinin (2). The sites having high affinity for losartan are designated as type 1 angiotensin II (AT1) receptors and those having a high affinity for PD123177 and its structural analogs such as PD123319 are designated as type 2 angiotensin II (AT2) receptor (3, 4). Both Northern (5) and Western (6) blot analyses have demonstrated AT1 receptor expression in human lung tissues. The renin-angiotensin system reportedly is activated in severe acute attacks of asthma, as evidenced by elevations of plasma renin and angiotensin II (7). Angiotensin II causes bronchoconstriction in patients with mild asthma (8). In subthreshold concentrations, angiotensin II enhances methacholine-evoked bronchoconstriction in vitro in isolated human bronchi and in vivo studies of patients with mild asthma (8). Furthermore, angiotensin II induces hypertrophy of human airway smooth muscle cells (9).

Animal studies have indicated that AT1 receptors are involved in angiotensin II effects including bronchoconstriction in guinea pigs (10), peptide leukotriene (LT) production in guinea pig airways (10), potentiation of endothelin-1-induced contractions in bovine bronchial smooth muscle (11), and Cl secretion by canine tracheal epithelium (12). These observations suggest that angiotensin II could be an important mediator in asthma, but despite these pharmacological characterizations, no evidence has been presented for a role of endogenous angiotensin II in asthma. We, therefore, examined the effect of candesartan cilexetil, an AT1 receptor antagonist (13), and PD123319, an AT2 receptor antagonist (14), on antigen-induced bronchoconstriction, eosinophil and neutrophil accumulation in airways, and airway hyperresponsiveness to methacholine in guinea pigs.

	METHODS

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Chemicals

The following chemicals were used: ovalbumin (Sigma, St. Louis, MO), aluminum hydroxide (Al[OH]₃; Wako Pure Chemical Ind., Osaka, Japan), candesartan ((±)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate; Takeda Chemical Ind., Ltd., Osaka, Japan), PD123319 ([S]-1-[[4-[dimethylamino]-3-methylphenyl]methyl]-5-[diphenylacetyl]- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid; Funakoshi Co. Ltd., Tokyo, Japan), sodium pentobarbitone (Abbot Laboratories, North Chicago, IL), and methacholine (Wako Pure Chemical Ind., Osaka, Japan).

Sensitization of Animals

Male albino Hartley strain guinea pigs were obtained from Sankyou Laboratory Service (Toyama, Japan) and were quarantined in the Animal Research Center of Kanazawa University for 1 wk before study. All animal procedures in this study complied with the standards specified in the Guidelines for the Care and Use of Laboratory Animals at the Takaramachi Campus of Kanazawa University.

Guinea pigs weighing 200-250 g were actively sensitized by a modification of the method reported by Andersson (15). Briefly, guinea pigs were pretreated with an intraperitoneal injection of 30 mg/kg of cyclophosphamide. Two days later the animals were immunized with 2.0 mg of ovalbumin and 100 mg of aluminum hydroxide (Al[OH]₃). A booster injection of 10 µg of ovalbumin together with 100 mg of Al(OH)₃ was given 3 wk after the primary immunization.

Antigen-induced Bronchoconstriction

Candesartan cilexetil (0.1, 1.0, or 10 mg/kg dissolved in dimethyl sulfoxide [DMSO] and then suspended in 5% gum arabic, n = 10 in each group) or PD123319 (30 mg/kg dissolved in saline, n = 6 in each group) was administered intraperitoneally 60 min before antigen challenge. Additional animals were studied in control groups receiving the intraperitoneal vehicle without candesartan cilexetil and either inhalation of saline instead of antigen (ovalbumin) as the challenge (negative controls) or inhalation of ovalbumin (positive controls).

