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Indirect evidence using nitric oxide (NO) synthase (NOS) inhibitors suggests that in guinea-pig airways bradykinin releases bronchoprotective NO. In this study ...
Detection of Nitric Oxide Release Induced by Bradykinin in Guinea Pig Trachea and Main Bronchi Using a Porphyrinic Microsensor Fabio L. M. Ricciardolo, Luciana Vergnani, Silke Wiegand, Franco Ricci, Nadia Manzoli, Axel Fischer, Silvia Amadesi, Renato Fellin, and Pierangelo Geppetti Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands; Department of Clinical and Experimental Medicine, University of Ferrara, Ferrara, Italy; and Institute for Anatomy and Cell Biology, Justus-Liebig University, Giessen, Germany

Indirect evidence using nitric oxide (NO) synthase (NOS) inhibitors suggests that in guinea-pig airways bradykinin releases bronchoprotective NO. In this study, using a recently developed electrochemical method of NO measurement based on a porphyrinic microsensor, we investigated whether bradykinin releases NO from guinea-pig airways and whether the epithelium is the main source of NO. Further, the Ca21-dependence of bradykinin-induced NO release was assessed stimulating airway preparations with bradykinin in Ca21-free conditions. We also studied the immunohistochemical distribution of the Ca21dependent constitutive isoforms of NOS (constitutive NOS [cNOS]: neuronal and endothelial [ecNOS]) in our preparations. The porphyrinic microsensor was placed in the bathing fluid onto the mucosal surface of tracheal or main bronchial segments. Addition of bradykinin vehicle (0.9% saline) did not cause any detectable change of the baseline signal. Addition of bradykinin caused an upward shift of the baseline that reached a maximum within 1 to 2 s. The amplitude of the response to bradykinin was concentration-dependent between the range 1 nM to 10 mM, with a maximum effect at 10 mM. Bradykinin-induced NO release was higher in tracheal than in main bronchial segments. The selective bradykinin B2 receptor antagonist 0 3 5 7 8 D-Arg -[Hyp , Thi , D-Tic , Oic ]bradykinin (1 mM) inhibited NO release induced by a submaximum concentration of bradykinin (1 mM). The ability of bradykinin to release NO was markedly reduced in epithelium-denuded segments, and abolished in Ca21-free conditions and after pretreatment with NG-monomethyl-L-arginine (100 mM), but not with NG-monomethyl-D-arginine. Both cNOS isoforms were present in trachea and main bronchi, ecNOS being the predominant isoform in the epithelium. The study shows that bradykinin via B2 receptor activation caused a rapid and Ca21-dependent release of NO, mainly, but not exclusively, derived from the epithelium. It also shows that both cNOS isoforms may be involved in bradykinin-evoked NO release. Ricciardolo, F. L. M., L. Vergnani, S. Wiegand, F. Ricci, N. Manzoli, A. Fischer, S. Amadesi, R. Fellin, and P. Geppetti. 2000. Detection of nitric oxide release induced by bradykinin in guinea pig trachea and main bronchi using a porphyrinic microsensor. Am. J. Respir. Cell Mol. Biol. 22:97–104.

Nitric oxide (NO) is a small diatomic, free-reactive, gaseous, lipophilic molecule with a very short half-life ( z 0.1 to 5 s) that is generated during the conversion of the (Received in original form February 22, 1999 and in revised form July 26, 1999) Address correspondence to: Fabio Ricciardolo, M.D., Lung Function Lab., C2-P, Leiden University Medical Center, P.O. Box 9600, NL-2300 RC Leiden, The Netherlands. E-mail: [email protected] Abbreviations: constitutive NOS, cNOS; N G-monomethyl-D-arginine, D-NMMA; endothelial NOS, ecNOS; maximal effect that an agonist can elicit in a given tissue under particular experimental conditions, Emax; femtomolar, fM; D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]bradykinin, HOE 140; inducible NOS, iNOS; immunoreactivity, IR; N G-monomethyl-L-arginine, L-NMMA; neuronal NOS, nNOS; nitric oxide, NO; NO synthase, NOS; phosphate buffer, PB; standard error of the mean, SEM. Am. J. Respir. Cell Mol. Biol. Vol. 22, pp. 97–104, 2000 Internet address: www.atsjournals.org

