INTRODUCTION

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Peroxynitrite is formed by the reaction of nitric oxide (NO) with superoxide anion (·O₂) (1). In many pathologic conditions including inflammation, simultaneous cellular production of ·O₂ and NO may occur, potentially leading to the continuous formation of peroxynitrite. Peroxynitrite is an extremely potent oxidant that can cause lipid peroxidation, DNA damage, and alterations of protein function in vitro (2). Recently, nitrogen-derived oxidants were shown to be formed in human acute lung injury, suggesting the possibility of an important role for peroxynitrite in inflammatory lung disease (3). Recent reports have suggested that single administration of peroxynitrite induces epithelial damage and hence airway hyperresponsiveness (4). However, we believed that the physiologic effects of peroxynitrite in the airway should be examined through the use of a compound that releases peroxynitrite in an in vivo experimental model. We had two reasons for this belief. The first was that peroxynitrite has a half-life of less than 1 s at pH 7.4 (5). Potentially greater and lesser production of peroxynitrite can be achieved in inflamed airways under conditions in which NO and superoxide anion (·O₂) production are stimulated, since a 100-fold increase in the rate of peroxynitrite formation should occur for every 10-fold increase in NO and ·O₂ concentration. The second reason for our use of a compound that provided prolonged release of peroxynitrite in studying the airway effects of this substance is that epithelial cells lining the respiratory airways may be an important source of antioxidant enzymes that protect the respiratory tract (6). These cells would be the first to contact oxidants and also the first to be exposed to oxidants generated by local inflammatory reactions in the airways. Moreover, it seems likely that the thin layer of epithelial lining fluid (ELF) may provide antioxidant protection and serve as a front-line defense for the alveolar epithelium. ELF contains various antioxidant substances, including vitamin E, reduced glutathione, and vitamin C (7). In addition, ELF contains a wide spectrum of plasma proteins, and some of these, such as ceruloplasmin and transferrin, may function as antioxidants in the lower respiratory tract (8). On this basis we felt that a single administration of peroxynitrite would be scavenged by several antioxidants, and would not produce sufficient physiologic effects for study.

₂-Adrenoceptors (₂-AR) are ubiquitous in body tissues, and are normally stimulated by endogenous circulating catecholamines such as adrenaline, or by exogenously administered ₂-AR agonists. Interaction of an agonist with a ₂-AR causes a conformational change in the receptor, resulting in activation of the G-protein complex, with stimulation of membrane-bound adenylate cyclase. In the airway, ₂-AR are present on smooth-muscle cells and epithelial cells, as well as on inflammatory cells such as mast cells, eosinophils, and mononuclear cells. Sustained receptor stimulation by ₂-agonists results in homologous desensitization, which occurs initially as a result of receptor uncoupling from the stimulatory G-protein, followed by a decrease in surface receptor number. However, previous study has suggested that oxygen radicals might be involved in the mechanism of airway hyperresponsiveness through decreases in adenylate cyclase activity (9). We thought that because it is a stronger oxidant than either NO or ·O₂ (10), peroxynitrite might influence ₂-AR responses in the airways. We therefore undertook the present study to determine whether peroxynitrite can alter airway ₂-AR function, using 3-morpholinosydnonimine (SIN-1), a compound that releases peroxynitrite, in anesthetized guinea pigs.

	METHODS

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Peroxynitrite Measurement

Authentic peroxynitrite readily oxidizes dihydrorhodamine 123, whereas ·O₂, H₂O₂, and NO alone do not (11). Peroxynitrite formation from SIN-1 was assayed by monitoring rhodamine formation at 500 nm in reaction mixtures containing dihydrorhodamine 123 (50 µM), potassium phosphate (20 mM, pH 7.4), and various amounts of SIN-1 for 30 min at 37° C. We utilized SIN-1 for generation of peroxynitrite in the following experiments.

