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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Inhibition of Nitric-Oxide Synthase Enhances Antigen-Induced Contractions and Increases Release of Cysteinyl-Leukotrienes in Guinea Pig Lung Parenchyma: Nitric Oxide as a Protective Factor

Experimental Asthma and Allergy Research, Division of Physiology, The Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Received April 6, 2005; accepted July 13, 2005.

	Abstract

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Nitric oxide (NO) in exhaled air is a biomarker of airway inflammation. However, the role of NO in the peripheral lung is not known. The aim of this study was to determine the role of endogenous NO in antigen-induced contractions of ovalbumin (OVA)-sensitized guinea pig lung parenchyma (GPLP). The contraction in this in vitro model of the peripheral lung closely resembles the corresponding response in human airways. Cumulatively increasing concentrations (10–10,000 µg/l) of OVA induced concentration-dependent contractions of the GPLP that were enhanced by the NO synthase (NOS) inhibitors N-nitro-L-arginine (L-NOARG; 100 µM), N-monomethyl-L-arginine (100 µM), N-nitro-L-arginine methyl ester (100 µM), and N-(3-(aminomethyl)benzyl)acetamidine (1400W; 1 µM). The enhancement induced by L-NOARG was reversed by coadministration with the 5-lipoxygenase inhibitor (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid (BAY x1005; 3 µM), whereas coadministration of L-NOARG with the cyclooxygenase inhibitor indomethacin (10 µM) did not change the effect of L-NOARG alone. L-NOARG (100 µM) did not affect the cumulative concentration-response relations for either leukotriene (LT) D₄ (0.1–100 nM) or histamine (1–30 µM). The NO donor NONOate (0.001–100 µM) was ineffective in GPLP but potently relaxed precontracted guinea pig pulmonary artery. Furthermore, L-NOARG enhanced the release of LTE₄ and decreased the release of prostaglandin E₂ induced by OVA. In conclusion, endogenous NO exerts an inhibitory effect on antigen-induced contractions in the peripheral lung. The action of NO apparently involves inhibition of the release of mediators rather than direct relaxation of airway smooth muscle. The findings support the belief that endogenous NO has a protective anti-inflammatory effect in the airways.

The major mediators of the early allergic airway response in humans are known to be cysteinyl-leukotrienes (CysLT) and histamine (Roquet et al., 1997), but also products of the cyclooxygenase (COX) pathway are involved (Manning et al., 1991). It has been shown that antigen-induced contractions in small airways are more severe (Wohlsen et al., 2003) than in larger airways. The aim of this study was to determine the role of NO in antigen-induced contractions of the peripheral part of the lung. The study was performed in the lung parenchyma obtained from actively sensitized guinea pigs (GPLP), an in vitro model for mast cell-driven antigen-induced contractions. The mediators of this particular contraction response (Wikström Jonsson and Dahlén, 1994) are similar to those established for the anaphylactic contraction of human airways in vitro and in vivo (Björck and Dahlén, 1993; Roquet et al., 1997). A recent study in the isolated perfused and ventilated guinea pig lung indicates that in addition to histamine and CysLT, one or several prostanoids contribute to the antigen-induced airway constriction in this particular species (Sundström et al., 2003).

Mast cells are able to produce NO (Gilchrist et al., 2002), and NO has been shown to inhibit the release of histamine from rat mucosal mast cells (Masini et al., 1991). A colocalization of NOS with 5-lipoxygenase (5-LO) along the nuclear membrane has recently been suggested, providing a potential for interaction between NO and leukotriene (LT) synthesis in human mast cells (Gilchrist et al., 2004). In airway macrophages, NO may in fact act on 5-LO and suppress LT synthesis (Coffey et al., 2000, 2002). The actions of NO on the COX pathway have not been completely elucidated but apparently involve both direct and indirect mechanisms (Salvemini et al., 1993; Watkins et al., 1997; Devaux et al., 2001).

One hypothesis to be tested was that NO in the peripheral lung also affects the antigen-induced contractions by relaxation of the airway smooth muscle. Another hypothesis was that NO affects the release of mast cell mediators, either globally or specifically via actions on COX or 5-LO pathways for arachidonic acid metabolism. Different selective inhibitors of endogenous NOS, COX, and 5-LO were used to study the impact of NO in the functional responses in the GPLP model. The tissue bath fluid was analyzed by enzyme immunoassays (EIA) to determine whether some of the key interventions affected the amount of released mediators during antigen-induced challenges.

