The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of non-insulin dependent diabetes mellitus (NIDDM).

The effects of N(omega-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, on airway responsiveness were studied in the spontaneously hyperresponsive Fischer and the control normoresponsive Lewis rat strains to investigate the role of the endogenous nitric oxide (NO) pathway in strain-related differences in airway responsiveness. Responsiveness to inhaled methacholine was significantly increased in L-NNA-treated Lewis rats but not in Fischer rats. L-NNA increased carbachol-induced tracheal contractions in vitro to a larger extent in Lewis rats compared with Fischer rats. The effect of L-NNA was abolished by removal of the epithelium. Carbachol induced a NO-dependent increase in guanosine 3',5'-cyclic monophosphate levels in tracheal tissues but to a lesser extent in Fischer (2.1-fold increase) than in Lewis (3.7-fold increase) rats. In conclusion, endogenous NO is involved in the regulation of airway responsiveness to cholinergic agonists in rats. A relatively ineffective NO-guanosine 3',5'-cyclic monophosphate regulatory mechanism in Fischer rats contributes, in part, to strain-related differences in airway responsiveness between Fischer and Lewis rats.

BACKGROUND: There is evidence that the cytokine IL-5 is a prominent feature of airway inflammation in asthma. OBJECTIVE: The aim of this study was to determine whether exogenous IL-5 could cause changes in lung physiology, the early and late airway response after antigen challenge, and airway inflammation in rats that do not have a propensity to develop these changes after sensitization and challenge. METHOD AND RESULTS: Intratracheal administration of IL-5 to ovalbumin sensitized Brown Norway SSN rats increased the airway responsiveness to methacholine (AHR) 20 hours after administration of IL-5 at the same time as an increase in neutrophils occurred in the lung lavage. This effect was dose dependent and was not caused by endotoxin. Concurrent intratracheal administration of 50 ng of anti-IL-5 monoclonal antibody with 10 microg of recombinant human IL-5 decreased the AHR and neutrophil influx. Pretreatment with 3 microg of IL-5 had no effect on the early and late airway response or on AHR after ovalbumin challenge. However, IL-5 increased lung re-sistance 20 hours after antigen challenge. Although total lung cells and differential counts did not differ significantly 8 hours after antigen challenge, the blood lymphocyte CD4/CD8 ratio decreased in IL-5 pretreated rats (P <.05). In addition, in situ hybridization showed a significant increase in cells within the airway wall expressing IL-4 and IL-5 mRNA in IL-5 treated/challenged rats compared to controls (P <.05). CONCLUSION: The intratracheal administration of IL-5 causes only part of the physiologic changes that are associated with asthma. Other factors are necessary to obtain the complete asthma phenotype.

Activated CD4+ helper T cells have been demonstrated in asthmatic airways and postulated to play a central role in eliciting allergic inflammation; direct evidence of their involvement seems to be lacking. We hypothesized that CD4+ T cells have the potential to induce allergic responses to antigen challenge, and tested this hypothesis in a model of allergic bronchoconstriction, the Brown Norway rat, using the approach of adoptive transfer. Animals were actively sensitized to either ovalbumin (OVA) or BSA and were used as donors of T cells. W3/25(CD4)+ or OX8(CD8)+ T cells were isolated from the cervical lymph nodes of sensitized donors and transferred to naive BN rats. 2 d after adoptive transfer recipient rats were challenged by OVA inhalation, and changes in lung resistance (RL), bronchoalveolar lavage (BAL) cells, and serum levels of antigen-specific IgE were studied. After OVA challenge recipients of OVA-primed W3/25+ T cells exhibited sustained increases in RL throughout the entire 8-h observation period and had significant bronchoalveolar lavage eosinophilia, which was detected by immunocytochemistry using an antimajor basic protein mAb. Recipients of BSA-primed W3/25+ T cells or OVA-primed OX8+ T cells failed to respond to inhaled OVA. OVA-specific immunoglobulin E was undetectable by ELISA or skin testing in any of the recipient rats after adoptive transfer. In conclusion, antigen-induced airway bronchoconstriction and eosinophilia were successfully transferred by antigen-specific W3/25+ T cells in Brown Norway rats. These responses were dependent on antigen-primed W3/25+ T cells and appeared to be independent of IgE-mediated mast cell activation. This study provides clear evidence for T cell mediated immune mechanisms in allergic airway responses in this experimental model.

