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Published ahead of print on November 3, 2003, doi:10.1164/rccm.200211-1372OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 227-234, (2004)
© 2004 American Thoracic Society

Effect of Phosphodiesterase-5 Inhibitor, Sildenafil (Viagra), in Animal Models of Airways Disease

Toby J. Toward, Nicola Smith and Kenneth J. Broadley

Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom

Correspondence and requests for reprints should be addressed to Kenneth J. Broadley, D.Sc., Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cathays Park, Cardiff CF10 3XF, UK. E-mail: BroadleyKJ{at}Cardiff.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphodiesterase (PDE)-5 degrades guanosine 3',5'cyclic monophosphate (cGMP) and its inhibitor sildenafil citrate (Viagra) treats erectile dysfunction by smooth muscle relaxation through elevated cGMP. Sildenafil was examined in two guinea pig models of airways disease: guinea pigs exposed to LPS or sensitized guinea pigs with atopy exposed to ovalbumen. Ovalbumen exposure caused early- and late-phase bronchoconstrictor responses, measured in conscious animals by whole-body plethysmography. Twenty-four hours after ovalbumen exposure there was airway hyperreactivity (AHR) to inhaled histamine and significantly elevated macrophages, eosinophils, and nitric oxide (NO) metabolites in bronchoalveolar lavage fluid. Sildenafil treatment (1 mg/kg, intraperitoneally) failed to affect the early and late responses but significantly reduced AHR, leukocyte influx, and elevated NO. LPS exposure (30 µg/ml) caused AHR to histamine at 1 hour and macrophage, eosinophil, and neutrophil influx at 24 hours with raised NO. Sildenafil pretreatment inhibited LPS-induced AHR, leukocyte influx, and NO generation. The effectiveness of sildenafil was not dependent on endogenous NO because inhibition of NO synthase with N{omega}-nitro-L-arginine methyl ester did not prevent its action. Inhibition of PDE5 by sildenafil was confirmed by elevated S-nitroso-N-acetylpenicillamine–induced cGMP generation in isolated lungs. These antiinflammatory actions of sildenafil in guinea pig models suggest that PDE5 inhibitors may have potential in treating airways disease.

Key Words: airway hyperreactivity • asthma • inflammation • LPS • phosphodiesterase-5 inhibitor • Viagra

The phosphodiesterase (PDE)-5 inhibitor, sildenafil citrate (Viagra), provides well tolerated pharmacotherapy for erectile dysfunction (1). PDE5 is responsible for guanosine 3',5' cyclic monophosphate (cGMP) degradation (2). Nitric oxide (NO), derived from nitric oxide synthase (NOS), stimulates soluble guanylate cyclase to generate intracellular cGMP (3). Elevated levels of cGMP, after PDE5 inhibition or NO stimulation, cause protein kinase G–dependent smooth muscle relaxation (3, 4). The consequent vasodilator activity is the basis of improved erectile function (1).

Features of asthma and chronic obstructive pulmonary disease (COPD) include bronchoconstriction, airway hyperreactivity (AHR), increased exhaled NO and pulmonary inflammation, characterized by eosinophil and neutrophil influx, respectively (5–7). Elevated intracellular levels of adenosine 3',5' cyclic monophosphate (cAMP) produced by inhibition of cAMP degradation via PDE4 or by ß2-adrenoceptor–mediated stimulation of adenylyl cyclase, causes protein kinase C– and protein kinase G–dependent bronchodilation (8) and suppression of inflammatory cell activity (9–12). Consequently, elevating cAMP levels with PDE4 inhibitors such as rolipram have been shown to reduce the airway inflammation, AHR, and early- and late-asthmatic responses induced by allergen challenge in animal models (9, 13–16). They have also been shown to alleviate some of the symptoms associated with asthma (17) and COPD (18), and this is the basis of their potential use in treating severe asthma and COPD (18, 19).

In primary airway epithelial cells, PDE4 and PDE5 predominate, hydrolyzing 60 to 75% and 80% of the total cAMP and cGMP, respectively (20). Cross talk exists between cAMP and cGMP because cAMP also inhibits PDE5, thereby allowing cGMP levels to rise and both cAMP (8) and cGMP to activate protein kinase G (4). Further similarities between the inhibitory activities of cAMP and cGMP on microvascular leakage (8, 13, 19) and inflammatory cell activity (2, 19, 21), raise the possibility of novel drug targets for asthma and COPD treatment via cGMP regulation.

Sildenafil selectively inhibits PDE5 with 80- to 8,500-fold greater affinity than PDE1 to PDE4 isozymes and with 240-fold greater potency than the earlier generation PDE5 inhibitor, zaprinast (22). The first aim of this study was to examine the effects of sildenafil in two well characterized conscious guinea pig models, whereby exposure to LPS induces neutrophil-dominated inflammation and AHR similar to COPD (10, 12, 23) or exposure of guinea pigs with atopy to allergen causes features of asthma (9, 24). In these models of inflammation, whole-body plethysmography was used to monitor airway function, AHR was assessed to inhaled histamine, and bronchoalveolar lavage fluid (BALF) was removed to determine levels of leukocyte infiltration and NO (metabolites) production in the airways. The second aim of this study, because the effectiveness of sildenafil to reverse erectile dysfunction is NO-dependent, was to evaluate whether any beneficial effects of sildenafil in these models were NO-dependent. Using the nonselective NOS-inhibitor, N{omega}-nitro-L-arginine methyl ester (L-NAME), to inhibit NO synthesis, the beneficial effects of sildenafil treatment on the lung after exposure to LPS were evaluated in the absence of airways NO (23). In this study, it is reported for the first time that the PDE5 inhibitor, sildenafil (Viagra), inhibits inflammation and airways reactivity in animal models of airways diseases and therefore may have therapeutic potential in the treatment of asthma and COPD.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
(Full details are available in the online supplement.)