amino acid L-arginine to L-citrulline by the enzyme NO synthase (NOS) in a reaction that requires nicotinamide adenine dinucleotide phosphate and molecular oxygen as cosubstrates, and tetrahydrobiopterin, thiol, and flavins (flavin adenine dinucleotide and flevin mononucleotide [FMN]) as cofactors (1, 2). Three distinct isoforms of NOS have been identified by protein purification and molecular cloning approaches. Neuronal (nNOS or NOS1), inducible (iNOS or NOS2), and endothelial (ecNOS or NOS3) isoenzymes are products of distinct genes located on different human chromosomes (3), and all of them are expressed in the airways (4– 7). Functionally, both constitutive NOS (cNOS) and iNOS have been described (8). In the airways, cNOS is expressed in neuronal (nNOS), endothelial (ecNOS) and epithelial cells (nNOS and ecNOS) (9). Different agonists, including

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bradykinin, acetylcholine, and histamine, may activate cNOS via a calmodulin-dependent rapid increase in the intracellular Ca21 concentration, resulting in the release of fM or pM concentrations of NO within seconds upon receptor stimulation (10). iNOS is not dependent for its activity on intracellular Ca21 or calmodulin, and its expression is regulated predominantly at a pretranslational level (11) and can be induced by proinflammatory cytokines, such as tumor necrosis factor-a, interferon-g, and interleukin-1b, and endotoxin (11). Once iNOS-gene transcription is induced, production of NO may increase to nanomolar concentrations several hours after exposure and may continue for days. In human airways, iNOS expression has been described in epithelial cells, macrophages, neutrophils, endothelial cells, and vascular smooth-muscle cells (9). Recent experimental evidence has suggested different roles of NO in the regulation of mammalian airway function (12). In fact, a high amount of NO formed by iNOS has deleterious effects and appears to be associated with airway inflammation (13), as determined by exhaled NO levels in humans and guinea pigs (14, 15). On the other hand, in low concentrations NO may act as a relaxant agent of airway smooth muscle, by activation of guanylyl cyclase and rise in cyclic guanosine monophosphate levels (12). NO has been identified as the transmitter of inhibitory nonadrenergic noncholinergic (iNANC) nerves that mediate relaxation of guinea pigs and human airways (16, 17) and as an endogenous modulator of cholinergic neural and excitatory NANC neural bronchoconstriction (18, 19). NO seems to be capable of modulating excitatory airway responses induced by different proinflammatory stimuli (20, 21). In particular, bronchoconstriction induced by bradykinin inhalation in guinea pigs was found to be markedly increased by NOS inhibitors (22). Functional in vitro evidence suggests that bronchorelaxant NO is released by bradykinin from the airway epithelium (23). In the present study we investigated the ability of bradykinin to release NO from guinea-pig airways by the direct measure of NO using a recently developed electrochemical method based on a porphyrinic microsensor (24). This method allows the measurement of NO concentration in the vicinity of a microsensor that is placed on the surface of the tissue under examination. To examine the

role of the epithelium, experiments were also performed in epithelium-denuded preparations. The role of bradykinin B2 receptors and NOS was studied using the bradykinin B2 receptor antagonist D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]bradykinin (HOE 140) and a NOS inhibitor, respectively. Ca21dependence of bradykinin-induced NO release was assessed stimulating either intact or epithelium-denuded airway preparations in Ca21-free conditions. Finally, we evaluated the immunohistochemical distribution of nNOS and ecNOS in sections of guinea-pig airways. Our results indicate that bradykinin, via activation of bradykinin B2 receptors, causes a Ca21-dependent rapid increase in NO concentration in the medium above the airway tissue. The release of NO derives mainly, but not exclusively, from the epithelial layer, and both cNOS isoforms may be involved in bradykinin-evoked NO release.