	Materials and Methods

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Animals and Ovalbumin Sensitization. For studies of antigen-induced contractions, male Dunkin Hartley guinea pigs (300–350 g b.wt.) were sensitized to ovalbumin (OVA) (chicken egg albumin) at least 4 weeks before the experiment. A stock solution of OVA was prepared by dissolving 500 mg of OVA in 10 ml of 0.9% NaCl and 10 ml of 2% aluminum hydroxide gel and shaking for 1 h. The guinea pigs were given an s.c. injection of 0.4 ml of OVA (10 mg) in the neck and an i.p. injection of 0.4 ml of OVA (10 mg). The regional committee of animal experimentation ethics (N14/02, N127/04) approved the study, which was completed in accordance with the Declaration of Helsinki.

Lung Parenchymal Strips. The animals were sacrificed with an overdose of inhaled CO₂, and the heart-lung package was quickly removed and placed in ice-cold Tyrode's solution (prepared each day, containing 149.2 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO₃, 5.5 mM glucose, 1.8 mM CaCl₂, 0.5 mM MgCl₂, and 0.4 mM NaH₂PO₄). The lung parenchyma was cut parallel to the peripheral margins, yielding four to eight strips, each having a size of 2 x 2 x 20 mm and a weight of approximately 50 mg.

Organ Bath Experiments. The lung parenchymal strips were set up at a resting tension of 2.5 mN (0.25 g) in 5-ml organ baths filled with Tyrode's solution, bubbled with carbogen gas (6.5% CO₂ in O₂) to keep a pH of 7.4, and the temperature was kept at 37°C. Changes in smooth muscle tension (i.e., contractions and relaxations) were recorded via isometric force-displacement transducers connected to a Grass polygraph (Grass Instruments, Quincy, MA), and responses were displayed via a chart recorder or by using the IOX data acquisition system (emka Technologies, Paris, France). Data were analyzed either manually from charts or using the software program DataAnalyst (emka Technologies). After an equilibration period of 90 min and washes every 15 min, 1 to 30 µM histamine was added cumulatively as a control of the GPLP reactivity. Preparations displaying contraction responses less than 1.0 mN to the highest concentration of histamine were excluded from further experiments. Another wash and equilibration period between histamine and treatment period was performed. The enzyme inhibitors indomethacin (10 µM) and BAY x1005 (3 µM) were given 30 min before the challenges, and all the other drugs were given 15 min before the challenges. OVA was added as cumulative challenge of increasing concentrations (1–10,000 µg/l) every 10 min without changing bath fluid. For study of NO donors, the GPLP was precontracted with a single dose of 10 nM LTD₄. Maximum contractions of the preparation were determined with 1 mM histamine, 1 mM acetylcholine, and 50 mM KCl at the end of each experiment, and other responses were expressed as the percentage of maximum contractions. Guinea pig pulmonary artery (GPPA) was prepared as rings with the endothelium gently removed and then mounted in the organ baths under a resting tension of 15 mN (1.5 g). After an equilibration period of 60 min and washes every 10 min, 10 µM noradrenaline was added, and the drugs were given at the plateau to study relaxations.

Drugs. NaCl, KCl, CaCl₂, MgSO₄, NaHCO₃,KH₂PO₄, and glucose were obtained from VWR (West Chester, PA). Histamine dihydrochloride, noradrenaline, acetylcholine, indomethacin, OVA (chicken egg albumin, grade II), N-monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine methyl ester (L-NAME), L-arginine, and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO). BAY x1005 was from Bayer AG (Wuppertal, Germany). N-Nitro-L-arginine (L-NOARG), 1400W, and diethylamine NONOate were purchased from Calbiochem (San Diego, CA). LTD₄ was from Cascade Biochemicals Ltd. (Reading, UK). Celecoxib (Celebrex) was obtained from Pfizer, Inc. (New York, NY). SC560, prostaglandin E₂ (PGE₂), and the EIA kits for LTE₄, thromboxane B₂ (TXB₂), LTB₄, PGD₂-mox, and PGE₂ were obtained from Cayman Chemical (Ann Arbor, MI).