Measurements of Pulmonary Resistance (RL)

Male Hartley guinea pigs (400 to 500 g; Nihon Keari Breeding Laboratory, Osaka, Japan) were used in the study. All guinea pigs were provided with food and water and kept in a temperature- and humidity-controlled environment at the Institute for Laboratory Animal Research of the Osaka City University Medical School. The guinea pigs were anesthetized using sodium pentobarbital (50 mg/kg intraperitoneally) 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 (VT) was set at 6 ml/kg. Airflow was monitored continuously with a pneumotachograph (TV241T; Nihon Koden Co., Tokyo, Japan) connected to differential pressure transducer (TP-602T; Nihon Koden). VT was determined by electrical integration of airflow. A fluid-filled polyethylene catheter was introduced into the esophagus to measure esophageal pressure (Pes) as an approximation of pleural pressure. Intratracheal pressure was measured with a polyethylene catheter inserted into a short tube connecting the tracheal cannula to the pneumotachograph. The transpulmonary pressure (Ptp; 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 neblizer, and were delivered to the airways by the ventilator. All protocols used in the study were approved by the Osaka City University Animal Research Committee, which is responsible for ensuring the proper use of experimental animals. All guinea pigs used in the study were anesthetized with pentobarbital repeated every 30 min throughout the experiments to suppress spontaneous breathing and pain.

Effect of SIN-1 on Acetylcholine-induced Bronchoconstriction

After measuring baseline RL (100% RL), we exposed guinea pigs to SIN-1 (10⁸ to ~ 10⁵ M, 40 breaths at each concentration). Thirty minutes after exposure to SIN-1, guinea pigs were exposed to acetylcholine (Ach; 5 × 10³ M, 40 breaths). The effect of each dose of SIN-1 was examined in different guinea pigs. Percentage bronchoprotection was calculated as follows: (% increase in maximal RL [without SIN-1]  % increase in maximal RL [with SIN-1])/% increase in maximal RL (without SIN-1) × 100.

Effect of SIN-1 on Isoprenaline-, Salbutamol-, and Forskolin-induced Bronchoprotection

Guinea pigs were exposed to SIN-1 (10⁷ M, 5 × 10⁸ M, and 10⁸ M, 40 breaths at each concentration). Thirty minutes after exposure to SIN-1, guinea pigs were exposed to isoprenaline, salbutamol, or forskolin (10⁷ M for each agent, 40 breaths) and then, 5 min later, to Ach (5 × 10³ M, 40 breaths). In another set of experiments, guinea pigs were exposed to N-acetylcysteine (NAC; 1 mM, 5 mM, and 10 mM, 60 breaths at each concentration) or glutathione (GSH; 1 mM or 10 mM, 60 breaths at each concentration) and then, 10 min later, to SIN-1 (10⁷ M, 40 breaths).

Effect of SNAP on Isoprenaline-induced Bronchoprotection

Guinea pigs were exposed to S-nitroso-N-acetyl-penicillamine (SNAP; 10⁷ M, 40 breaths). Five minutes after exposure to SNAP, guinea pigs were exposed to isoprenaline (10⁷ M, 40 breaths) and then, 5 min later, to Ach (5 × 10³ M, 40 breaths).

Sources of Materials

Pentobarbital was purchased from Abbott Laboratories (North Chicago, IL). Ach, isoprenaline, salbutamol, forskolin, NAC, and GSH were purchased from Sigma Chemical Co. (St. Louis, MO). SIN-1, SNAP, and peroxynitrite were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Rhodamine 123 and dihydrorhodamine 123 were purchased from Molecular Probes (Eugene, OR). SIN-1 was dissolved in water, and further dilutions were made in 0.9% saline.

Statistical Analysis

All values are expressed as mean ± SEM. The statistical significance was determined by analysis of variance (ANOVA) followed by Scheffe's test. Values of p < 0.05 were considered significant.

