INTRODUCTION

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Nitric oxide (NO) is synthetized by a family of NO synthases (NOS) that are expressed by different cell types in the lung and the airways, including bronchial epithelial cells (1) and airway smooth muscle cells (2). This endogenously generated NO, which is the product of the constitutive NOS isoforms (cNOS), type 1 NOS (NOS1), or neural NOS (nNOS), and type 3 NOS (NOS3), or endothelial NOS (eNOS), is an important modulator of airway responses to exogenous contractile agents, such as histamine, and to vagal stimulation (3). Among the two cNOS isoforms, NOS1 is the main contributor to this modulatory activity on airway tone (7, 8). The NO pathway may also play a role in the onset of airway inflammation, particularly via the expression of the inducible NOS (NOS2, 9).

Bronchial hyperreactivity (BHR) and airway inflammation are characteristic features of asthma (10). Substantial evidence supports the concept that both phenomena are related (11). Experimental studies, using pharmacological tools, have demonstrated the contribution of endogenous pulmonary NO to the development of BHR measured both in vitro (12) and in vivo (3). Clinical relevance has been attributed to these observations, as a deficiency in cNOS-derived NO was recently found to contribute to BHR in patients with severe asthma (13). However, none of these studies has clearly established which of the two cNOS isoforms, NOS1 or NOS3, mainly contributes to the decreased production of endogenous NO.

Therefore, the aim of the present study was to evaluate NOS1, NOS3, and also NOS2 protein expression and activity in lung homogenates and in the tracheal smooth muscle collected from OVA-immunized and multiple challenged guinea pigs sacrificed 6 and 24 h after the last challenge. This model reproduces several of the characteristic features of asthma, including airways infiltration by inflammatory cells, particularly eosinophils, and BHR to intravenously injected histamine (14). Exhaled NO levels, a reliable index for monitoring pulmonary NO production (15), were also measured in the different groups of animals in order to characterize fully the pattern of NO generation in the airways. Finally, the time course of in vivo bronchoconstrictor response to intravenous histamine was evaluated in order to correlate changes in NOS expression and function with airway hyperresponsiveness in the present experimental model.

	METHODS

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Guinea Pig Immunization and Challenge

Pathogen-free male Hartley guinea pigs (250-300 g body weight; Charles River, France) were housed in individual cages in climate-controlled animal quarters and were given water and food ad libitum. The experiments conducted in the present study were approved by the local Institutional Animal Care and Use Committee and the experimental protocol was in agreement with the recommendations related to animal studies of the French Law (Ministère des Affaires Sociales et de la Solidarité Nationale, Paris, France). The animals were immunized with 0.5 ml of 0.9% wt/vol NaCl (saline) containing 100 mg ovalbumin (OVA) (Sigma Immunochemical, St. Quentin, France), injected subcutaneously on the neck, and another 0.5 ml intraperitoneally on Day 1. On Days 8, 9, 10, 12, 13, and 14, the animals were challenged in a 5-L plastic chamber by exposure to aerosolized OVA (OVA animals). The OVA solution (0.1% OVA in 10 ml saline) was delivered using a Devilbiss nebulizer (Sunrise, Devilbiss Medical, Nantes, France) for 10 min. The time of exposure was determined by the appearance of respiratory distress signs (polypnea, bronchospasm, contraction of accessory respiratory muscles, and cyanose). In case these reactions were absent, the dose of OVA was increased to 0.2% or 0.3%, leading to the development of respiratory distress in all animals. Another group of animals (OVAi animals) were immunized to OVA as described above and exposed to aerosolized saline. Control guinea pigs (C animals) received saline intraperitoneally and they were subsequently exposed to aerosolized saline for 10 min on Days 8, 9, 10, 12, 13, and 14.