Indomethacin was dissolved in equal parts of ethanol and 1 M Tris, pH 8.0, and then diluted in 0.9% NaCl. Stock solutions of 1 mM LTD₄ were dissolved in 50% ethanol-water and then diluted in 20% ethanol-water. The concentration and purity of LTD₄ was checked by UV spectroscopy. L-Arginine was dissolved in 1 M HCl. BAY x1005, SC560, and celecoxib were dissolved in dimethyl sulfoxide. OVA and L-NOARG were dissolved in 0.9% NaCl. The other drugs were dissolved and diluted in Tyrode's solution or distilled water. Dilutions of drugs were freshly made from the stocks for each experiment. The drugs were present in the organ bath fluid during the remaining experiment.

Measurements of Released Mediators. One milliliter of fluid was collected from each organ bath and immediately frozen at -20°C. The samples were taken at the end of the equilibration period to obtain basal mediator release from the tissue and at the obtained contractile plateau after challenge with 100 µg/l of OVA. EIA analyses of the different mediators CysLT, LTB₄, TXA₂, PGD₂, and PGE₂ were performed according to the manufacturer's instructions. TXA₂ was measured as the stable metabolite TXB₂. CysLT were measured as LTE₄, the end metabolite of LTC₄ and LTD₄. The assay detection limits in the bath fluid levels for the different mediators were 7.8 pg/ml for TXB₂, LTE₄, PGE₂, and PGD₂ and 3.9 pg/ml for LTB₄. Results below detection limits were set as zero in the statistical evaluation. The EIA specificity for the different mediators to interfere with each other was less than 0.01%, with the exception of the TXB₂ EIA that cross-reacted with PGD₂ (0.53%) and with PGE₂ (0.09%). The LTE₄ EIA was performed with the CysLT antiserum and cross-reacted with both LTC₄ (50%) and LTD₄ (100%). Histamine was measured as described previously (Shore et al., 1959; Bergendorff and Uvnäs, 1972). Duplicates of 300 µl were placed in 96-well plates, and the amount of histamine was analyzed by a fluorometer at the 450-nM wavelength. The detection limit for histamine was 3.9 ng/ml. The data were expressed as molar amounts per gram wet tissue (fmol/g or pmol/g).

Calculations and Statistics. All the data are presented as mean ± S.E.M. Statistical analyses were made for paired and unpaired observations by Student's t test or analyses of variances followed by Tukey's t test or Bonferroni t test. A p value of less than 0.05 was considered significant.