Animals were placed in a whole-body, double-chamber plethysmograph for measurement of specific airway resistance (sRaw) by a modification of the method described by Pennock and coworkers (16). A respiratory mechanics analyzer (model PMUA+SAR; Buxco Electronics, Troy, NY) was used to measure the phase shift between nasal and thoracic air flow and to compute sRaw on a breath by breath basis. A bias flow of air (20 ml/s) was maintained through the nasal chamber to ensure a constant supply of fresh air to the animal. The analyzer subtracted this flow from its calculations. Sixty minutes after administration of candesartan cilexetil or PD123319, animals were challenged by 60-s exposure to an aerosol of ovalbumin generated from a 10 mg/ ml solution in saline by a DeVilbiss 646 nebulizer (DeVilbiss Co., Somerset, PA) operated by compressed air at 5 L/min and passed through the nasal chamber. The nebulizer output was 0.14 ml/min. The sRaw was measured for 5 min before candesartan cilexetil or PD123319 and antigen challenge (baseline measurement) and for 15 min after challenge.

To evaluate the effect of chronic pretreatment of candesartan cilexetil on antigen-induced early phase bronchoconstriction, antigen challenge was performed as previously described after 3 d pretreatment of intraperitoneal candesartan cilexetil at a dose of 1.0 or 10 mg/ kg twice a day with last dosing 1 h before the challenge (n = 7 in each group).

Antigen-induced Airway Hyperresponsiveness

Antigen-induced airway hyperresponsiveness was measured 24 h after antigen challenge. Guinea pigs were anesthetized by an intraperitoneal injection of 75 mg/kg of sodium pentobarbitone and were placed in a supine position. After the trachea was cannulated with a polyethylene tube (outside diameter, 2.5 mm; inside diameter, 2.1 mm), the animals were ventilated artificially using a small-animal respirator (Model 1680, Harvard Apparatus Co., Inc., South Natick, MA) adjusted to deliver a tidal volume of 10 ml/kg at a rate of 60 strokes/min. Changes in lung resistance to inflation, specifically the lateral pressure of the tracheal tube (pressure at the airway opening; Pao, cm H₂O), were measured using a pressure transducer (TP-603T, Nihon Koden Kogyo Co., Ltd., Tokyo, Japan) according to the modification of the method of Konzett and Roessler (17) described by Jones and co-workers (18). After confirming that the change in Pao following inhalation of histamine and LTC₄ represented the average of the changes in pulmonary resistance (RL) and reciprocal dynamic pulmonary compliance (1/Cdyn) (19), we use the change in Pao as an overall index of bronchial response to bronchoactive agents (20). After completion of all surgical procedures, the lungs were overinflated by two times tidal volume for two breaths by closing the outlet port of the respirator. This was done to standardize the volume history of the lungs. Five minutes after initiation of artificial respiration, when the Pao had stabilized, a succession of doubling concentrations of methacholine from 25 to 1,600 µg/ml was given for 20 s at intervals of 5 min using an ultrasonic nebulizer until a 200% increase in Pao was recorded or the dose sequence was completed. The nebulizer generated the aerosol at a rate of 15.2 µl/min during the 20-s period, and 46.4% of the generated aerosol was deposited in the lungs as measured by a radioaerosol technique (21). The median aerodynamic diameter of the particles of normal saline was 3.59 ± 1.96 µm (mean ± SD) (22).

To evaluate the effect of candesartan on airway responsiveness to methacholine in nonsensitized guinea pigs (weight, 350-400 g), methacholine responsiveness was measured as previously described 60 min after intraperitoneal candesartan injection at a dose of 1.0 or 10 mg/kg (n = 8 in each group).

Analysis of Bronchoalveolar Cells

After the examination of antigen-induced airway hyperresponsiveness, 10 ml of sterile saline at 37° C was infused twice through the tracheal cannula. The bronchoalveolar lavage fluid (BALF) was recovered manually by gentle aspiration with a disposable syringe after each infusion. The total cell number was determined using a Turk solution. Differential cell counts were performed on cytospin preparations (Cytospin 2, Shandon, UK) by counting 300 cells after staining by the May-Grünwald-Giemsa method.