Typically, pharmacokinetic studies in mice require one animal per time point, thus resulting in differences due to dosing error, animal to animal variation and more importantly the euthanasia of a large number of animals. A method for the determination of pharmacokinetic data from serially bled mice to support early drug discovery is described. Sample analysis relies on liquid chromatography coupled with tandem mass spectrometry permitting robust and reproducible analysis requiring approximately 3 min per sample. Several parameters are discussed including the method of sample collection, preparation and analysis. The use of serially bled mice has lead to a remarkable reduction in animal usage and a corresponding reduction in compound required for such experiments. Using conventional methodology, a nine-point pharmacokinetic curve with four animals per time point would require 36 mice. With the method described below, only four mice in total are used and euthanasia is not required, permitting reuse after several weeks recovery and washout. Also, pharmacodynamic-pharmacokinetic correlation is possible and is demonstrated using a mouse model of diabetes.

The purpose of the study was to investigate the relationships between upper airways responses and pulmonary responses of two strains of highly inbred rats to inhaled antigen. To do this we measured the upper and lower airways resistance for 60 min after challenge of Brown-Norway rats (BN; n = 13) and an inbred rat strain (MF; n = 11), derived from Sprague-Dawley, with aerosolized ovalbumin (OA). Rats were actively sensitized with OA (1 mg sc) using Bordetella pertussis as an adjuvant. Two weeks later the animals were anesthetized and challenged. Tracheal pressure, esophageal pressure, and airflow were measured, from which total pulmonary resistance was partitioned into upper airway and lower pulmonary resistance (RL). The peak upper airway response to inhaled OA was similar in BN (1.89 +/- 0.66 cmH2O.ml-1.s; n = 7) and MF (2.85 +/- 0.68 cmH2O.ml-1.s; n = 6). The lower airway response to OA challenge was substantially greater in BN, and RL changed from 0.07 +/- 0.01 to 0.34 +/- 0.13 (n = 6; P < 0.05). The MF did not have any significant increase in RL after challenge; the baseline RL was 0.12 +/- 0.02 and only reached a peak value of 0.15 +/- 0.05 (n = 5; P = NS). Lower airway responsiveness of BN (n = 10) to serotonin, an important mediator early allergic airway responses, was similar to MF (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of the present investigation was to examine the effects of an inhaled glucocorticoid, budesonide, on antigen-induced production of cysteinyl leukotrienes (cys-LTs) and pulmonary inflammatory cell infiltration in the Brown Norway rat, an animal model of asthma. Two weeks after sensitization to ovalbumin, rats were treated with budesonide (2.5 mg/kg) 18 and 1 h before challenge with antigen. Budesonide abolished the late response to ovalbumin (P<0.02) and strongly inhibited the in vivo synthesis of N-acetyl-leukotriene E(4), an indicator of cys-LT synthesis, during this period (P<0.005). Both total bronchoalveolar lavage (BAL) cells (P<0.01) and BAL macrophages (P<0.005) were markedly reduced to approximately 25% of their control levels after treatment with budesonide. It can be concluded that inhibition of the antigen-induced late response in Brown Norway rats by budesonide is associated with reductions in both BAL macrophages and cys-LT synthesis. It is possible that the effect of budesonide on cys-LT synthesis is related to its effects on pulmonary macrophages.

The COX-2 inhibitor DFP [5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone] was found to have a long half-life in humans. Analogues have been characterized in order to optimize pharmacokinetics. This has lead to the discovery of 5(S)-(5-ethyl-5-methyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone analogue 11 a potent and selective COX-2 inhibitor which is metabolized to a greater extent than DFP upon incubation with rat and human hepatocytes, suggesting a shorter half-life in humans.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1)(IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration.(ABSTRACT TRUNCATED)

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50)= 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50)= 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50)> 50 microM in U937 cells and IC(50)> 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50)= 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50)= 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50)= 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50)= 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50)= 0.24 mg/kg), and adjuvant-induced arthritis (ID(50)= 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.