Airway function (specific airways conductance: sGaw) was monitored in conscious Dunkin–Hartley guinea pigs (male, 300–400 g), using wholebody plethysmography (25) to measure airflow across a pneumotachograph and box pressure with a Biopac data acquisition system and AcqKnowledge software (Biopac Systems Inc., Santa Barbara, CA).

Guinea pigs actively sensitized with ovalbumin (OA, intraperitoneal, 10 µg) and aluminum hydroxide (100 mg) received nebulized OA (100 µg/ml, 1 hour) or pathogen-free saline 14 days later. Nonsensitized guinea pigs were exposed to nebulized LPS (30 µg/ml, 1 hour) or pathogen-free saline. sGaw was measured before OA or LPS exposure and at regular intervals up to 12 hours and at 24 hours afterwards. Sildenafil (1 mg/kg), based on human oral doses (26), or pathogen-free saline were administered (intraperitoneally) 24 and 0.5 hours before OA, LPS, or saline exposure. OA-exposed animals were further treated at 6 hours. Nebulized L-NAME (12 mM, 15 minutes) (27) was delivered to untreated or sildenafil-treated nonsensitized animals or at 105 minutes after completing exposures to LPS or vehicle. AHR was assessed with a threshold dose of nebulized histamine (1 mM, 20 seconds), which 24 hours before challenges gave negligible bronchoconstriction. This was repeated in the same animal 24 hours after OA or 1 or 4 hours after LPS exposure. Bronchodilator activity of sildenafil was assessed with a higher bronchoconstrictor dose of inhaled histamine (3 mM, 20 seconds) administered before and after sildenafil treatment. sGaw was measured before and at 0, 5, and 10 minutes after histamine exposures.

Twenty-four hours after exposure to OA and 1, 4, or 24 hours after LPS (or their saline equivalents), animals were overdosed with pentobarbitone sodium for BAL to determine total (cells/sample) and differential cell counts (macrophages, eosinophils, and neutrophils) after cytospin centrifugation and staining with Leishman's stain (1.5% in 100% methanol).

The remaining BALF was centrifuged (1,200 rpm, 6 minutes) and the supernatant frozen (-70°C) for determination of nitrite and nitrate by the Griess reaction (28) as described previously (10). BALF (100 µl) was incubated (37°C) for 30 minutes with N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid buffer (50 mM, pH 7.4), flavin adenine dinuucleotide (5 µM), nicotinamide adenine dinucleotide phosphate reduced (0.1 mM), distilled water (290 µl), and nitrate reductase (0.2 U/ml) for the conversion of nitrate to nitrite. Nitrate reductase was omitted for nitrite determination. Unreacted nicotinamide adenine dinucleotide phosphate reduced was oxidized by incubating (25°C, 10 minutes) with potassium ferricyanide (1 mM). The samples were then incubated (25°C, 10 minutes) with Griess reagent (N-(1-naphthyl)-ethylenediamine: 0.2% [wt/vol], sulfanilamide: 2% [wt/vol], solubilized in double-distilled water: 95% and phosphoric acid: 5% [vol/vol]) (1 ml) and the absorbance measured at 543 nm. Levels of nitrite were calibrated against a sodium nitrite (0–150 µM) standard curve.

cGMP generation in lungs from saline- or sildenafil-treated animals was measured in chopped lungs suspended in phosphate-free Krebs solution (0.3 mg/ml), gassed with 5% CO2/95% O2 at 37°C. S-nitroso-N-acetylpenicillamine (10-3 M) was added for 20 minutes and the reaction terminated by adding 0.5 ml of the suspension to 0.5 ml hydrochloride (1 N), which was homogenized (20,000 rpm, 20 seconds) and centrifuged (13,000 rpm, 3 minutes). Supernatant cGMP levels were determined by ELISA and expressed as picomoles per milligram of wet-lung weight.

Mean changes in sGaw are presented as a percentage of the baseline value preceding a challenge. BALF cell counts and NO metabolites were compared using analysis of variance followed by Scheffe's post hoc analysis. Airway function was compared using analysis of variance over the entire time course followed by paired or unpaired Student's (two-tailed) t test. Differences were considered significant when p values were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Sildenafil Treatment on cGMP Generation in Isolated Chopped Lung
As a measure of the effectiveness of sildenafil treatment in inhibiting PDE5, cGMP generation by S-nitroso-N-acetylpenicillamine (10-3 M) was examined in lungs taken 30 minutes after the second dose of sildenafil. The basal levels of cGMP were not significantly different between saline-treated and sildenafil-treated guinea pig lungs (1.91 ± 0.54 and 2.43 ± 0.34 pmol/mg). However, the S-nitroso-N-acetylpenicillamine–generated cGMP was significantly greater in lungs from sildenafil-treated guinea pigs (16.4 ± 3.6 pmol/mg, n = 6) (p < 0.05) than after saline treatment (5.48 ± 0.43 pmol/mg, n = 5).