Materials and Methods Tracheal and Bronchial Segment Preparation Male guinea pigs (350 to 500 g) were killed with sodium pentobarbital (80 mg/kg, intraperitoneally) and subsequently exsanguinated. The trachea together with the main bronchi were rapidly removed, isolated, carefully cleared of adhering periadventitial fat, and placed in a petri dish with a Krebs solution of the following composition (in mM): NaCl (118), KCl (4.7), CaCl 2 (2.5), MgSO4 (1.2), KH2PO4 (1.2), NaHCO3 (25), and glucose (8.3). The solution was maintained at 378C and was aerated continuously by bubbling with a mixture of 95% O 2–5% CO2, which maintained a pH of 7.4. We prepared tracheal and main bronchial segments, three to four cartilage rings wide, that were opened by a longitudinal cut of the anterior surface. These segments were placed in a petri dish containing an oxygenated Krebs solution (10 ml) maintained at 378C. In a separate set of experiments, the epithelial layer of tracheal and main bronchial segments was removed by a cotton swab (20). To verify that the tissues were denuded of epithelium, histologic examinations were performed. The tissues were fixed by immersion in formaldehyde (4%) and embedded in paraffin blocks. Sections measuring 5 mm were cut and stained with hematoxylin and eosin for histologic evaluation. Histologic examination showed that the

Figure 1. Calibration curve used for NO determination by porphyrinic microsensor. This curve was obtained by measuring, by chronoamperometric method, the produced current during time at increasing concentrations of NO standard solutions. C, Coulomb.

Ricciardolo, Vergnani, Wiegand, et al.: Detection of Bradykinin-Induced NO Release in the Airways

epithelial layer was completely removed in the preparations that were treated with the cotton swab, whereas no damage was observed to the lamina propria (data not shown). In Ca21-free experiments, CaCl2 was omitted from the Krebs solution to which ethyleneglycol-bis-(b-aminoethyl ether)-N,N9-tetraacetic acid (1 mM) was added. Isotonicity of the medium was maintained by adding an appropriate concentration of NaCl. Determination of NO Release Using a Porphyrinic Microsensor After a resting period of 30 min, a porphyrinic microsensor was placed in the bathing fluid onto the mucosal surface of the tracheal or main bronchial segment. Vehicle or drug was then added (50 ml) with a Hamilton syringe and the NO release measurement performed. Currents proportional to NO concentration were measured by a chronoamperometric method by using a voltametric analyzer Autolab 20 electrochemical work station (ECHO-Chemie, Utrecht, The Netherlands) and a porphyrinic microsensor (24). A calibration curve (Figure 1) was obtained with NO standard solution (2 nmol ? l21) prepared as described previously (25). Detected NO represented a local concentration that was established on the tissue surface or in close proximity (0.2 to 1 mm). The response time of the microsensor was about 1 ms. Therefore, the sensor could only detect a concentration of NO that was not consumed by the extremely fast intracellular chemical reaction of NO with superoxide anion (26). Experimental Design After a 30-min resting period, concentration-response curves to bradykinin (1 nM to 10 mM) were constructed in either intact or epithelium-denuded tracheal and main bronchial segments in a noncumulative manner. Each curve was obtained by the addition of increasing concentrations of bradykinin (from 1 nM to 10 mM) at 10-min intervals between concentrations. The same procedure was followed for the experiments in Ca21-free conditions. To investigate the role of bradykinin B2 receptor and of NOS, we tested the effect of the bradykinin B2 receptor antagonist HOE 140 (1 mM, for 15 min) or its vehicle (0.9% saline for 15 min) and of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 mM for 30 min) or its inactive enantiomer NG-monomethyl-D-arginine (D-NMMA; 100 mM for 30 min) on NO release induced by bradykinin at the submaximum concentration (1 mM) in either intact or epitheliumdenuded tracheal and main bronchial segments. At least five experiments were performed in each condition. Immunohistochemistry Tissues were fixed by immersion in Zamboni’s solution (2% formaldehyde, 15% saturated picric acid in 0.1 M phospate buffer, pH 7.4) for 6 h at 4 8C. After several washes in 0.1 M phosphate buffer (PB), tissues were stored in PB containing 18% sucrose for cryoprotection and then frozen in liquid nitrogen. Sections of 8 mm were cut on a cryostat (Leica LM 1900; Leica Instruments, Nussloch, Germany) and air-dried for 30 min. Sections were incubated (30 min at room temperature) with a blocking solution containing 10% normal swine serum and 1% bovine