Animals of different groups were sacrificed 6 h (groups C-6, OVA-6, and OVAi-6, respectively) and 24 h after the last challenge (groups C-24, OVA-24 and OVAi-24, respectively). At these time points they were anesthetized with sodium pentobarbital (50 mg/kg of body weight intraperitoneally), the thoracic and abdominal cavities were opened immediately, and the animals were exsanguinated via the abdominal aorta. Lung samples from two different regions were dissected: one region encompassing 0.5 cm around the hilium, and the other including the terminal 1 cm extremity of the lower region of the lungs (proximal and distal specimens, respectively). Proximal and distal samples reflected predominantly bronchial and parenchymal tissue, respectively. Samples were stored at 80° C until use. Tracheas were also rapidly excised and placed in a Krebs-Henseleit solution. Tracheal smooth muscle (TSM) was dissected under a binocular dissecting microscope and cleaned of fat, blood, and connective tissue. Epithelial layer was left intact. Samples were then immediately frozen in liquid nitrogen and stored at 80° C until use. Each experimental group included 8-15 animals.

In a separate subset of animals in which lung and tracheal tissue were not sampled, exhaled NO was quantified 6 and 24 h after the last challenge (n = 8 in each group), as described below.

In another subset of animals (n = 6-8 in each group), airway contractile responses were evaluated by measuring changes of pulmonary inflation pressure after intravenous administration of increasing doses of histamine in anesthetized, mechanically ventilated guinea pigs (see below).

Biochemical Assays

Frozen lung samples from each animal were homogenized at 4° C using an Ultraturrax T25 (Janke and Kunkel, IKA Works, Cincinnati, OH) in six volumes (wt/vol) of homogenization buffer (pH 7.4) composed of 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml phenylmethylsulfonyl fluoride, 0.32 mM sucrose, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A. Frozen TSM samples were homogenized at 4° C using a Potter homogenizer (Stir-park Laboratory Mixer; Colle Parmer Instrument, Niles, IL) in six volumes (wt/vol) of the same homogenization buffer than that used for lung samples. In both cases, the crude homogenates were centrifuged at 4° C for 15 min at 10,000 rpm. The supernatant was used for the different biochemical assays.

Protein concentration was measured spectrophotometrically in 96-well plates using the Bio-Rad protein reagent (Bio-Rad Laboratories, Richmond, CA), with bovine serum albumin as standard.

Western blot analysis. Western blot experiments were performed as described previously (16). Proteins (50 and 150 µg per lane for the lung and for the TSM homogenates, respectively) in the tissue homogenates were denatured by boiling for 5 min in sample buffer (0.5 M Tris-HCl, pH 6.8, 10% [wt/vol] sodium dodecyl sulfate [SDS], 10% [vol/vol] glycerol, 5% [vol/vol] 2-mercaptoethanol, and 0.05% [wt/vol] bromophenol blue) and separated by electrophoresis on precasted 7.5% SDS-polyalcrylamide gel (Bio-Rad). They were transferred overnight at 4° C to polyvinylidene difluoride membranes (Bio-Rad) in 25 mM CAPSO buffer (pH 10) and 20% methanol. Subsequent steps were performed at room temperature. The membranes were then blocked with 10% nonfat dry milk in Tris-buffered saline (25 mM Tris, pH 7.5, 150 mM NaCl) + 0.05% (vol/vol) Tween 20 (TBST solution). After washing in TBST, the membranes were incubated 1 h with an anti-NOS1 or NOS3 monoclonal antibody used at a 1:1000 dilution, or a monoclonal anti-NOS2 antibody, used at a 1:500 dilution, all from Transduction Laboratories (Lexington, KY) These antibodies were generated against amino acids 195-1289, 961-1144, and 1030-1209 of human NOS1, NOS2, and NOS3 proteins, respectively (catalog numbers N31020, N30020, and N32020, respectively); they have been used previously for detection of NOS isoforms in guinea pigs (17). Different positive controls were used: lysates of guinea pig cerebellum and human pituitary cells for NOS1, lysates of guinea pig aorta and human endothelial cells for NOS3, and lysates of liver from lipopolysaccharide (LPS)-inoculated guinea pigs (10 mg/kg intraperitoneally) and cytokine-activated murine macrophages for NOS2. The lysates of human pituitary cells, human endothelial cells, and cytokine-activated murine macrophages were from Transduction Laboratories. After washing, the membranes were incubated for 1 h with a 1:3000 dilution of a goat anti-mouse immunoglobulin G (IgG) conjugated to alkaline phosphatase (Bio-Rad). The blots were washed with TBST, followed by detection of immunoreactive proteins by the enhanced chemiluminescence method using AMMPD (Tropix, Inc.) as a substrate (Bio-Rad).