Effect of Sildenafil after Allergen Exposure
Airway function.
Aerosolized OA exposure of OA-sensitized guinea pigs treated with vehicle caused the development of an early-phase antigen-induced bronchoconstrictor response (EAR), indicated by an immediate fall in sGaw from baseline values (-51.7 ± 12.5% decrease from baseline sGaw, p < 0.001) (Figure 1) . This early bronchoconstrictor response recovered to baseline values 6 hours later. A late-phase antigen–induced bronchoconstrictor response (LAR: -33.6 ± 9.5 mean peak % change from baseline sGaw, p < 0.01) followed between 7 and 11 hours. Airway function recovered to baseline values at 12 hours after OA exposure and was unaltered at 24 hours. Exposure to saline failed to alter airway function from baseline sGaw values (Figure 1). The airway function response in OA-sensitized guinea pigs exposed to OA was not significantly different in animals treated with and without vehicle (data not shown).



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  		Figure 1. Airway function of conscious ovalbumin (OA)-sensitized guinea pigs after exposure (1 hour) to saline (OA vehicle, open triangles) and saline with sildenafil treatment (1 mg/kg, closed diamonds) or OA (100 µg/ml) and treatment with sildenafil (1 mg/kg, open circles) or the sildenafil vehicle (saline, open squares). Treatment was administered (intraperitoneally) 24 and 0.5 hours before exposure to OA and 6 hours afterwards. Each point represents the mean ± SEM (n = 6) change in specific airways conductance (sGaw) expressed as a percentage of the baseline sGaw values. Negative values represent bronchoconstriction. The early antigen–induced response (EAR) is shown from 0 to 6 hours after OA exposure, and the mean peak late antigen–induced response (LAR) is depicted from 7 to 11 hours, as individual animals exhibited a LAR at different times. +p Values less than 0.05, ++p values less than 0.01, and +++p values less than 0.001 denote significant difference between exposure to OA with vehicle treatment and saline; **p values less than 0.01 and ***p values less than 0.001 denote significant difference between OA with vehicle treatment and OA with sildenafil treatment as determined by analysis of variance of the entire time course (single factor) followed by the Student's t test (paired). The airway's response to OA was not significantly different in OA-sensitized guinea pigs with and without vehicle treatment (data not shown).

 
Treating the OA-exposed guinea pigs with sildenafil failed to affect the development of an EAR and LAR (Figure 1). The peak developments of bronchoconstriction during the OA-induced EAR (-43.9 ± 12.0% decrease from baseline sGaw, p < 0.001) and LAR (-22.5 ± 5.5 mean peak % change from baseline sGaw, p < 0.01) were not significantly (p < 0.05) affected in guinea pigs treated with sildenafil. After exposure of sildenafil-treated animals to saline, airway function was not altered from baseline sGaw values, and the profile of response was not different from that observed in the absence of sildenafil (Figure 1).

Airway reactivity to inhaled histamine.
Inhaled histamine (1 mM, 20 seconds, nose only) before saline or OA exposure failed to cause a significant bronchoconstriction (Figures 2A and 2B) . At 24 hours after saline exposure, the airway responsiveness to histamine (1 mM, 20 seconds) was not significantly different (p < 0.05) to that before saline (Figure 2B). However, at 24 hours after exposure to OA, there was a significant (p < 0.01) bronchoconstriction to inhaled histamine (1 mM, 20 seconds), indicating AHR (Figure 2A). Treating the OA-sensitized guinea pigs with vehicle failed to affect the development of AHR at 24 hours after OA exposure (Figure 2B). However, in OA-sensitized guinea pigs treated with sildenafil there was no significant difference between inhaled histamine responses before and 24 hours after OA exposure, indicating that the AHR was inhibited (Figure 2B). There was no AHR to histamine in sildenafil-treated sensitized guinea pigs before OA exposure. A higher dose of inhaled histamine (3 mM, 20 seconds) that produces a significant bronchoconstriction (-17.2 ± 11.8 mean % decrease from baseline sGaw) (p < 0.01) was used 24 hours before sildenafil treatment and saline exposure (Figure 2B, histogram C). When repeated after treating with sildenafil and 24 hours after saline exposure, the bronchoconstriction (-17.6 ± 11.9%) was not significantly (p < 0.05) altered.



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  		Figure 2. Changes in airway responsiveness to histamine (1 or 3 mM, nose only, 20 seconds) in conscious OA-exposed (100 µg/ml, 1 hour) OA-sensitized guinea pigs. (A) Time course for airways response to inhaled histamine (1 mM) as the mean ± SEM change in sGaw expressed as a percentage of the baseline (BL) (n = 6) before (open circles) and 24 hours after OA (closed triangles). (B) Mean ± SEM peak change in sGaw expressed as a percentage of the BL in response to inhaled histamine (1 or 3 mM) (n = 6) in OA- and saline (OA vehicle)-exposed (1 hour) OA-sensitized guinea pigs. Histamine was administered 24 hours before (open bars) and in the same animal 24 hours after (solid bars): OA and vehicle treatment (1 mM, B); OA and sildenafil treatment (1 mM, D); or saline with (3 mM, C) and without (1 mM, A) sildenafil treatment. Sildenafil (1 mg/kg) or the sildenafil vehicle (saline) was administered (intraperitoneally) 24 and 0.5 hours before exposure to OA and at 6 hours afterwards. BL sGaw (s-1 cm of H2O-1) values before histamine challenge are as follows. (A) Before OA = 0.41 ± 0.02 and 24 hours after = 0.38 ± 0.02. (B) Before OA and sildenafil = 0.41 ± 0.02 and 24 hours after = 0.38 ± 0.03; before OA and vehicle = 0.39 ± 0.01 and 24 hours after = 0.40 ± 0.03; saline = 0.35 ± 0.02 and 24 hours after = 0.32 ± 0.02; before saline and sildenafil = 0.35 ± 0.02 and 24 hours after = 0.38 ± 0.03. Negative values represent bronchoconstriction. **p Values less than 0.01 and ++p values less than 0.01 denote significant difference from BL or between before or after exposure, respectively, as determined by analysis of variance (single factor) followed by a Student's paired t test.