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serum albumin in PB to prevent unspecific protein binding. Two polyclonal antisera from rabbits raised against nNOS (type I, purified from porcine cerebellum) or ecNOS (type III, recombinant protein) were used as primary antisera (overnight at room temperature, dilutions 1:1,000 and 1:400, respectively; both kindly provided by Dr. B. Mayer, Graz, Austria). After washes in 0.1 M PB (three times for 15 min each at room temperature), sections were incubated with a fluoroisothiocyanate-conjugated antirabbit immunoglobulin antiserum from goat (ICN, Aurora, OH; dilution 1:400, 1 h at room temperature). Slides were washed again in 0.1 M PB and coverslipped in carbonate-buffered glycerol. For control of the specificity of the antisera, the antisera were preincubated with the corresponding antigen (concentration 20 mg protein/ml diluted antiserum) and used as the primary antisera as described earlier. As an additional control, the primary antisera were replaced by normal rabbit serum. All pictures of the nNOS and ecNOS incubations were taken at identical exposure times. Microscopic evaluation was performed using a photomicroscope (Olympus BX-50; Olympus, Hamburg, Germany) equipped with an epifluorescence filter module (excitation filter BP 450-490, barrier filter 515-565). Drugs Bradykinin was purchased from Peninsula Laboratories Inc. (Belmont, CA). L-NMMA and D-NMMA were obtained from Sigma Chemical (St. Louis, MO). HOE 140 (27) was kindly provided by Dr. K. J. Wirth (Hoechst AG, Frankfurt, Germany). All drugs were dissolved in 0.9% saline. Stock solution of 10 mM in distilled water of bradykinin and HOE 140 were stored at 2208C until use. L-NMMA and D-NMMA were freshly prepared for each experiment. Statistical Analysis The pharmacologic terminology adopted in this paper follows the recent International Union of Pharmacology recommendation (28). Emax is the maximal effect that an agonist can elicit in a given tissue under particular experimental conditions. Values in the text and figures are the means 6 standard error of the mean (SEM). Statistical comparisons were performed using Student’s t tests for unpaired values or the one-way analysis of variance and Dunnett’s test. In all cases, a P value of less than 0.05 was considered significant. Emax values were calculated via a software package.

Results Bradykinin-Induced NO Release Addition of bradykinin vehicle (0.9% saline, 50 ml) to tracheal and main bronchial segments did not cause any detectable change in baseline signal. Addition of bradykinin caused an upward shift of the baseline, which reached a maximum within 1 to 2 s and then diminished. The response to bradykinin was concentration-dependent between the range of 1 nM to 10 mM (Figures 2 and 3). In intact tracheal segments the threshold concentration for stimulation was 10 nM, whereas in epithelium-denuded tracheal segments it was 100 nM (Figure 2). In intact main bronchial segments the threshold concentration for stimulation was 10 nM, whereas in epithelium-denuded main

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TABLE 1

The effect of bradykinin (10 mM) on NO (fM) release from guinea-pig intact or epithelium-denuded tracheal and main bronchial segments in Ca21-free conditions Baseline Bradykinin

Trachea 1E

Trachea 2E

Bronchi 1E

Bronchi 2E

36 6 11 48 6 14

29 6 9 32 6 8

26 6 8 35 6 9

21 6 7 25 6 6

1E and 2E indicate intact and epithelium-denuded segments, respectively. Each value is the mean 6 SEM of at least five experiments.