The membranes were also immunoblotted with a monoclonal anti--actin antibody (Sigma Immunochemical, St. Quentin, France) in order to verify the amounts of loaded proteins.

Quantification of Western-Blot staining was performed using a Perfect Image 2.01 image analysis system (ICONIX, Courtaboeuf, France). Optical density (OD) was expressed in arbitrary units. The OD for each NOS isoform was normalized to the OD of -actin measured in the same lane.

Nitric oxide synthase activity measurement. Nitric oxide synthase activity was measured by the conversion of L-[³H]arginine to L-[³H]citrulline according to the method described by Bredt and Snyder (18). The supernatant of tissue homogenate (50 µl) was added to 10-ml prewarmed (37° C) tubes containing 100 µl of reaction buffer of the following composition: 50 mM KH₂PO₄, 60 mM valine, 1.5 mM NADPH, 10 mM FAD, 1.2 mM MgCl₂, 2 mM CaCl₂, 1 mg/ml bovine serum albumin, 1 µg/ml calmodulin, 10 µM tetrahydrobiopterin, and 25 µl of 120 µM stock L-[2,3-³H]arginine (150-200 cpm/pmol). The samples were incubated for 30 min at 37° C, and the reaction was terminated by the addition of 500 µl of cold (4° C) stop buffer (pH 5.5, 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 12 mM EDTA). To obtain free L-[³H]citrulline for the determination of enzyme activity, 2 ml of Dowex 50w resin (8% cross-linked, Na⁺ form, Bio-Rad) was added to eliminate excess L-[2,3-³H]arginine. The supernatant was removed and examined for the presence of L-[³H]citrulline by liquid scintillation counting. Enzyme activity was expressed in picomoles of L-citrulline produced per minute per milligram of total protein. To differentiate between NOS2 activity, which is independent of Ca²⁺ and calmodulin (19), and constitutive NOS isoform activity (Ca²⁺ and calmodulin dependent), NOS activity was also measured in the presence of 1.5 mM each ethylene glycol-bis(aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and EDTA, which replaced CaCl₂ and calmodulin in the reaction buffer, and in the presence of 1 mM N^G-nitro-L-arginine methyl esther (NOS inhibitor). Ca²⁺/calmodulin-dependent NOS activity was calculated as the difference between activity measured in the presence of CaCl₂ and that measured in EDTA/EGTA buffer. Ca²⁺/calmodulin-independent NOS activity was calculated as the difference between samples assayed in the presence of EGTA/ EDTA and in the presence of N^G-nitro-L-arginine methyl ester.

Nitrite and nitrate (NOx) assay. NO_x measurement was performed according to the method described by Giovannoni and coworkers (20). The nitrate present in the sample was stoichiometrically reduced by incubating for 15 min at 37° C in the presence of reduced nicotinamide adenine dinucleotide and the enzyme nitrate reductase. After incubation at room temperature for 15 min, total nitrite formed reacts with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride to give a red-violet diazo dye that is measured on the basis of its absorbance at 550 nm. The nitrite concentration was determined from a linear standard curve between 6.25 and 150 µM potassium nitrate. Assays were performed in quadruplicate.

Concentration of NO in the Exhaled Air

NO was measured on a chemiluminescent analyzer (NOA 280; Sievers Instruments, Boulder, CO) with a detection threshold of 1 part per billion (ppb) (21, 22). Quantification of NO is based on the gas-phase interaction between NO and ozone (O₃) that forms nitrogen dioxide (NO₂) and oxygen (O₂): NO + O₃  NO₂* + O₂. Some of the NO₂ produced by this reaction is in the excited state (NO₂*) and emits light as electrons return to ground state: NO₂* + O₂  NO₂ + light. Light is measured by a cooled, photomultiplier tube that gives the signal for the presence of NO (23).