Statistical Analysis

Airway responsiveness to inhaled methacholine was expressed as the dose of methacholine required to provoke a 200% increase (PC₂₀₀) in the Pao. Values for PC₂₀₀ were logarithmically transformed for analysis and reported as the geometric mean (geometric standard error of the mean [GSEM]). All measurements except for PC₂₀₀ were expressed as mean ± standard error of the mean (SEM). All results were compared using analyses of variance (ANOVA) followed by Fisher's protected least significant difference. A value of p < 0.05 was accepted as an indication of significance.

	RESULTS

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No significant changes were apparent in sRaw after administration of candesartan cilexetil or PD123319. Although aerosolized antigen caused acute bronchoconstriction that peaked within 5 min in all guinea pigs, candesartan cilexetil (either single or 3 d treatment) or PD123319 caused no inhibition of antigen-induced acute bronchoconstriction (Figures 1 and 2). Antigen inhalation significantly increased bronchoconstrictor responses to methacholine (Figure 3) and airway accumulation of neutrophils and eosinophils (Figure 4). Candesartan cilexetil, but not PD123319, dose dependently prevented antigen-induced airway hyperresponsiveness to methacholine (Figure 3) and also prevented eosinophil accumulation (Figure 4). Baseline airway responsiveness to methacholine was not altered by candesartan cilexetil (Figure 5).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Effects of single treatment with candesartan cilexetil (top panel ) (n = 10 in each group) and PD123319 (bottom panel ) (n = 6 in each group) on antigen-induced early phase airway obstruction in guinea pigs. Error bars indicate mean ± SEM. Significance is indicated as follow: p < 0.01 compared with the negative control group.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Effects of 3 d treatment with candesartan cilexetil (n = 7 in each group) on antigen-induced early phase airway obstruction in guinea pigs. Error bars indicate mean ± SEM.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effects of candesartan cilexetil (top panel ) (n = 10 in each group) and PD123319 (bottom panel ) (n = 6 in each group) on bronchial hyperresponsiveness to methacholine 24 h after antigen inhalation in guinea pigs. Error bars indicate mean ± SEM. PC200, provocative concentration of methacholine producing a 200% increase above the baseline pressure of airway opening. Significance is indicated as follows: p < 0.01 compared with the negative control group; *p < 0.05 and **p < 0.01 compared with the positive control group.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Effects of candesartan cilexetil (top panel ) (n = 10 in each group) and PD123319 (bottom panel ) (n = 6 in each group) on eosinophil and neutrophil accumulation in BALF 24 h after antigen inhalation in guinea pigs. Error bars indicate mean ± SEM. P, positive control; N, negative control. Significance is indicated as follows: p < 0.05 and p < 0.01 compared with the negative control group; *p < 0.05 and **p < 0.01 compared with the positive control group.

View larger version (11K): [in this window] [in a new window]   Figure 5.   Effects of candesartan on airway responsiveness to methacholine in nonsensitized guinea pigs (n = 8 in each group). Error bars indicate mean ± SEM. PC200, provocative concentration of methacholine producing a 200% increase above the baseline pressure of airway opening.

	DISCUSSION

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In this study we investigated the effects of candesartan cilexetil and PD123319 in guinea pigs on antigen-induced early phase bronchoconstriction, and then on airway inflammation and airway hyperresponsiveness 24 h after antigen inhalation. Our results showed that candesartan cilexetil but not PD123319 reduced both antigen-induced eosinophil accumulation in BALF and airway hyperresponsiveness to methacholine. These effects occurred despite the fact that the immediate response to antigen was not blocked.