Figure 2. The effect of graded concentrations of bradykinin (from 1 nM to 10 mM) on the NO release in intact tracheal segments (open columns) and in epithelium-denuded tracheal segments (filled columns) of guinea pigs. Each column is the mean 6 SEM of at least five experiments. *P , 0.05 versus vehicle; **P , 0.01 versus vehicle. #Significant difference between the two groups stimulated with the maximum concentration of the stimulus (P , 0.05).

bronchial segments it was 1 mM (Figure 3). Maximum response in either intact or epithelium-denuded tracheal and main bronchial segments was obtained with 10 mM bradykinin. Emax values were significantly higher in tracheal segments (intact: 1,848.5 6 323.5 fM, n 5 5; epitheliumdenuded: 946.8 6 157.5 fM, n 5 5) than in bronchial segments (intact: 410.3 6 62.1 fM, n 5 5, P , 0.01; epithelium-denuded: 155.9 6 22.4 fM, n 5 5, P , 0.01). Maximum increase in NO release induced by bradykinin was significantly higher in intact tracheal and main bronchial segments than in epithelium-denuded preparations (Figures 2 and 3).

In Ca21-free conditions, bradykinin (10 mM) failed to increase NO levels in intact and epithelium-denuded segments of guinea-pig trachea and main bronchi (Table 1). The bradykinin B2 receptor antagonist HOE 140 (1 mM for 15 min) or its vehicle (0.9% saline) did not affect the baseline level of NO. In the presence of HOE 140 (1 mM), bradykinin (1 mM)-induced NO release was abolished in all the four different preparations tested (Table 2). In another series of experiments, pretreatment with L-NMMA (100 mM, for 30 min) reduced the baseline NO level in either tracheal (intact: 239 6 7%; epithelium-denuded: 229 6 5%; n 5 10, P , 0.05) or main bronchial segments (intact: 236 6 6%; epithelium-denuded: 227 6 5%; n 5 10, P , 0.05). L-NMMA also blocked the bradykinin-induced increase in NO release in intact and epithelium-denuded segments of trachea and main bronchi (Figure 4). The presence of the inactive enantiomer of L-NMMA, D-NMMA (100 mM, for 30 min), did not affect the action of bradykinin (Figure 4). Immunohistochemical Distribution of nNOS and ecNOS Immunoreactivity (IR) for nNOS (type 1) was seen in nerve fibers innervating the smooth-muscle layer of the trachea (Figures 5a and 5b) and main bronchi. Additional sites of nNOS-IR occurred in extraneuronal structures such as the respiratory epithelium and the airway smooth muscle. In tracheal (Figure 5b) and main bronchial preparations denuded of the epithelium, nNOS-IR was restricted to nerve fibers and to the airway smooth muscle. ecNOS-IR (type 3) was seen in endothelial cells of mucosal blood vessels. In addition, the respiratory epithelium of the trachea (Figure 5c) and the main bronchi showed a strong staining for ecNOS. In epithelium-denuded preparations, ecNOS-IR was found only in endothelial cells of blood vessels of the airway wall (Figure 5d). Preabsorption TABLE 2

NO (fM) levels in the bathing fluid over mucosal surface of guinea-pig intact or epithelium-denuded tracheal and main bronchial segments measured by a porphyrinic microsensor Trachea 1E

Trachea 2E

Bronchi 1E

Bronchi 2E

Vehicle 52 6 31* BK 1 HOE 140 139 6 46* BK 1 Vehicle 1,684 6 262

48 6 28* 121 6 39* 733 6 91

33 6 21* 91 6 35* 342 6 64

30 6 20* 61 6 19* 154 6 31

Treatment

Figure 3. The effect of graded concentrations of bradykinin (from 1 nM to 10 mM) on the NO release in intact main bronchial segments (open columns) and in epithelium-denuted main bronchial segments (filled columns) of guinea pigs. Each column is the mean 6 SEM of at least five experiments. *P , 0.05 versus vehicle; **P , 0.01 versus vehicle. #Significant difference between the two groups stimulated with the maximum concentration of the stimulus (P , 0.05).