Before each study, the analyzer was calibrated using NO-free air and a certified gas cylinder of 107 ppb of NO. The animals were placed in a body box tailored made to keep them still during the measurement. Inspired air was delivered from a tank containing 21% O₂ balanced in 79% N₂ and freed of NO. This normoxic, NO-free air was continuously gassed through the box in order to keep ambient NO below 1 ppb. The animals spontaneously breathed, with their muzzle directly applied in a small sealed aperture from which exhaled air was sampled and NO concentration measured. NO was measured in the exaled air during a continuous period of 1 min and the average value was obtained. Three different measurements separated by a 3-min period were performed. The mean value of these three measures was calculated and used as the value of exhaled NO for each animal.

Measurement of Pulmonary Inflation Pressure

Guinea pigs were anesthetized by an intraperitoneal injection of 75 mg/kg of sodium pentobarbitone and were placed in a supine position. A catheter was then placed into a jugular vein for drugs administration. The body temperature of the animal was maintained at 37° C using a heating blanket.

The animals were paralyzed with succinylcholine (infused at 10 µg/ kg/min), tracheostomized, and ventilated using a positive-pressure, constant-volume animal ventilator (Harvard Apparatus; South Natick, MA; tidal volume 2.5-3.5 ml, 100 breaths/min). Pulmonary inflation pressure (Ppi) was measured with a Grass FT 03 force transducer (Grass Instruments, Quincy, MA). All signals were displayed on a Grass polygraph.

A positive pressure of 35-50 mm H₂O was needed for adequate ventilation of the animals. Given constant flow and volume, bronchoconstriction was measured as the increase in Ppi over the baseline inflation pressure, as previously described by Jacoby and coworkers (24). Histamine (1-30 µg/kg) was injected intravenously at 10-min intervals between each dose, and the increase in Ppi was recorded.

Statistical Analysis

Values are given as mean ± SEM. Dose-response curves of histamine-induced bronchoconstriction in the different groups of animals were compared using two-way analysis of variance (ANOVA) for repeated measures. The other data were analyzed by one-way ANOVA; differences between means were analyzed with the Duncan new multiple range. Significance for all statistics was accepted at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Data obtained in groups C-6 and C-24 were similar. Therefore, they were combined and presented as group C.

Pulmonary NOS Expression and Activity

Distal lung homogenates from C animals expressed NOS1 protein, as assessed by Western blot, with a molecular weight identical to the NOS1 protein detected in a lysate of human pituitary cells and guinea pig cerebellum (Figure 1A). Expression of NOS1 decreased 6 h after OVA challenge and returned to basal levels at 24 h. Accordingly, densitometric analysis showed a significant reduction in the NOS1 -actin ratio in group OVA-6, as compared with group C (p < 0.05), whereas no significant difference was observed between OVA-24 and C animals (Figure 1B). No changes in NOS1 expression were observed in OVAi as compared with C animals.

View larger version (34K): [in this window] [in a new window]   Figure 1.   (A) Representative Western blot analysis of NOS1 and -actin expression in "proximal" and "distal" (P and D, respectively) lung homogenates. C, control animals; OVA-6, animals immunized and multiple aerosol challenged with OVA and sacrificed 6 h after the last challenge; OVA-24, same as OVA-6, except that the animals were sacrificed at 24 h; OVAi-6, same as OVA-6 except that the animals were challenged with saline; OVAi-24, same as OVA-24, except that the animals were challenged with saline. H+, lysate of human pituitary cells; GP+, lysate of guinea pig cerebellum. (B and C ) Densitometric analysis of NOS1 and -actin bands. Abbreviations are the same as those in A. Each bar represents the mean ± SEM of each group. *p < 0.05 versus group C.