To evaluate the role of lymphocytes in the pathogenesis of allergic bronchoconstriction, we investigated whether allergic airway responses are adoptively transferred by antigen-primed lymphocytes in Brown Norway (BN) rats. Animals were actively sensitized to ovalbumin (OA) or sham sensitized, and 14 d later mononuclear cells (MNCs) were isolated from intrathoracic lymph nodes, passed through a nylon wool column, and transferred to naive syngeneic rats. Recipients were challenged with aerosolized OA or bovine serum albumin (BSA)(5% wt/vol) and analyzed for changes in lung resistance (RL), airway responsiveness to inhaled methacholine (MCh), and bronchoalveolar lavage (BAL) cells. Recipients of MNCs from sensitized rats responded to OA inhalation and exhibited sustained increases in RL throughout the 8-h observation period, but without usual early airway responses. Recipients of sham-sensitized MNCs or BSA-challenged recipients failed to respond to antigen challenge. At 32 h after OA exposure, airway responsiveness to MCh was increased in four of seven rats that had received sensitized MNCs (p = 0.035). BAL eosinophils increased at 32 h in the recipients of both sensitized and sham-sensitized MNCs. However, eosinophil numbers in BAL were inversely correlated with airway responsiveness in the recipients of sensitized MNCs (r =-0.788, p = 0.036). OA-specific immunoglobulin E (IgE) was undetectable by enzyme-linked immunosorbent assay (ELISA) or passive cutaneous anaphylaxis (PCA) in recipient rats following adoptive transfer. In conclusion, allergic late airway responses (LAR) and cholinergic airway hyperresponsiveness, but not antigen-specific IgE and early responses, were adoptively transferred by antigen-primed lymphocytes in BN rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Even though the eosinophil is potentially an important contributor to airway narrowing during the late allergic airway response, direct evidence of its participation is lacking. Therefore, we examined the effects of eosinophilia induced by Sephadex on the magnitude of the late airway response of sensitized rats following allergen challenge. Brown Norway rats were actively sensitized to ovalbumin (OA). At the same time and 14 days later, a test group was administered Sephadex G200 (0.5 mg intravenously). The animals were challenged with an aerosol of OA and pulmonary resistance (RL) was measured over 6 h. The early response to OA reached a peak more rapidly and the magnitude of the late response, measured as the area under the curve of RL against time, was significantly greater in the Sephadex-treated group (48.3; geometric mean) compared to the control animals (18.9; p < 0.02). The percentage of eosinophils was increased in the bronchoalveolar lavage of Sephadex-treated animals (4%) compared to the controls (0.9%; p < 0.02) following OA challenge. These results demonstrate that Sephadex induces eosinophilia in Brown Norway rats and is associated with an increase in the late allergic airway response. This is consistent with the hypothesis that the eosinophil is an important determinant of the late response.

To investigate if airway-parenchymal interdependence may account for differing bronchial responsiveness between inbred rat strains, Fisher and Lewis 12-wk-old male rats were anesthetized, tracheostomized, and placed in a pressure plethysmograph. Functional residual capacity, total lung capacity [lung volume at transpulmonary pressure (PL) of 30 cmH2O], and specific compliance were determined and were found to be similar. Rats were paralyzed and mechanically ventilated. Concentration-response curves were constructed by calculating lung resistance (RL) and lung elastance (EL) after nebulization of saline and then doubling doses of methacholine (0.0625-512 mg/ml). In Fisher (n = 8) and Lewis (n = 7) rats RL and EL were again determined at a lung volume corresponding to 2 cmH2O PL above FRC. The doubling, maximal, and half-maximal effective concentrations were determined for RL and EL. The doubling of effective concentrations of RL and EL were significantly less for Fisher rats. Other groups of Fisher (n = 5) and Lewis (n = 5) rats were similarly exposed to three concentrations of methacholine (64, 128, and 256 mg/ml), and determinations of RL and EL were made at lung volume corresponding to PL of 0, 2, 4, and 8 cmH2O. In both groups, Lewis rats exhibited a significant effect of volume on maximal RL and EL, whereas Fisher rats did not. The absence of volume effect on bronchoconstriction in the hyperresponsive Fisher strain is consistent with the hypothesis that altered airway-parenchymal interdependence contributes to bronchial hyperresponsiveness.

The purpose of this study was to determine the relative contributions of airway wall edema and smooth muscle contraction to the early response (ER) of allergic bronchoconstriction. Brown Norway rats, 6 to 7 wk old, were sensitized with ovalbumin (OA). Anesthetized rats were challenged with either OA or saline 2 wk later. Pulmonary resistance (RL) was measured every minute until either it increased to 150% of the baseline, defined as a significant ER, or until 15 min elapsed. Eight OA-challenged test rats with a significant ER and eight saline-challenged control rats were used for morphometric studies. The lungs were quick-frozen with liquid nitrogen, processed with freeze substitution, and sagittal sections (5 microns) were stained with hematoxylin and eosin. The airway lumen subtended by the epithelial basement membrane (LuB) and cross sectional airway wall area (AW) of all airways were measured by camera lucida and digitization. The LuB and AW of each airway was standardized for size by dividing by the ideal airway lumen (LuBideal), which was calculated from the length of basement membrane, assuming a perfect circle in the unconstricted state. The cumulative frequency distribution of the LuB/LuBideal for the airways from test rats was shifted to the left compared with the control rats (p less than 0.01), indicating airway narrowing after challenge. Airway narrowing increased as a function of airway size. Cumulative frequency distributions of AW/LuBideal showed that there was a significant increase in the wall thickness of only the small airways of test animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of the study was to investigate strain dependence and mechanisms of airway responses to dry-gas hyperpnea challenge in the rat. We studied responses in a strain that is hyperresponsive to methacholine, Fischer 344 (F-344); in two normoresponsive strains, Lewis and ACI; and in an atopic but normoresponsive strain, Brown Norway (BN). We examined the effects of a neurokinin (NK) 1-receptor (CP-99994), an NK2-receptor (SR-48968), and a leukotriene D4 (LTD4)-receptor antagonist (pranlukast) on responses to hyperpnea challenge in BN rats. The animals were ventilated with a tidal volume of 8 ml/kg and a frequency of 150 breaths/min with either a dry or humidified mixture of 5% CO2-95% O2 for 5 min for hyperpnea challenge, whereas responses to challenge were measured during spontaneous breathing. Pulmonary resistance increased after dry-gas challenge in BN and ACI but not in F-344 and Lewis rats. CP-99994, SR-48968, and pranlukast significantly attenuated the increase in pulmonary resistance after dry-gas challenge. There were no significant differences in responsiveness to airway challenge with LTD4 among the BN, F-344 and ACI rats. We conclude that responses to dry-gas hyperpnea challenge are strain dependent in rats and are mediated by NKs and LTD4.