	RESULTS

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The formation of peroxynitrite by the autoxidation of SIN-1 was confirmed by the oxidation of dihydrorhodamine 123 (Figures 1a and 1b). SIN-1, in a time- and concentration- dependent manner, oxidized dihydrorhodamine 123 to rhodamine 123, and NAC (1 mM) completely inhibited this oxidation. The oxidation of dihydrorhodamine 123 is mediated by peroxynitrite and not by either NO or ·O₂ alone (13). SNAP, a potent NO donor, did not oxidize dihydrorhodamine 123. The baseline RL was 0.18 ± 0.02 cm H₂O/ml/s, and administration of Ach (5 × 10³ M) resulted in a significant increase in RL (0.50 ± 0.05 cm H₂O/ml/s). SIN-1 inhibited Ach-induced bronchoconstriction in a dose-dependent manner (Figure 2). SIN-1 (10⁷ M) alone had no effect on baseline RL or Ach- induced bronchoconstriction. However, pretreatment with SIN-1, but not with the solvent for SIN-1, significantly attenuated isoprenaline-induced bronchoprotection against Ach- induced bronchoconstriction (mean inhibition by SIN-1 = 79.2%) (Figure 3). This inhibitory effect of SIN-1 was exhibited in a dose-dependent manner. Pretreatment with SIN-1 also had this effect on salbutamol-induced bronchoprotection (mean inhibition by SIN-1 = 85.0%) (Table 1). In addition, forskolin (10⁷ M) inhibited Ach-induced bronchoconstriction, but pretreatment with SIN-1 attenuated forskolin-induced bronchoprotection (mean inhibition by SIN-1 = 93.7%). NAC and GSH had no effect on baseline RL, Ach-induced bronchoconstriction, or isoprenaline-induced bronchoprotection. However, NAC significantly reversed the inhibitory effect of SIN-1 on isoprenaline-induced bronchoprotection against Ach in a dose-dependent manner (Figure 4). GSH also reversed this inhibitory effect of SIN-1. On the other hand, a subthreshold concentration of SNAP, a potent NO donor, significantly enhanced isoprenaline-induced bronchoprotection against Ach (Figure 5).

View larger version (11K): [in this window] [in a new window]   Figure 1.   (a) Peroxynitrite formation induced by SIN-1 or SNAP was assayed by monitoring rhodamine formation. A standard curve of oxidizing activity of dihydrorhodamine 123 was constructed, using peroxynitrite per se. SIN-1 (solid circles; 103 M) produced 50 µM of peroxynitrite after a 30-min incubation time, but SNAP (open circles; 103 M) did not. Results are expressed as means (n = 8, SD was < 5% in all cases). (b) Dose-response curve of peroxynitrite formation from SIN-1. SIN-1 (solid circles) produced peroxynitrite after a 30-min incubation time in a dose-dependent manner. SIN-1-derived peroxynitrite was eliminated by addition of NAC (open circles; 103 M). Results are expressed as means (n = 8, SD was < 5% in all cases).

View larger version (8K): [in this window] [in a new window]   Figure 2.   Bronchoprotective effects of SIN-1 against Ach. Each point represents the mean ± SEM for six animals (*p < 0.05, **p < 0.01 versus solvent alone).

View larger version (36K): [in this window] [in a new window]   Figure 3.   Effect of SIN-1 on isoprenaline-induced bronchoprotection against Ach. Each bar represents the mean ± SEM for seven animals. Iso = isoprenaline, Ach = acetylcholine.

View larger version (30K): [in this window] [in a new window]   Figure 4.   Effect of SIN-1 on isoprenaline-induced bronchoprotection in the presence of NAC or GSH. Each bar represents the mean ± SEM for six animals. Iso = isoprenaline, Ach = acetylcholine (*p < 0.05, **p < 0.01 versus solvent alone).

View larger version (35K): [in this window] [in a new window]   Figure 5.   Effect of SNAP on isoprenaline-induced bronchoprotection. SNAP (107 M) did not have a significant bronchoprotective effect against Ach. Each bar represents the mean ± SEM for six animals. Iso = isoprenaline, Ach = acetylcholine. N.S. = not significant.