1E and 2E indicate intact and epithelium-denuded segments, respectively. Pretreatments, HOE 140 (1 mM) or its vehicle (0.9% saline), were added 15 min before the stimulus (BK: bradykinin, 1 mM). Each value is the mean 6 SEM of at least five experiments. *P , 0.05 versus control (BK 1 Vehicle group).

Ricciardolo, Vergnani, Wiegand, et al.: Detection of Bradykinin-Induced NO Release in the Airways

Figure 4. Effect of incubation with L-NMMA (100 mM for 30 min; hatched columns) or D-NMMA (control: 100 mM for 30 min; open columns) on the maximum increase in NO release induced by bradykinin (1 mM) in tracheal and main bronchial segments of guinea pigs. Filled columns indicate preparations treated with the vehicle of bradykinin (0.9% saline). Each column is the mean 6 SEM of at least five experiments. *P , 0.05 versus control; **P , 0.01 versus control.

of the antisera with the corresponding antigen (Figure 5e) or replacement of the primary antiserum by a normal rabbit serum (Figure 5f) resulted in absence of labeling.

Discussion Bradykinin is a bronchoconstrictor agent in a variety of mammals, including guinea pigs, ferrets, and others (29). Inhalation of bradykinin in humans causes bronchoconstriction only in asthmatic patients (29). Multiple mechanisms are activated by bradykinin to increase bronchial tone. These mechanisms depend upon the experimental conditions and the route of administration of the peptide. In guinea pigs, the animal species most commonly used in airway studies, intravenous administration of bradykinin causes bronchoconstriction by an indomethacin- and atropine-sensitive pathway (30). However, if bradykinin is given by aerosol, bronchoconstriction is substantially mediated by tachykinin release from peripheral endings of airway primary sensory neurons (30, 31). Bradykinin may also activate bronchodilator pathways, including release of relaxant prostaglandins from the airway epithelium (32). More recently, the ability of bradykinin to activate an additional and more potent bronchodilator pathway has been reported in guinea pigs. This mechanism relies on the ability of bradykinin to release NO, inasmuch as NOS inhibitors markedly potentiated bradykinin-induced bronchoconstriction (22). In vivo evidence for the existence of this protective pathway was confirmed by in vitro investigation (23). The relaxant response produced by bradykinin, injected inside isolated tracheal tube preparations, was changed into a contraction after pretreatment with L-NMMA or epithelium removal (23). These findings suggest that bradykinin releases bronchodilator NO from the guinea-pig airway epithelium. A similar bronchodilator mechanism has been shown to be activated by other medi-