Distal lung homogenates from C animals expressed NOS3 protein, as assessed by Western blot, with a molecular weight identical to the NOS3 protein detected in a lysate of human endothelial cells and guinea pig aorta (Figure 2A). NOS3 protein expression was unmodified both in OVA and OVAi animals, either at 6 or 24 h after challenge (Figure 2A and 2B).

View larger version (37K): [in this window] [in a new window]   Figure 2.   (A) Representative Western blot analysis of NOS3 and -actin expression in lung homogenates. H+ lysate of human endothelial cells; GP+, lysate of guinea pig aorta. Other abbreviations are the same as those in Figure 1A. (B and C ) Densitometric analysis of NOS1 and -actin bands. Abbreviations are the same as those in Figure 1A. Each bar represents the mean ± SEM of each group.

Constitutive NOS activity, measured by the conversion of L-[³H]arginine to L-[³H]citrulline, paralleled changes in NOS1 protein expression. Indeed, L-[³H]citrulline formation from L-[³H]arginine was significantly decreased in group OVA-6, as compared with group C (p < 0.05), whereas no significant modification was observed in group OVA-24 (Figure 3A). No difference in NOS activity was observed between OVAi and C animals (Figure 3A).

View larger version (42K): [in this window] [in a new window]   Figure 3.   (A) Constitutive NOS (cNOS) activity in the lungs of the different groups of animals. cNOS activity was expressed as nanomoles of L-[3H]citrulline produced by milligram of protein and by minute of incubation. Abbreviations are the same as those in Figure 1A. Each bar represents the mean ± SEM of each group. *p < 0.05 versus group C. (B) Nitrite and nitrate (NOx) concentration in the lungs of the different groups of animals. NOx concentration was expressed as micromolar by microgram of protein. Abbreviations are the same as those in Figure 1A. Each bar represents the mean ± SEM of each group. *p < 0.05 versus group C.

Similar to NOS activity, NO_x concentrations in distal lung samples were significantly lower in the OVA-6 group than in group C (p < 0.05) and were not different between OVA-24 and C groups (Figure 3B).

NOS2 protein was detected in a lysate of cytokine-activated murine macrophages and of liver from LPS-inoculated guinea pigs (Figure 4). In contrast, no NOS2 protein and activity was detected in any of the groups of animals evaluated in the present study (data not shown). Comparable results concerning NOS1, NOS2, and NOS3 protein expression, NOS activity, and NO_x levels were obtained in proximal lung samples (Figures 1, 2, and 3).

View larger version (26K): [in this window] [in a new window]   Figure 4.   Representative Western blot analysis showing positive controls of NOS2 expression. H+, lysate of cytokine-activated murine macrophages for NOS2; GP+, lysate of liver from LPS-inoculated guinea pigs.

NOS Expression and Activity in Tracheal Smooth Muscle

NOS1 protein was expressed in the tracheal smooth muscle from C animals (Figure 5A). As observed in lung samples, this expression was significantly reduced in the OVA-6 group (p < 0.05 versus group C), and was unmodified in OVA-24, as compared with the C group. Accordingly, constitutive NOS activity was significantly decreased in OVA-6, as compared with the C group (Figure 5B). NOS3 and NOS2 proteins and activities were undetectable in the tracheal smooth muscle sample of any animal, irrespective for their treatment (saline or OVA) or time point after challenge.

View larger version (30K): [in this window] [in a new window]   Figure 5.   (A) Representative Western blot analysis of NOS1 and -actin expression in the tracheal smooth muscle. Abbreviations are the same as those in Figure 1A. (B) Densitometric analysis of NOS1 and -actin bands. Abbreviations are the same as those in Figure 1A. Each bar represents the mean ± SEM of each group. *p < 0.05 versus group C. (C ) Constitutive NOS (cNOS) activity in the tracheal smooth muscle. cNOS activity was expressed as nanomoles of L-[3H]citrulline produced by milligram of protein and by minute of incubation. Abbreviations are the same as those in Figure 1A. Each bar represents the mean ± SEM of each group. *p < 0.05 versus group C.