 
Leukocyte infiltration.
OA-sensitized guinea pigs exhibited increased levels of macrophages in the BALF compared with nonsensitized guinea pigs (Figure 3A) . Exposure of OA-sensitized guinea pigs to saline failed to affect the levels of leukocytes in the airways at 24 hours, and the cellular content of the BALF was not significantly (p < 0.05) different from that observed before exposure in naive OA-sensitized animals (Figure 3A). Treating saline-exposed OA-sensitized animals with sildenafil also failed (p < 0.05) to affect the levels of BALF leukocytes, compared with naive OA-sensitized guinea pigs (macrophages: 9.9 ± 2.0; eosinophils: 0.6 ± 0. 1 x 1 06 cells/BALF sample) (Figure 3A). However, exposure of OA-sensitized guinea pigs to OA caused a significant influx of macrophages (p < 0.001) and eosinophils (p < 0.001) into the airways at 24 hours (data not shown). Treating the OA-sensitized animals with vehicle did not alter the significant airways infiltration of macrophages (34.9 ± 2.6 cells/BALF sample) and eosinophils (33.2 ± 4.4 cells/BALF sample), at 24 hours after OA (Figure 3A). However, treating the animals with sildenafil significantly reduced the OA-induced influx of macrophages and eosinophils at 24 hours, by 22% (p < 0.05) and 38% (p < 0.01), respectively (Figure 3A).



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  		Figure 3. (A) Differential cell (macrophage, eosinophil, and neutrophil) counts and (B) levels of combined nitric oxide (NO) metabolites (nitrate and nitrite) recovered in the bronchoalveolar fluid (BALF) from naive nonsensitized and OA-sensitized guinea pigs. Sensitized guinea pigs were exposed (1 hour) to saline (OA vehicle), saline with sildenafil treatment, and OA (100 µg/ml) with vehicle or sildenafil treatment and lavage performed 24 hours later. Each point represents the mean ± SEM (n = 6) of (A) the differential cells per BALF sample (x106) and (B) the concentration of combined NO metabolites (µM) per 100 µl of BALF. **p Values less than 0.01 and ***p values less than 0.001 indicate a significant difference from naive animals, and +p values less than 0.05 and ++p values less than 0.01 denote significance of sildenafil treatment, as determined by analysis of variance (single factor) followed by Scheffe's post hoc analysis.

 
NO.
The variation in concentrations of the individual NO metabolites (nitrate and nitrite) in BALF were synchronous. However, as described previously (23), lower levels of nitrate were analyzed due to the rate-limiting nitrate reductase conversion of nitrate to measurable nitrite (data not shown). Therefore, to describe changes in the NO metabolite levels, the combined nitrate and nitrite values are shown in Figure 3B.

At 24 hours after exposing OA-sensitized guinea pigs to saline, there was no significant (p < 0.05) difference in the levels of BALF NO metabolites, compared with naive OA-sensitized animals (Figure 3B). However, at 24 hours after exposure to OA, the BALF NO metabolites were significantly elevated. Treating the OA-exposed animals with vehicle failed (p < 0.05) to alter the significant increase in combined NO metabolites (159.3 ± 14.6 µM/100 µl of BALF sample) at 24 hours (Figure 3B). However, treatment with sildenafil significantly reduced the raised NO metabolite levels at 24 hours after OA challenge by 48% (p < 0.05) (Figure 3B). Interestingly, treating saline-exposed animals with sildenafil caused a 73% rise in combined NO metabolites at 24 hours, compared with exposure to saline only (Figure 3B).

Effect of Sildenafil after LPS Exposure
Airway function.
Exposure of nonsensitized guinea pigs to LPS (30 µg/ml) or the LPS vehicle (saline) caused an immediate fall in sGaw, indicating bronchoconstriction, that recovered to baseline values 0.5 hours later. This response to LPS or saline exposure was no different in nonsensitized animals treated with vehicle or sildenafil (Figure 4) .



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  		Figure 4. Airway function of conscious nonsensitized guinea pigs after exposure (1 hour) to saline (LPS vehicle, open diamonds) and saline with sildenafil treatment (1 mg/kg, closed squares) or LPS (30 µg/ml, closed circles) and treatment with sildenafil (1 mg/kg, closed triangles) or the sildenafil vehicle (saline, open circles). Sildenafil was administered (intraperitoneally) 24 and 0.5 hours before exposure to LPS. Each point represents the mean ± SEM (n = 6) change in sGaw expressed as a percentage of the baseline sGaw values. Negative values represent bronchoconstriction. No significant difference was identified between any groups as determined by analysis of variance of the entire time course (single factor).