T cell cytokines are important in asthma. Interleukin (IL)-3, an important growth factor for mast cells and eosinophils has been shown to be increased in the airways of asthmatic subjects, but its precise functions are uncertain. The aim of this study was to determine whether recombinant human (rh) IL-3 affected airway responses, inflammation and leukotriene production after antigen challenge in Brown Norway (BN) rats. Having established that rhIL-3 (>12.5 microg subcutaneously b.i.d. for 4 days) caused a doubling of mast cell numbers in the airways of BN rats, sensitized rats were pretreated with rhIL-3 (50 microg) or vehicle subcutaneously b.i.d. for 4 days. Ovalbumin (OA) challenge was performed and the early (EAR), and late (LAR) airway response and the associated biliary leukotriene (LT) excretion measured. The pulmonary cellularity was evaluated by means of lung digestion 8 h after challenge. IL-3 increased the number of eosinophils isolated from the lungs after antigen challenge (0.77+/-0.23 versus 0.38+/-0.12 x 10(6) cells, p=0.03). However, there were no effects on the numbers of neutrophils, lymphocytes and macrophages. Neither the EAR nor the LAR after OA challenge were altered by IL-3. Likewise biliary cysteinyl-LT excretion was similar in IL-3-treated animals and controls after challenge. In conclusion, interleukin-3 caused an increase in the numbers of mast cells and eosinophils around the airways without affecting the magnitude of either early or late airway responses or mediator release after antigen challenge. The present results suggest that airway inflammation can occur in rats without increasing the allergic asthmatic response.

A series of novel 2-pyridinyl-3-(4-methylsulfonyl)phenylpyridines has been synthesized and evaluated with respect to their ability to inhibit the isozymes of cyclooxygenase, COX-1, and COX-2. Optimum COX-2 activity is observed by introduction of a substituent at C5 of the central pyridine. 5- Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine 33 was identified as the optimum compound in this series.

By inserting an oxygen link between the 3-fluorophenyl and the lactone ring of 5,5-dimethyl-3-(3fluorophenyl)-4-(4-methanesulfonylphenyl)-2 (5H)-furanone 1 (DFU), analogs with enhanced in vitro COX-2 inhibitory potency as well as in vivo potency in models of inflammation were obtained.

Isocapnic dry gas hyperpnea-induced bronchoconstriction (HIB) in the guinea-pig is mediated by both tachykinin release from airway sensory nerve C-type fiber terminals and secondary synthesis of cysteinyl-leukotrienes, in particular LTD(4). Beta (beta)(2)-agonists are potent bronchodilators but potentially could also inhibit the airway response to hyperpnea challenge via effects on the release of LTD(4)from airway cells in vivo. The purpose of this study was to test the hypothesis that beta(2)agonists attenuate HIB in guinea-pigs, in part, by reduction in LTD(4)release in vivo. Twenty-six guinea-pigs (400-550 g) were anesthetized with xylazine (7 mg/kg) and pentobarbital (65 mg/kg), tracheotomized and mechanically ventilated with a small animal ventilator using a tidal volume of 3 ml and a breathing frequency of 60 breaths/min. Dry gas (95%O(2)/5%CO(2)) with a 4 ml tidal volume and a breathing frequency of 150/min was used for hyperpnea challenge. Challenge with isocapnic dry gas triggered a significant increase in pulmonary resistance (0.3 +/- 0.02 vs. 0.57 +/- 0.06 cmH(2)O/ml per s; P=0. 017; n=13) and excretion of LTD(4)in the bile (baseline: 2.43 vs. HIB: 4.66 pmol/h; P=0.04). Salbutamol pretreatment completely blocked the airway response to the challenge (0.3+/-0.02 vs. 0.3+/-0. 05 cmH(2)O/ml per s; n=13) and reduced the biliary excretion of LTD(4)(baseline: 2.42 pmol/h; vs. HIB: 2.40 pmol/h). We conclude that salbutamol inhibited the airway responses to dry gas hyperpnea challenge and LTD(4)synthesis by the airway cells.