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ators, including histamine (20), substance P (33), endothelin (34), and others (35). The present study was undertaken to obtain biochemical evidence that bradykinin causes NO release and that the airway epithelium may be the source of the bradykinin-induced NO release. Results indicate that bradykinin caused an increase in NO concentration, measured by means of a porphyrinic microsensor placed in the vicinity of the mucosal surface of guinea-pig airway segments. This effect of bradykinin was concentration-dependent and was blocked by the bradykinin B2 receptor antagonist HOE 140 and by L-NMMA. Thus, the graded effect of bradykinin appears to be mediated by bradykinin B2 receptors via the activation of the L-Arg-NOS pathway. Functional or morphologic evidence for the presence of B2 receptors has been obtained in a variety of cells of the guinea-pig airways (29, 31). Sensory nerve fibers, smooth-muscle cells, endothelial cells, fibroblasts, and epithelial cells may be included in this list. In addition, our data indicate that although very low levels of NO are released in baseline conditions, L-NMMA inhibited the endogenous production of NO, reflecting a tonic activation of NOS pathway. This study also shows a reduced ability of bradykinin to release NO in bronchial as compared with tracheal segments. This observation suggests a decreasing distribution of bradykinin-stimulated NOS isoform(s) from proximal to distal airways. Epithelium removal markedly decreased bradykininevoked NO release, thus suggesting that most of the NO released by bradykinin originated from epithelial structures. However, although reduced, a significant and concentration-dependent increase in NO level was also detected in epithelium-denuded segments by the porphyrinic microsensor. This observation suggests that bradykinin may release NO, a gaseous molecule well known for its rapid diffusion and ability to permeate cell membranes (36), from subepithelial cells, including nerve fibers, smoothmuscle, and endothelial cells. It must be underlined, however, that airway epithelium remains the main source (about 60%) of the bradykinin-evoked NO release, and that functional studies indicate that only epithelial NO is capable of mediating the bradykinin-induced relaxation in tracheal smooth muscle (23). Regarding the airway cell type(s) responsible for the bradykinin-evoked NO release, the present data do not offer any firm conclusions. In guinea-pig airways, NO has been recognized as the main mediator of the iNANC relaxation (16). There is also evidence that sensory nerves express NOS activity (37). However, a previous study showed that bronchoconstriction induced by bradykinin, after vagotomy, but not by capsaicin is potentiated by the inhibition of the L-Arg-NOS pathway in guinea pigs, suggesting that bronchorelaxant NO does not derive from neural structures (22). Other cells, including gland cells or fibroblasts, might theoretically play a role, although no evidence supports their contribution to bradykinin-induced NO release. Airway mucosa consists mainly of epithelial cells. Epithelial cells express functional bradykinin B 2 receptors whose stimulation results in intracellular Ca21 mobilization (38). Airway epithelial cells may also express cNOS (in the two isoforms of nNOS and ecNOS) as well as iNOS

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Figure 5. Immunohistochemical localization of IR for nNOS (a and b) and ecNOS (c and d) in intact (a and c) and epithelium-denuded (b and d) guinea-pig trachea. (a) nNOS-IR in intact trachea is localized to a nerve fiber (arrow) and in extraneuronal structures such as the respiratory epithelium (ep) and also the airway smooth muscle (asm). (b) In epitheliumdenuded tracheal preparations, the nNOS-IR occurs only in a nerve fiber and in airway smooth muscle. (c) ecNOS-IR is present in the endothelium (e) of a small mucosal blood vessel and in the respiratory epithelium (ep). (d) In epitheliumdenuded trachea, ecNOS-IR is restricted to the endothelium (e). Preabsorption of the antiserum (e) or replacement of the primary antiserum by a normal rabbit serum ( f) results in absence of labeling. Note that all pictures were taken at identical exposure times. Bar 5 20 mm in a–f.

(9). These observations suggest that the epithelial cell is a good candidate as the main source of NO release after exposure of airway segments to bradykinin. Several pieces of evidence suggest that in the present experiments the increase in NO caused by bradykinin is due to cNOS activation. First, NO increase was rapid in onset. cNOS is known to produce rapid (s) increases in NO, as compared with the slowly developing (h or d) augmentation of NO associated with the expression/stimulation of iNOS. Second, exposure of airway segments to bradykinin resulted in increased NO release that was in the fM–pM range. These concentrations are compatible with the amounts of NO usually released by cNOS, and these amounts are much lower than those released by

iNOS. Third, and more importantly, experiments performed in a Ca 21-free medium demonstrated that the presence of extracellullar Ca21 was a prerequisite for the bradykinin-evoked NO release. Because cNOS, but not iNOS, is a Ca21/calmodulin-dependent enzyme we may hypothesize that cNOS mediated bradykinin-induced increase in NO in guinea-pig airway segments in vitro. As an extension of this hypothesis, it is possible that release of bronchorelaxant NO by bradykinin in in vitro (guinea-pig tracheal tube preparations) (23) or in vivo (anesthetized guinea pigs) (22) conditions is also mediated by cNOS stimulation. The immunohistochemical analysis of cNOS performed in the present study showed the presence of both the neu-