 
Airway reactivity to inhaled histamine.
Inhalation of a threshold dose of histamine (1 mM,) before saline or LPS exposure failed to produce a significant bronchoconstriction (Figure 5A) . Twenty-four hours later, at 1 hour after saline there was no change in reactivity (Figure 5A, histogram A). However, at 1 hour after LPS exposure, in guinea pigs treated with the sildenafil vehicle, there was a significant bronchoconstriction to inhaled histamine (1 mM) (Figure 5A, histogram B). The reactivity to histamine had recovered by 4 hours after the LPS challenge (Figure 5A, histogram D). Treatment with sildenafil itself did not induce AHR to histamine administered before LPS, but it inhibited the AHR to inhaled histamine at 1 hour after the LPS exposure (Figure 5A, histogram C). To determine whether sildenafil exerted a bronchodilator action, a higher dose of histamine (3 mM) that causes significant bronchoconstriction was used 24 hours before and 1 hour after a saline exposure. Sildenafil treatment before the saline exposure did not affect the bronchoconstriction by histamine after saline (-25.6 ± 5.49% decrease in peak sGaw values from baseline) compared with before (-26.5 ± 5.4%) (Figure 5C, histogram H).



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  		Figure 5. Changes in airway responsiveness to histamine (1 or 3 mM, nose only, 20 seconds) in conscious nonsensitized guinea pigs exposed (1 hour) to LPS (30 µg/ml)- or saline (LPS vehicle). Responses to inhaled histamine 24 hours before (open bars) and in the same animal 1 or 4 hours after LPS (solid bars) are shown as the mean (n = 6) peak change in sGaw expressed as a percentage of the baseline. (A) Effects of sildenafil treatment (C) or vehicle (B) on histamine (1 mM) responsiveness 1 hour after LPS exposure compared with 1 hour after saline exposure (A) and 4 hours after LPS exposure (D, no vehicle). (B) Effect on histamine (1 mM) responsiveness 4 hours after saline or LPS exposure of an N{omega}-nitro-L-arginine methyl ester (L-NAME) challenge (12 mM, 15 minutes) given 2 hours after the saline vehicle exposure (E) or LPS exposure, without (F) and with sildenafil treatment (G). (C) Effect of sildenafil treatment on responsiveness to the bronchoconstricting dose of histamine (3 mM) 1 hour after saline exposure (H) or 4 hours after saline after L-NAME challenge at 2 hours (I). Sildenafil (1 mg/kg) or the sildenafil vehicle (saline) was administered (intraperitoneally) 24 and 0.5 hours before exposure to LPS. Negative values represent bronchoconstriction. Baseline sGaw (s-1 cm of H2O-1) values before histamine challenge are as follows. Before saline = 0.46 ± 0.04 and 1 hour after = 0.36 ± 0.02; before LPS and vehicle treatment = 0.33 ± 0.02 and 1 hour after = 0.33 ± 0.01; before LPS and sildenafil treatment = 0.36 ± 0.02 and 1 hour after = 0.34 ± 0.02; before LPS = 0.33 ± 0.01 and 4 hours after = 0.30 ± 0.02; before saline and L-NAME = 0.38 ± 0.01 and 4 hours after (LPS) = 0.35 ± 0.02; before LPS and L-NAME = 0.43 ± 0.04 and 4 hours after (LPS) = 0.39 ± 0.01; before LPS, L-NAME and sildenafil = 0.43 ± 0.03 and 4 hours after (LPS) = 0.39 ± 0.04; before saline and sildenafil = 0.43 ± 0.01 and 1 hour after = 0.41 ± 0.03; before saline, L-NAME and sildenafil = 0.33 ± 0.04 and 4 hours after (saline) = 0.36 ± 0.03. ++p Values less than 0.01 denote significant difference between before or after exposure as determined by analysis of variance (single factor) followed by a Student's paired t test.

 
Leukocyte infiltration.
Exposure of nonsensitized guinea pigs to saline failed to significantly affect the BALF levels of leukocytes in the airways at 24 hours (p < 0.05) compared with naive nonsensitized animals (Figure 6A) . Treating saline-exposed animals with sildenafil also failed (p < 0.05) to alter the BALF leukocyte content, from that observed in naive guinea pigs (macrophages: 6.7 ± 0.1; eosinophils: 0.6 ± 0.5; neutrophils: 0.0 ± 0. 0 x 1 06 cells/BALF sample) (Figure 6A). However, 24 hours after exposure of guinea pigs to LPS there was a significant influx of macrophages (p < 0.001), eosinophils (p < 0.01), and neutrophils (p < 0.001) into the airways. Treating the LPS-exposed animals with vehicle did not alter this significant airways infiltration of macrophages (27.1 ± 2.6, p < 0.001), eosinophils (1.3 ± 0.2, p < 0.05), and neutrophils (34.2 ± 2.8, p < 0.001) at 24 hours after LPS (Figure 6A). However, treating the animals with sildenafil significantly reduced the LPS-induced influx of macrophages, eosinophils and neutrophils at 24 hours, by 30% (p < 0.05), 46% (p < 0.05), and 65% (p < 0.001), respectively (Figure 6A).



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  		Figure 6. (A) Differential cell (macrophage, eosinophil, and neutrophil) counts and (B) levels of combined NO metabolites (nitrate and nitrite) recovered in the BALF from nonsensitized guinea pigs 24 hours after exposure to LPS (30 µg/ml, 1 hour) or saline. Guinea pigs were exposed to saline (LPS vehicle), saline with sildenafil treatment, and LPS with vehicle or sildenafil treatment. Each point represents the mean ± SEM (n = 6) of (A) the differential cells per BALF sample (x106) and (B) the concentration of combined NO metabolites (µM) per 100 µl of BALF. *p Values less than 0.05, **p values less than 0.01, and ***p values less than 0.001 indicate a significant difference from naive animals and +p values less than 0.05, ++p values less than 0.01, and +++p values less than 0.001 denote significance of sildenafil treatment as determined by analysis of variance (single factor) followed by Scheffe's post hoc analysis.