	Results

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Effects of NOS Inhibitors on Antigen-Induced Contractions. Challenge with cumulative concentrations of OVA (10–10,000 µg/l) induced concentration-dependent contractions of sensitized GPLP (Fig. 1). Pretreatment with the unselective NOS inhibitor L-NOARG (100 µM) did not affect the basal tone of the preparation nor the maximal contractile response (control, 2.29 ± 11 mN, n = 5; L-NOARG, 2.43 ± 21 mN, n = 5; N.S.). However, L-NOARG (100 µM) significantly (p < 0.001) shifted the concentration-response relation for OVA to the left (Fig. 1A). Three additional and structurally different NOS inhibitors, L-NAME (100 µM), L-NMNA (100 µM), and 1400W (1 µM), respectively, mimicked the enhancement of the OVA response induced by L-NOARG (100 µM) (Fig. 1B).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Drug effects on the concentration-response to 1 to 10,000 µg/l of OVA in sensitized lung parenchymal strips. A, effect of pretreatment with NOS inhibitor 100 µM L-NOARG (n = 5), 100 µM L-arginine (n = 5), and the combination 100 µM L-NOARG and 100 µM L-arginine (n = 5) on the concentration response to 10 to 10,000 µg/l of OVA compared with the control (n = 5). B, effect of the NOS inhibitors 100 µM L-NAME (n = 5), 100 µM L-NOARG (n = 9), 100 µM L-NMMA (n = 5), and 1 µM 1400W (n = 5) on the concentration response to 1 to 10,000 µg/l of OVA compared with the control (n = 9). Data are expressed as mean ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Direct Effects of NO on Smooth Muscle. L-NOARG (100 µM) did not alter the contractions induced by cumulative challenge with either 0.1 to 100 nM LTD₄ (Fig. 2A) or 1 to 30 µM histamine (Fig. 2B). Nor did the NO donor diethylamine NONOate (0.01–100 µM) relax the parenchyma precontracted with 10 nM LTD₄ (Fig. 3A), whereas 0.0001 to 100 µM NONOate dose-dependently relaxed the GPPA precontracted by 10 µM noradrenaline (Fig. 3B).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Drug effects on concentration responses to agonists in nonsensitized GPLP. A, effect of pretreatment with 100 µM L-NOARG (n = 5), 100 µM L-arginine (n = 5), and the combination 100 µM L-NOARG and 100 µM L-arginine (n = 5) on cumulatively increasing doses (0.1–100 nM) of LTD4 (n = 5). B, effect of pretreatment with 100 µM L-NOARG (n = 5), 100 µM L-arginine (n = 5), and the combination 100 µM L-NOARG and 100 µM L-arginine (n = 5) on cumulatively increasing doses (1–30 µM) of histamine (n = 5). Data are expressed as mean ± S.E.M.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of NO donor NONOate (0.0001–100 µM) on precontracted nonsensitized GPLP and GPPA. A, effect of cumulative concentrations of NONOate (0.01–100 µM) (n = 5) on 10 nM LTD4 precontracted GPLP compared with the control (n = 6), N.S. B, effect of cumulative concentrations of NONOate (0.0001–100 µM) (n = 4) on 10 µM noradrenaline precontracted GPPA compared with the control (n = 4). Data are expressed as mean ± S.E.M. N.S., p > 0.05; ***, p < 0.001.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effect of pretreatment with 100 µM L-NOARG (black, n = 5) compared with the control (white, n = 6) on mediator release from sensitized GPLP after challenge with 100 µg/l of OVA. Release of A, LTE4; B, LTB4; C, TXB2; D, PGE2; E, PGD2; and F, histamine. All the samples were collected at baseline and then at the plateau after 100 µg/l of OVA. The parenchymal strips had been pretreated 15 min with either Tyrode's solution or 100 µM L-NOARG. All the data are expressed as mean ± S.E.M. Concentrations are expressed as mole/gram wet lung parenchymal strip. *, p < 0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Drug effects on the concentration response to 1 to 10,000 µg/l of OVA in sensitized lung parenchymal strips. A, effect of pretreatment with 10 µM indomethacin (n = 10) compared with the control (n = 12). B, effect of pretreatment with 5 µM SC560 (n = 6) and 3 µM celecoxib (n = 7) compared with the control (n = 7). C, effect of combined treatment with 10 µM indomethacin and 100 µM L-NOARG (n = 10) compared with the control (n = 12) and 100 µM L-NOARG (n = 10). D, effect of pretreatment with 100 nM PGE2 (n = 6) and combined treatment with 10 µM indomethacin and 100 nM PGE2 (n = 6). Data are expressed as mean ± S.E.M. *, p < 0.05; **, p < 0.01.

Effect of 5-LO and L-NOARG on OVA-Induced Contractions. The enhanced contractions to OVA by 100 µM L-NOARG were reversed by coadministration with 3 µM BAY x1005. The 5-LO inhibitor BAY x1005 (3 µM) alone had no significant effect on the cumulative concentration response to 1 to 10,000 g/l of OVA (Fig. 6). The release of LTE₄ during the antigen-induced contractions (19 ± 6.1 fmol/g) was abolished after pretreatment with 3 µM BAY x1005 (<1.5 fmol/g, n = 4, p = 0.001) at 1000 µg/l of OVA.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Drug effects on the concentration response to 1 to 10,000 µg/l of OVA in sensitized lung parenchymal strips. Effect of pretreatment with 3 µM BAY x1005 (n = 8), 100 µM L-NOARG (n = 10), and combined treatment with 3 µM BAY x1005 and 100 µM L-NOARG (n = 5) compared with the control (n = 10). Data are expressed as mean ± S.E.M. ***, p < 0.001.