Ricciardolo, Vergnani, Wiegand, et al.: Detection of Bradykinin-Induced NO Release in the Airways

ronal and endothelial isoforms in trachea and main bronchi of guinea pigs. Confirming a previous report (4), nNOS was found within nerve fibers. However, positive staining also occurred in extraneuronal structures, including epithelial and smooth-muscle cells of guinea-pig airways. As discussed earlier, functional evidence seems to exclude any involvement of nNOS present in nerve terminals in bradykinin-induced bronchoprotective NO release. An intense staining for ecNOS was also seen in the respiratory epithelium of guinea pigs, as previously observed in rats and human samples (5, 6), as well as in endothelial cells of the airway blood vessels. Thus, the present experiments cannot discriminate as to which isoform is responsible for the bradykinin-evoked NO release in guinea-pig airways, and theoretically both nNOS and ecNOS may be involved. Most immunoreactivity for ecNOS and nNOS was confined to the epithelial layer. However, the presence of subepithelial ecNOS and nNOS justifies the ability of bradykinin to release NO in epithelium-denuded preparations. The relevance of the present findings is not confined to the guinea-pig model. In mild asthmatics PD20FEV1 to inhaled bradykinin was decreased by 3.2 doubling doses after L-NMMA pretreatment (39). In severe asthmatics, who showed in control conditions a remarkable hyperresponsiveness to bradykinin, L-NMMA was unable to decrease further PD 20FEV1 to inhaled bradykinin (40). These findings led to the proposal that in human subjects, as already observed in guinea pigs, bronchoconstriction by bradykinin is reduced by the ability of bradykinin to release bronchorelaxant NO, and that this NO release is reduced or abolished in severe asthma. Respiratory viral infection in guinea pigs decreased the ability to activate the release of bronchorelaxant NO (41). Thus, it is tempting to speculate that bronchoprotection produced by bradykinin via Ca21-dependent NO release is reduced or lost in virusinfected guinea pigs as well as in patients with severe asthma. There is compelling evidence that in asthma, and particularly in severe asthma, airway epithelium progressively changes to a proinflammatory phenotype characterized by inflammatory cell infiltration, epithelial cell shedding and damage, and expression of proinflammatory proteins, including cytokines, chemokines, enzymes, and others. iNOS has been found to be upregulated in the epithelium of asthmatic patients (13) and this upregulation seems to contribute to the elevated NO levels measured in the exhaled air of asthmatics. However, these increased NO levels do not result in increased bronchodilatation but rather are considered a marker of inflammation and to correlate with the severity of the disease (13). The multiple biologic functions of NO span from bronchoprotection to inflammation. At present it is not known whether different chemical species related to NO are involved in these diverse functions, and the precise role of the various cells of the airway tissue in the release of protective and detrimental NO has not been defined. However, there is evidence that nitrosothiols, NO-adduct compounds, could be responsible for bronchodilatation in asthmatic children (42), whereas increased formation of peroxynitrite, a potent oxidant formed by the rapid reaction of the free radical NO with superoxide anions (13), is associated with induction of iNOS (13). The

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present experiments do not distinguish between these different chemical species, and consequently they cannot discriminate between a NO-related chemical species that causes bronchodilatation and the detrimental one. Indirect pharmacologic evidence indicates the airway epithelium as the source of the bradykinin-evoked release of bronchorelaxant NO (23). Present data offer biochemical evidence that favors this view and point toward the Ca21-dependent cNOS activation as the mechanism of bronchoprotective NO release in guinea pigs. Further studies may clarify whether Ca21-dependent cNOS activation exists in human airway epithelium and whether this pathway is downregulated in asthma as the Ca21-independent iNOS pathway appears to be upregulated. Acknowledgments: This work was supported by a grant from Azienda Ospedale S. Anna, Ferrara, Italy.

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