 
NO.
The combined BALF NO metabolites removed at 24 hours after exposing nonsensitized guinea pigs to saline were not significantly (p < 0.05) different from that removed from naive nonsensitized animals (Figure 6B). However, in guinea pigs exposed to LPS or treated with vehicle and exposed to LPS, NO metabolite levels were significantly elevated at 24 hours (Figure 6B). Treating saline-exposed guinea pigs with sildenafil failed to alter the levels of NO metabolites in BALF (Figure 6B). However, treating LPS-exposed animals with sildenafil significantly attenuated the raised NO metabolite levels at 24 hours by 59% (p < 0.01) (Figure 6B).

Effect of Inhibiting NOS with L-NAME on the Activity of Sildenafil
Airway function after exposure to L-NAME.
Exposure to nebulized L-NAME (12 mM, 15 minutes) caused an immediate bronchoconstriction in unchallenged animals (-20.6 ± 8.4% decrease from baseline sGaw) or 2 hours after an exposure to LPS (-23.7 ± 7.3%) or 2 hours after saline exposure (-18.7 ± 7.3%). This recovered to baseline sGaw values 0.5 hours later (data not shown). This immediate bronchoconstriction was not significantly (p < 0.05) different, however, from that observed after saline exposure alone.

Airway reactivity to inhaled histamine after exposure to LPS followed by L-NAME.
Two hours after exposure to nebulized LPS, guinea pigs were further exposed to inhaled L-NAME. A further 2 hours later (4 hours after LPS), exposure to inhaled histamine (1 mM) revealed AHR (Figure 5B, histogram F). This contrasts with the lack of AHR to histamine, at 4 hours after exposure to LPS alone (Figure 5A, histogram D). There was no AHR to inhaled histamine (1 mM) 4 hours after exposure to saline and 2 hours after exposure to L-NAME (Figure 5B, histogram E). In animals treated with sildenafil, the inhaled histamine (1 mM) responses before and 4 hours after exposure to LPS followed by L-NAME were not significantly different (Figure 5B, histogram G). Thus, sildenafil inhibited the extension of AHR by L-NAME to 4 hours after LPS exposure. The airway responses to the higher bronchoconstricting dose of inhaled histamine (3 mM) before (-23.7 ± 8.6 peak % change from baseline sGaw) and 4 hours after exposing sildenafil-treated guinea pigs to saline and (2 hours after) L-NAME (-28.6 ± 9.2%) were not significantly (p < 0.05) different (Figure 5C, histogram I).

Leukocyte infiltration after exposure to LPS followed by L-NAME.
Compared with naive and saline-exposed animals (macrophages: 2.1 ± 0.5 and neutrophils: 0.0 ± 0.0), there was an increase in macrophages (6.5 ± 3.5) and neutrophils (30.1 ± 6.1, p < 0.001) at 4 hours after LPS exposure (Figure 7A) . Leukocyte levels in the BALF removed 4 hours after saline exposure, with or without L-NAME exposure at 2 hours, were not significantly (p < 0.05) different from those observed in naive animals (Figure 7A). Pretreating of animals exposed to both saline and L-NAME with sildenafil also failed to significantly (p < 0.05) affect the BALF leukocyte content at 4 hours. In guinea pigs exposed to LPS, exposure to L-NAME at 2 hours caused a further increase in macrophages (24.4 ± 5.9) at 4 hours but not in neutrophils (24.0 ± 3.0) (Figure 7A). In sildenafil-treated guinea pigs, the increases in macrophages and neutrophils in the BALF removed at 4 hours after exposure to LPS and followed at 2 hours by L-NAME were significantly attenuated by 57% (p < 0.01) and 90% (p < 0.001), respectively (Figure 7A).



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  		Figure 7. Effects of L-NAME on (A) differential cell (macrophage, eosinophil, and neutrophil) counts and (B) levels of combined NO metabolites (nitrate and nitrite) recovered in the BALF from nonsensitized guinea pigs 1 or 4 hours after exposure to LPS (30 µg/ml, 1 hour) or saline. Guinea pigs were either not exposed (naive) or exposed to saline (LPS vehicle) or LPS and lavaged 1 or 4 hours later. Effects of L-NAME challenge (12 mM, 15 minutes) given 2 hours after saline or LPS with or without sildenafil treatment were examined on lavage performed 4 hours after the saline or LPS exposures. Treatment with sildenafil (1 mg/kg) or the sildenafil vehicle (saline) was administered (intraperitoneally) 24 and 0.5 hours before exposure to LPS. Each point represents the mean ± SEM (n = 6) of (A) the differential cells per BALF sample (x106) and (B) the concentration of combined NO metabolites (µM) per 100 µl of BALF. **p Values less than 0.01 and ***p values less than 0.001 indicate a significant difference from naive animals and +p values less than 0.05, ++p values less than 0.01, and +++p values less than 0.001 denote significance of sildenafil treatment as determined by analysis of variance (single factor) followed by Scheffe's post hoc analysis.