Exhaled NO Levels

Exhaled NO levels measured in conscious, not tracheotomized, guinea pigs were significantly lower in OVA-6, as compared with the C group (3.53 ± 0.31 versus 2.52 ± 0.25 ppb, n = 8, p < 0.05), but were not statistically different from values of the OVAi-6 group (3.86 ± 0.40 ppb, n = 8). Values of the OVA-24 group (3.78 ± 0.42 ppb) were not different from values of the C group and of the OVA-i 24 group (3.60 ± 0.32 ppb).

Pulmonary Inflation Pressure

Intravenous injection of increasing doses of histamine caused a dose-dependent bronchoconstriction in C animals (Figure 6; p < 0.0005). A similar degree of bronchoconstriction was observed in the OVA-i 6 h and OVA-i 24 h groups (data not shown). Histamine-induced bronchoconstriction was potentiated in both OVA-6 and OVA-24 animals, with respect to C animals (Figure 6; p < 0.01, respectively). Furthermore, the degree of bronchoconstriction was 25-60% higher in OVA-6 animals as compared with OVA-24 animals (p < 0.05).

View larger version (10K): [in this window] [in a new window]   Figure 6.   Dose-response increase in pulmonary inflation pressure (Ppi), expressed in cm H2O, in response to intravenous injection of increasing doses (1-30 µg/kg) of histamine. Abbreviations are the same as those in Figure 1A. Each point represents the mean ± SEM of each group. Intravenous injection of histamine caused a dose-dependent increase in Ppi in C animals (p < 0.0005). This increase in Ppi was potentiated in both OVA-6 and OVA-24 animals with respect to C animals (p < 0.01, respectively). Furthermore, the increase in Ppi was 25-60% higher in OVA-6 animals as compared with OVA-24 animals (*p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study investigated NOS expression and activity in the respiratory tract of OVA-immunized and multiple aerosol-challenged guinea pigs. The main results are that NOS1 protein expression decreased significantly in lung homogenates 6 h after the last challenge and returned to basal levels at 24 h, whereas NOS3 expression was unmodified, and NOS2 protein was undetectable at both time points. Decreased NOS1 expression was associated with a significant reduction in NOS activity, evaluated directly by the conversion of L-[³H]arginine to L-[³H]citrulline and, indirectly, by the measurement of NO_x concentration in lungs. This reduction in NOS1 expression and activity in the lung was accompanied by a significant decrease in the amounts of exhaled NO. Furthermore, NOS1 protein was expressed in tracheal smooth muscle of control animals and changed in parallel with pulmonary NOS1 in response to antigen stimulation. BHR was observed at both 6 h and 24 h after the last challenge, being however more important at 6 h than at 24 h. Collectively, these results demonstrate for the first time that OVA challenge in immunized guinea pigs induced a transient reduction in NOS1 protein expression and activity in the respiratory system, which parallels early BHR. Interestingly, antigen immunization alone, a condition that has been shown to induce phenotypical modifications in tracheal smooth muscle (25), produced no modification in NOS1 expression and activity.