	DISCUSSION

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SIN-1 is thought to generate equimolar amounts of superoxide anion and NO, which may rapidly interact to form peroxynitrite. Though there are no data showing that SIN-1 causes the release of peroxynitrite in guinea pigs in vivo, SIN-1 has been utilized for exposure to peroxynitrite of various kinds of pulmonary cells in previous studies (14, 15).

Recent studies have suggested that peroxynitrite stimulates guanylate cyclase and induces accumulation of cyclic guanosine monophosphate (cGMP) in vascular endothelial and smooth-muscle cells (16, 17). Thus, peroxynitrite was postulated to cause relaxation of isolated canine coronary arteries and bovine pulmonary arteries through a cGMP-dependent mechanism (18, 19). These findings suggested that peroxynitrite has a potent vascular relaxant activity. However, no previous studies have examined the relaxant effect of peroxynitrite on airway smooth muscle. Recent studies suggested that relaxation of airway smooth muscle elicited by SIN-1 was inhibited by a soluble guanylate cyclase inhibitor (20, 21). The present study showed that SIN-1 is a potent peroxynitrite- releasing compound and that it produced significant bronchoprotection against Ach-induced bronchoconstriction in a dose-dependent manner. These findings provide strong evidence that peroxynitrite generated from SIN-1 exerts bronchoprotective effects via a guanylate cyclase-dependent pathway.

In this study we showed that the bronchoprotective effect of isoprenaline was significantly attenuated by a subthreshold concentration of SIN-1, and that SIN-1 exhibited this inhibitory effect on isoprenaline-induced bronchoprotection in a dose-dependent manner. Moreover, pretreatment with SIN-1 also had inhibitory effects on salbutamol (another ₂-AR agonist)- and forskolin (an adenylate cyclase activator)-induced bronchoprotection. These findings suggest that the effects of peroxynitrite are directed against either adenylate cyclase activity or against effects downstream of such activity (i.e., protein kinase A [PKA] or the targets of PKA). In addition, NAC inhibited the formation of peroxynitrite from SIN-1 in vitro, and pretreatment with NAC reversed the inhibitory effect of SIN-1 on isoprenaline-induced bronchoprotection in a dose-dependent manner. GSH also reversed this inhibitory effect of SIN-1. Our findings suggest that these antioxidants might be useful as therapeutic agents for impaired ₂-AR function induced by peroxynitrite. In this study we also found that SNAP, a potent NO donor, did not have the inhibitory effect of SIN-1, and that it instead significantly enhanced isoprenaline-induced bronchoprotection. We have already determined that increased cyclic adenosine monophosphate (cAMP) levels synergistically enhance NO-mediated bronchoprotection (22).

The airways of asthmatic patients are often inflamed, and it has been shown that the production of superoxide anion by alveolar macrophages of allergic asthmatic patients is increased after segmental antigen challenge (23). The concentration of NO in exhaled air of asthmatic patients is increased (24), and we previously found a higher than normal concentration of NO derivatives in induced sputum of patients with asthma (25). According to the existing evidence, it is likely that peroxynitrite is formed in the respiratory tract. A previous hypothesis suggested that peroxynitrite may indirectly exacerbate the airway inflammatory response by inducing the shedding of airway epithelial cells (26)which may occur even in patients with mild asthmathereby exposing afferent nerve endings. This might induce the release of sensory neuropeptides through axon reflexes and result in bronchoconstriction, mucus hypersecretion, and microvascular leakage, leading to edema of the airway wall and extravasation of plasma into the airway lumen (27). In the present study we found that peroxynitrite may play a direct role in the regulation of ₂-AR function in the airways.

In conclusion, this is the first report that peroxynitrite is an important mediator of alterations of ₂-AR function in the airways. However, further studies will be required to determine whether peroxynitrite has an important role in the regulation of airway responses.