Candesartan cilexetil is the prodrug form of candesartan (CV-11974), a new AT1 receptor antagonist shown to have high affinity in a receptor-binding assay and high potency in a rabbit aorta contraction assay (13). Completely inhibiting specific binding to the AT1 receptor in a concentration-dependent, monophasic manner, CV-11974 has a K_i value of 0.64 nM (13). This drug shows no affinity for the AT2 receptor (13). The affinity of CV-11974 for these AT1 receptors is about 80 and 10 times higher, respectively, than those of losartan (another AT1 receptor antagonist) and EXP3174 (an active metabolite of losartan) (13). Losartan interacts with thromboxane A₂ (TXA₂)/prostaglandin H₂ (PGH₂) receptors (23) and inhibits induction of platelet aggregation and vasoconstriction in rats by the TXA₂ analog U46619 (24). This inhibition is specific for losartan. CV-11974 does not block platelet aggregation or vasoconstriction caused by U46619 (24). Furthermore, CV-11974 has no effect on vascular contraction induced by norepinephrine, potassium chloride, serotonin, prostaglandin F₂ (PGF₂), or endothelin (25), indicating that CV-11974 is a high selective inhibitor of angiotensin II. Although candesartan cilexetil at 1 mg/kg orally has no effect on basal plasma aldosterone concentrations or blood pressure in normotensive rats (26), candesartan cilexetil at similar doses (0.1-10 mg/kg orally) produces a clear, dose-dependent reduction of blood pressure in several rat models of hypertension (26).

On the other hand, Macari and coworkers (27) reported that infusion of the AT2 receptor antagonist PD123319 at doses of 30 mg/kg/d in rats produces plasma concentrations of approximately 200 nM. Since the concentration that inhibits 50% (IC₅₀) of PD123319 for the AT2 receptor is approximately 17 nM (28), infusion of 30 mg/kg/d of PD123319 should result in an effective AT2 blockade without affecting the AT1 receptor. Since PD123319 had no effect in our observations, antigen- induced bronchial hyperresponsiveness and eosinophil accumulation appear to be mediated via the AT1 receptor.

Airway hyperresponsiveness following antigen challenge occurred at a time when Pao was identical between all groups, thus reflecting a true increase in airway responsiveness and not results of residual bronchoconstriction from the initiation of methacholine inhalation. The effect of candesartan cilexetil appeared to be specific to antigen-induced airway hyperresponsiveness, since the drug alone showed no effect on sRaw or baseline airway responsiveness. Therefore, the effects of candesartan cilexetil on antigen-induced airway hyperresponsiveness cannot be attributed to anticholinergic or bronchodilatory effects of the compound.

The airway eosinophil influx in response to antigen challenge was completely blocked by the AT1 receptor antagonist. Because direct effects of angiotensin II on chemotaxis have not yet been described, one can only speculate on mechanisms by which candesartan cilexetil blocked inflammatory cell influx. Possibilities include a modulatory effect of angiotensin II on the release or action of chemotactic mediators such as platelet-activating factor (PAF) (29), LT (10), or TXA₂ (30).

Although alternative angiotensin II-forming pathways independent of ACE are known to exist in various tissues (31), details of such pathways remain unclear. Several serine proteinases such as chymase, cathepsin G, and tonin appear to be involved in ACE-independent angiotensin II formation in vivo (32). Among these enzymes, chymase has been shown by biochemical analysis to be a highly efficient angiotensin II-forming enzyme with a high substrate specificity for angiotensin I (33). Chymase is abundant in various human tissues including the lung (33). Mast cells store active chymase in their secretory granules, which they release upon IgE-mediated activation (34). Human neutrophil cathepsin G has been reported to potentiate angiotensin II-generating activity (35). This ability to produce angiotensin II may be of significance in the development of biochemical events associated with allergic inflammation, but further studies are required to test the hypothesis. For example, it is important to explore the effects of ACE inhibitors on the same phenomenon to help establish the source of the endogenous angiotensin II either by ACE-dependent or -independent pathways.

In summary, we found in guinea pigs that angiotensin II played an important role in antigen-induced airway hyperresponsiveness and eosinophil accumulation via the AT1 receptors. This report is the first to suggest involvement of AT1 receptors in allergic airway reactions.