 
Airways NO after exposure to LPS followed by L-NAME.
During the AHR at 1 hour after LPS exposure, there was a deficiency in the combined NO metabolites, which recovered to baseline levels at 4 hours (Figure 7B) together with recovery of airway reactivity (Figure 5A, histogram D). Exposure to saline failed to affect the levels of NO metabolites at 1 hour (Figure 7B). L-NAME exposure, 2 hours after exposure to saline or LPS, inhibited the generation of NO metabolites at 4 hours (Figure 7B). However, in sildenafil-treated guinea pigs exposed to LPS or saline followed by L-NAME, there was no deficiency in NO metabolites and levels of NO were increased above that found in naive animals (Figure 7B).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Exposure of OA-sensitized guinea pigs with atopy to aerosolized antigen (OA) caused the development of an EAR (0–6 hours) and LAR (7–11 hours), as reported previously by us (24) and by others (13). Treating the OA-sensitized guinea pigs with sildenafil failed to affect the EAR or LAR. Allergen-induced mast cell degranulation and subsequent release of bronchoconstrictor mediators (e.g., histamine, tryptase, and prostanoids) into the airways appears to contribute to the EAR (21, 29). Although elevated cGMP can attenuate rat peritoneal mast cell degranulation (21), it is not known whether mast cells of the lungs are similarly affected. It would appear that mediators contributing to the EAR in the guinea pig are not modulated by elevated cGMP.

Exaggerated airways responsiveness to spasmogenic stimuli (e.g., histamine) is a common feature of patients with asthma or COPD (5). Similarly, in the sensitized guinea pig model of allergic asthma, 24 hours after allergen exposure, there was AHR to a threshold dose of histamine (1 mM), which was inhibited by sildenafil. Because sildenafil did not inhibit a larger bronchoconstricting dose of histamine (3 mM), it could be concluded that the inhibition of AHR was not via persistent bronchodilation arising from the inhibition of PDE5 (1). This finding may indicate that sildenafil inhibited the OA-induced AHR through the NO–cGMP pathway (1, 27) or by suppression of inflammatory cell activation (2, 21) and recruitment (13).

BALF removed from OA-sensitized animals 24 hours after OA exposure revealed raised levels of eosinophils and macrophages, which were attenuated by sildenafil and could be attributed to the consequent elevated intracellular cGMP levels. Although eosinophils predominately express cAMP-specific PDE4 (2), elevated cGMP levels can indirectly increase the cAMP concentration by directly inhibiting PDE3 degradation of cAMP (4, 30). Therefore, in this study, sildenafil may have suppressed eosinophil influx via a cGMP–cAMP ‘cross talk’ mechanism, causing an elevation in intracellular cAMP, which has been shown to suppress inflammatory cell activity (2, 19). Ortiz and coworkers (13) have also demonstrated in guinea pigs that the PDE5 inhibitor, zaprinast (10 mg/kg, intraperitoneally), attenuated antigen-induced microvascular leakage. The reduced airways penetration of leukocytes by sildenafil in the present study could similarly be due to attenuation of microvascular permeability.

Basal (physiologic) levels of NO are antiinflammatory through suppression of leukocyte activation and microvascular leakage and oppose AHR by maintaining bronchodilatory tone (31–33). Excessive NO, derived from inducible NOS (iNOS), however, favors inflammation through the development of Th2-lymphocyte responses (e.g., eosinophilia and IgE ‘priming’ of mast cells) and microvascular leakage (31). Inflammation may also be exacerbated through the formation of cytotoxic peroxynitrite from excess NO and inflammatory-derived superoxide (3, 31, 34). Individuals with asthma exhale increased NO levels (6, 34). The BALF removed from OA-sensitized animals 24 hours after OA exposure also contained elevated NO metabolite levels, which were significantly reduced by sildenafil. Suppression of proinflammatory mediators responsible for iNOS induction (6, 31) or elevated cGMP levels inhibiting NO overproduction directly are possible explanations. Interestingly, 24 hours after saline exposure, sildenafil treatment caused elevated NO levels in OA-sensitized but not in nonsensitized guinea pigs. This is probably derived from constitutive NOS sources because there was a lack of leukocyte influx and therefore proinflammatory mediators responsible for iNOS induction in saline-exposed animals. It would appear to be a feature of the sensitized state, and airways mast cells are a likely source of constitutive NOS because they are known to be upregulated in OA-sensitized animals (2, 21) and individuals with asthma (17, 31).

As described previously (10, 12, 23), acute exposure of guinea pigs to inhaled LPS causes features associated with COPD where neutrophilia correlates with AHR (5–7). At 1 hour after LPS exposure, guinea pigs displayed AHR to histamine, which recovered at 4 hours. Sildenafil treatment inhibited this LPS-induced AHR. Antagonism of histamine responses by bronchodilatation could be discounted. Sildenafil also significantly attenuated the LPS-induced airways infiltration of macrophages, eosinophils, and neutrophils and inhibited the increased generation of NO, presumably iNOS-derived (10), measured 24 hours after exposure. Resident macrophages are likely to orchestrate neutrophil recruitment through chemotactic factors such as tumor necrosis factor-{alpha} and interleukin-8 (35). Tumor necrosis factor-{alpha} also stimulates the transcription of iNOS, responsible for NO overproduction (33). In LPS-exposed rats, inhibiting tumor necrosis factor-{alpha} attenuates AHR and neutrophil influx (36). Fuhrmann and coworkers (20) suggest that elevating cGMP downregulates tumor necrosis factor-{alpha} expression, which would therefore suppress neutrophil influx and AHR. Thus, sildenafil, through raising intracellular cGMP levels, would attenuate this LPS-induced overproduction of NO.