	Discussion

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In the present study, inhibition of endogenous NO enhanced antigen-induced contractions of peripheral airways. The effect of NO in the peripheral lung appears to be at the level of mediator release because inhibition of endogenous NO or application of exogenous NO had insignificant relaxant activity at the level of the airway smooth muscle. The mechanism apparently involves increased release of contractile CysLT and decreased release of PGE₂.

Pretreatment with the unselective NOS inhibitor L-NOARG caused an enhancement of the contraction evoked by challenge with cumulative doses of OVA. This enhancement by the competitive substrate analog inhibitor L-NOARG was reversed when L-NOARG was given together with the substrate L-arginine. Moreover, the enhancement of the antigen response by L-NOARG was reproduced by three other NOS inhibitors, L-NAME, L-NMMA, and 1400W. L-NAME, L-NMMA, and L-NOARG are unselective inhibitors of eNOS, neuronal NOS, and iNOS, whereas 1400W has been documented to selectively inhibit iNOS (Garvey et al., 1997). There was no significant difference in effect of the four different NOS inhibitors tested. Taken together, the main enzyme generating NO in this particular model may be iNOS, as supported by the effect of 1400W.

Furthermore, the role of NO in the early allergic airway response appears to be protective by reducing the contraction response in the peripheral lung. Studies in a transgenic mouse model with an overexpressed iNOS also indicated that NO had no proinflammatory effects on the lung and decreased the airway responsiveness (Hjoberg et al., 2004). However, data from murine models with different deletions of NOS are conflicting (De Sanctis et al., 1999; Xiong et al., 1999), and these particular mice studies also used measurements of pulmonary function that presumably measure central airway function. It remains to establish how these in vivo observations in mice relate to our current findings in the peripheral lung of the guinea pig.

In any event, one possible explanation of the inhibitory effect of endogenous NO on the antigen-induced contraction would be relaxation of airway smooth muscle, as has been shown in more proximal airways (Persson et al., 1993). However, endogenous or exogenous NO did not induce any significant relaxant effect in the GPLP model, suggesting that the peripheral lung might be less responsive to the direct smooth muscle relaxant effects of NO. L-NOARG had no significant effect on contractions induced by either of the direct smooth muscle stimulants histamine and LTD₄. The lack of effect on LTD₄ contractions was also seen in a previous study (Sakata and Bäck, 2002) and is in distinct contrast to the effect of NO inhibition on the responses induced by agonists in vascular preparations (Bäck et al., 2002). The finding that NO is devoid of smooth muscle relaxing properties in the peripheral lung was further strengthened by the fact that the NO donor NONOate had no relaxant activity even at 100 µM bath concentration in LTD₄-precontracted GPLP. In contrast, 30 nM NONOate caused 50% relaxation of GPPA precontracted by noradrenaline. Taken together, it is not likely that the inhibitory effect of NO on the antigen-induced contractions was related to effects at the level of the airway smooth muscle. In fact, there is one report in which nitroglycerin did not relax bovine lung parenchyma (Gruetter and Lemke, 1985), suggesting that the lack of NO sensitivity on airway smooth muscle is a general characteristic of the peripheral lung.

Based on the initial findings, the study therefore tested the other hypothesis that the effects of the NO inhibitors were exerted at the level of release of proinflammatory mediators. Challenge with OVA markedly increased release of LTE₄, LTB₄, PGD₂, TXB₂, PGE₂, and histamine from GPLP. The NOS inhibitor L-NOARG had no significant effect on the release of histamine after OVA challenge. Other studies have shown that inhibition of endogenous NO caused increased release of histamine from activated mast cells (Masini et al., 1991) and that NOS inhibition enhanced the antigen-induced mast cell degranulation (Coleman, 2002). However, a recent study indicated that NOS inhibition had no effect on the onset of mast cell degranulation (Swindle et al., 2004). Therefore, we speculated that NO, in this particular lung model, predominantly inhibits a more specific pathway than mast cell activation in general.

