Sulfanilamide

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^sup [omega]^-nitro-L-arginine methyl ester did not prevent its action. Inhibition of PDES 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.

Keywords: 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 [beta]2-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^sup [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

(Full details are available in the online supplement.)

Airway function (specific airways conductance: sG^sub aw^) was monitored in conscious Dunkin-Hartley guinea pigs (male, 300-400 g), using whole-body 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. sG^sub aw^ 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 wore 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. sG^sub aw^ 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% CO^sub 2^/95% O2 at 37°C. S-nitroso-N-acetylpenicillamine (10^sup -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 sG^sub aw^ 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

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^sup -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

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 sG^sub aw^ from baseline values (-51.7 ± 12.5% decrease from baseline sG^sub aw^, p

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 sG^sub aw^, p

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

Leukocyte infiltration. OA-sensitized guinea pigs exhibited increased levels of macrophages in the BALF compared with non-sensitized 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

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

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 sG^sub aw^, 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).

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 sG^sub aw^ values from baseline) compared with before (-26.5 ± 5.4%) (Figure 5C, histogram H).

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

NO. The combined BALF NO metabolites removed at 24 hours after exposing nonsensitized guinea pigs to saline were not significantly (p

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 sG^sub aw^) 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 sG^sub aw^ values 0.5 hours later (data not shown). This immediate bronchoconstriction was not significantly (p

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 sG^sub aw^) 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

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

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

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 over-production (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 bronchodilalation 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.

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

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

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

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

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@ Cardiff.ac.uk

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.

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

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