In confirmation of our previous studies (23), during the LPS-induced AHR (1 hour after LPS), the generation of NO metabolites in the BALF was reduced. The resolution of AHR coincided with a recovery in NO generation, 4 hours later. Inhibiting NO recovery with the nonselective NOS-inhibitor, L-NAME, prolonged AHR to at least 4 hours, suggesting the involvement of a NO-dependent mechanism in the recovery from LPS-induced AHR. L-NAME alone failed to cause AHR, indicating that NO-deficiency alone was not responsible for the LPS-induced AHR. Previous studies from this laboratory and by others have also described this phenomenon of NO-deficiency during AHR followed by recovery in comparable guinea pig models of lung inflammation induced by allergen (24, 27), parainfluenza virus (3), and ozone (37). The PDE4-inhibitor, rolipram, or the corticosteroid, dexamethasone, prevented both NO-deficiency and AHR after LPS exposure (10). Furthermore, the LPS-induced AHR coincided with reduced lung levels of cAMP and cGMP (38). Together this data suggests that in this model, the LPS-induced AHR and associated NO-deficiency share common proinflammatory mechanisms, presumably interrelated with the associated deficiency in lung cyclic nucleotide generation.

The effectiveness of sildenafil in reversing male erectile dysfunction is due to potentiation of endogenous NO-mediated vascular relaxation in the corpus cavernosum (1). This is due to cGMP, generated by NO, being elevated through PDE5 inhibition. Thus, it was of interest to determine whether the beneficial effects of sildenafil observed against the inflammatory consequences of an LPS challenge were dependent on endogenous levels of NO. Two hours after L-NAME (4 hours after saline), there was a predictable elimination of NO (metabolites) confirming the effectiveness of NOS inhibition. At 4 hours after LPS followed by L-NAME, however, the NO levels were markedly reduced, and there was now AHR to histamine at 4 hours after LPS. Sildenafil treatment of guinea pigs challenged with L-NAME, prevented the L-NAME–induced reduction of NO, in both saline- and LPS-exposed animals at 4 hours. The levels of NO were elevated above that for LPS exposure alone at 4 hours, and the extension of AHR to 4 hours after LPS seen after L-NAME treatment was inhibited by sildenafil. No inhibition of the bronchoconstrictor response to the larger dose of histamine (3 mM) was observed after sildenafil treatment in L-NAME–exposed animals. Therefore, loss of AHR was not due to bronchodilatation from elevation of cGMP levels by sildenafil. Therefore, increasing cGMP with sildenafil reversed the associated AHR and L-NAME–induced NO deficiency. A possible mechanism for this is via a feedback mechanism of cGMP on NOS-derived NO. A deficiency in NO, below normal physiologic levels, may activate the machinery to induce the expression of iNOS and other nuclear transcription factor-KB–dependent proinflammatory mediators (31, 33). In this study, inhibiting NO production with L-NAME appeared to elevate BALF macrophages at 4 hours after LPS or saline exposure by fourfold and fivefold, respectively, but failed to further increase the elevated levels of neutrophils after LPS exposure. However, in animals treated with sildenafil and exposed to LPS followed by L-NAME, the BALF content of macrophages and neutrophils were significantly attenuated. These findings suggest that NO deficiency may contribute to an airways influx of leukocytes after inflammatory provocation. Thus, as sildenafil restores the L-NAME–induced NO deficiency in both saline- and LPS-exposed animals, it is plausible that sildenafil suppresses leukocyte recruitment into the airways (and AHR) by restoring the NO or cGMP pathways. These opposing effects of lowered NO (L-NAME) and raised cGMP levels (sildenafil) on leukocyte infiltration may also arise from changes in microvascular permeability and adhesion of leukocytes (2, 13, 32).

In summary, sildenafil attenuated AHR, inflammation, and NO dysfunction in two conscious guinea pig models. The effectiveness of sildenafil does not appear to depend on endogenous NO levels as it does in erectile dysfunction because inhibition of NOS with L-NAME does not prevent the action of sildenafil. That sildenafil, in the dose administered, was inhibiting PDE5 was demonstrated by the enhanced generation of cGMP by S-nitroso-N-acetylpenicillamine in lungs removed from guinea pigs treated with sildenafil. This confirmed that cGMP generated by the NO donor was prevented from breakdown by PDE5, thus allowing its levels to accumulate. The results of this study suggest that sildenafil and future PDE5 inhibitors with increased selectivity may have potential in treating COPD and asthma. The proven safety of sildenafil (Viagra) and freedom from major adverse effects in patients with erectile dysfunction makes this an attractive possibility. Recently, sildenafil has been shown to inhibit AHR to inhaled methacholine in individuals with asthma; however, the authors did not measure inflammation in the airways (39). Currently, there are no reports in the literature describing the antiinflammatory actions of sildenafil, either in clinical trials or animal models. It would therefore be of interest to determine whether asthma symptoms amongst users of Viagra are improved.


   Acknowledgments
 
The authors are grateful to Pfizer, Sandwich, UK for the generous gift of sildenafil.


   FOOTNOTES
 
Supported by a GlaxoSmithKline studentship (T.J.T).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: T.J.T. has no declared conflict of interest; N.S. has no declared conflict of interest; K.J.B. has no declared conflict of interest.

Received in original form November 25, 2002; accepted in final form October 30, 2003


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 RESULTS
 DISCUSSION
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