The decrease in NOS activity was associated with an attenuation in NOS1, but not NOS3, protein expression, and with a reduction in exhaled NO. These findings suggest that the reduction in NOS activity presently reported was related to a decrease in the activity of NOS1. The contribution of NOS1 to expired NO gas is in agreement with results in mice harboring a homozygous targeted disruption of the NOS1 gene, showing that NOS1 is responsible for approximately 40% of the NO measured in mixed expired air (7). The decrease in constitutive NO production in OVA-challenged guinea pigs could have been compensated or even overwhelmed by the induction of NOS2. However, we were unable to detect any NOS2 protein expression and activity at the two time points evaluated (6 h and 24 h after the last OVA challenge). This finding is in agreement with data in studies of guinea pigs (9) but is in contrast with results obtained in rats (26, 27), showing NOS2 expression 4-8 h after a single OVA challenge. The absence of NOS2 activity at 24 h presently described is also in contrast with previous findings demonstrating the ability of the NOS2 inhibitor aminoguanidine to interfere with changes in airway tone at the same time point in OVA-immunized and single-challenged guinea pigs (9). These discrepancies could be related to species differences (rats versus guinea pigs) in pulmonary NOS2 characteristics (28), or to a lack of cross-reactivity of the anti-human NOS2 antibody with the guinea pig isozyme. However, this latter possibility is unlikely as we demonstrated the ability of this same antibody to react with NOS2 protein in the liver from LPS-inoculated guinea pigs (Figure 4). Alternatively, different inflammatory mechanisms involved in single as opposed to multiple OVA challenges could explain the aforementioned discrepancies. Indeed, preliminary experiments in our model based on multiple OVA challenges showed a marked eosinophilia in bronchoalveolar lavage fluid both at 6 h and 24 h after the last allergen challenge (the proportions of eosinophils being 45% and 60% of the total cell counts at each time point, respectively) with a small neutrophilia (6% of total cell counts) only at 6 h. In contrast, the model of a single-challenged guinea pig was characterized by a 3-fold increase in the number of neutrophils at 24 h (9). This different cellularity may reflect differences in the inflammatory pathways in the lung, and thus may explain the discrepancies concerning NOS2 induction. Whatever the reason(s) of these differences, the parallelism between the results of the L-[³H]citrulline assay and the measurements of NO_x concentration in the lungs and NO levels in exhaled air, further confirms the absence of a significant NOS2 induction in this model.

NOS1 protein expression and activity were similar in both proximal and distal lung samples (representing bronchi and parenchymal lung tissue, respectively). This is in agreement with previous data obtained by Yan and coworkers (29) in guinea pig bronchi and lung preparations. Indeed, NOS1 is expressed by different cell types distributed both in the airways and lungs, such as nonadrenergic noncholinergic nerves, airways smooth muscle (2), and epithelial cells (1). The transient decrease in NOS1 expression and activity in OVA-immunized and challenged animals was detected in both proximal and distal lung samples as well as in the tracheal smooth muscle. This similarity makes unlikely a pathogenic role of certain types of cells infiltrating either the bronchi or lung parenchyma, such as the eosinophils. Furthermore, the transitory character of the decreased NOS1 expression supports a role of cells recruited and/or activated during the early response to antigen challenge. However, the nature of the cells and/or mediators involved in the genesis of the present findings remains to be established.

Whatever the mechanism(s) involved in the early and reversible decrease in NOS1 expression and activity, this phenomenon may explain the transient deficiency in endogenous pulmonary NO previously demonstrated after allergen challenge in immunized guinea pigs (3, 12) and rats (30). Furthermore, the present results are in line with those reported in a model of viral infection-induced airway inflammation in guinea pigs, showing a decrease in endogenous NO production (31). Because this deficiency in endogenous NO contributed to the development of BHR, a role for decreased NOS1 protein expression and activity in airway hyperresponsiveness appears very likely. This conclusion is supported by the temporal association between the decrease in NOS1 expression and the maximal BHR to histamine observed in the present experimental model. Indeed, as shown previously by Schuiling and coworkers (3), BHR was 25-60% more important 6 h than 24 h after the last antigen challenge, the time point when NOS1 expression was decreased. These results are in agreement with a recent study from Kakuyama and coworkers (8) showing that NOS1-derived NO attenuated cholinergic bronchiolar contraction in mice, by a mechanism dependent upon NO interaction with prostaglandins. Interestingly, NOS3-deficient mice failed to exhibit abnormal cholinergic responses demonstrating a unique role of NOS1 in modulating airways reactivity (8). It should be noted, however, that BHR is a multifactorial and complex process that is not exclusively dependent on NOS1, as shown by the persistence of some degree of hyperresponsiveness to histamine 24 h after the last OVA challenge, a time point in which NOS1 expression and activity returned to basal levels.

We conclude that a deficiency in NOS1 expression and activity in the respiratory system accompanies bronchopulmonary alterations after antigen challenge in immunized guinea pigs. Interestingly, recent studies have established a linkage of the diagnosis of asthma to a region that maps near the human NOS1 gene (32), stressing, therefore, the implications of NOS1 in the pathophysiology of asthma.