Influence of 5-LO and 5-LO Products on Effect of NOS Inhibition. Pretreatment with L-NOARG significantly enhanced the OVA-induced release of LTE₄. To further investigate the role of CysLT and NO, the 5-LO inhibitor BAY x1005 was used. Pretreatment with BAY x1005, as expected, abolished the release of LTE₄. Moreover, the enhancement of the OVA response by L-NOARG was not seen in preparations also treated with BAY x1005, suggesting that the effect of NO inhibition is exerted at the level of 5-LO. This interaction is supported by the findings, suggesting that eNOS are colocalized with 5-LO in human mast cell nucleus and that endogenous NO acts as a regulator of LT synthesis (Gilchrist et al., 2004). Likewise, NO appears to be able to suppress 5-LO activity in airway macrophages (Coffey et al., 2000, 2002).

It may seem surprising that BAY x1005 alone did not significantly affect the contractile response to OVA. However, this lack of effect of 5-LO inhibition alone has been explained previously in detailed studies of antigen-induced contractions in the guinea pig airways (Wikström Jonsson and Dahlén, 1994; Sundström et al., 2003). Neither inhibition of LT, histamine, or prostanoids separately affects the antigen-induced contraction, whereas combined inhibition of these synergistically acting mediators almost completely abolishes the antigen-induced contraction.

Influence of COX Products on Effect of NOS Inhibition. The unselective COX inhibitor indomethacin (10 µM) enhanced the concentration-response relation to OVA. The selective COX-1 inhibitor SC560 mimicked the enhancement, whereas the selective COX-2 inhibitor celecoxib had no effect, suggesting that it is a COX-1-generated product that is involved in the observed modulation of the early allergic airway response. Exogenous PGE₂ shifted the concentration-response curve for OVA to the right and reversed the enhancement induced by indomethacin, suggesting that endogenous PGE₂ inhibits the antigen response in this particular model. The effect may accordingly in part relate to relaxation of airway smooth muscle, but inhibition of mast cell mediator release (Raud et al., 1988; Tilley et al., 2001) may also be involved.

The release of PGE₂ was significantly decreased in preparations treated with L-NOARG, which raised the possibility that endogenous NO stimulated PGE₂ release. However, on the functional level, when L-NOARG was given to indomethacin-treated preparations, the enhancement of the concentration-response relation to OVA caused by L-NOARG was the same. Therefore, the current observations suggest that the two regulatory mechanisms (NO and PGE₂) in this particular model predominantly represent different or at least not immediately connected pathways. The effect of L-NOARG on PGE₂ release may be indirect, and additional studies are required to resolve the relation between COX and NO pathways in this particular model.

In conclusion, inhibition of endogenous NO enhanced the antigen-induced contractions in this model of the peripheral lung. The mechanism apparently involves modulation of the release of mediators rather than direct relaxation of airway smooth muscle. Accordingly, the NO donor did not relax the airway smooth muscle; however, there were increased release of contractile CysLT and reduced release of PGE₂ after inhibition of NOS in the GPLP. Based on these findings, we propose that endogenous NO in the peripheral lung exerts an inhibitory effect on antigen-induced contractions by reducing the release of CysLT. Studies of human airways in vitro indicate that the early airway response after antigen stimulation is more severe and long-lasting in the smaller airways (Wohlsen et al., 2003), which further implies that NO has an important protective role in the peripheral lung as a beneficial modulator of the early allergic airway response.

	Footnotes

 
This work was supported by the Swedish Heart Lung Foundation, the Swedish Medical Research Council, Biolipox AB, and Karolinska Institutet.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.086694.

ABBREVIATIONS: NO, nitric oxide; NOS, nitric-oxide synthase(s); eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; CysLT, cysteinyl-leukotriene(s); COX, cyclooxygenase; GPLP, guinea pig lung parenchyma; 5-LO, 5-lipoxygenase; LT, leukotriene; EIA, enzyme immunoassay(s); OVA, ovalbumin; BAY x1005, (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid; GPPA, guinea pig pulmonary artery; L-NMMA, N-monomethyl-L-arginine; L-NAME, N-nitro-L-arginine methyl ester; L-NOARG, N-nitro-L-arginine; 1400W, N-(3-(aminomethyl)benzyl)acetamidine; SC560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; PG, prostaglandin; TX, thromboxane.

Address correspondence to: Anna-Karin Larsson, Experimental Asthma and Allergy Research, Division of Physiology, The Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 287, SE-17177 Stockholm, Sweden. E-mail: anna-karin.larsson{at}imm.ki.se

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