Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

A prostaglandin D2 receptor antagonist modifies experimental asthma in sheep.

BACKGROUND: Prostaglandin (PG) D(2) is the major cylooxygenase metabolite released by mast cells upon allergen stimulation, and elicits responses through either the prostanoid DP1 receptor and/or the chemoattractant receptor homologous molecule expressed on T-helper type 2 (Th2) cells (CRTH2/DP2). Experimental evidence suggests that stimulation of one or both these receptors contributes to asthma pathophysiology. OBJECTIVE: The aim of this study was to test the hypothesis that the prostanoid DP1 receptor contributes to asthma pathophysiology by determining the efficacy of an orally active antagonist for this receptor, S-5751, on allergen-induced bronchoconstriction, airway hyperresponsiveness (AHR) and cellular inflammation in the sheep model of asthma. METHODS: PGD(2)-induced cyclic adenosine monophosphate (cAMP) production in platelet-rich plasma was used to establish the in vitro efficacy of S-5751. In vivo, sheep naturally allergic to Ascaris suum were challenged with an aerosolized antigen with and without S-5751 treatment (given 4 days before and for 6 days after the challenge). RESULTS: S-5751 inhibited PGD(2)-induced cAMP production in platelet-rich plasma with an IC(50) value of 0.12 microm. S-5751 at 30 mg/kg, but not at 3 mg/kg, reduced the early bronchoconstriction and inhibited the late bronchoconstriction. AHR and inflammatory cell infiltration in bronchoalveolar lavage fluid at days 1 and 7 were also inhibited with the 30 mg/kg dose. The responses observed with S-5751 at 30 mg/kg were comparable with those with montelukast treatment (0.15 mg/kg, twice a day, intravenous); however, S-5751 did not block inhaled leukotrieneD(4)-induced broncoconstriction. CONCLUSION: Prostanoid DP1 receptor inhibition may represent an alternative target for asthma therapy.

Alpha 4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.

Eosinophils and T lymphocytes are thought to be involved in allergic airway inflammation. Both cells express the alpha 4 beta 1-integrin, very late antigen-4 (VLA-4, CD49d/CD29); alpha 4-integrins can promote cellular adhesion and activation. Therefore, we examined the in vivo effects of a blocking anti-alpha 4 monoclonal antibody, HP 1/2, on antigen-induced early and late bronchial responses, airway hyperresponsiveness, inflammatory cell influx, and peripheral leukocyte counts in allergic sheep. Sheep blood lymphocytes, monocytes, and eosinophils expressed alpha 4 and bound HP 1/2. In control sheep, Ascaris antigen challenge produced early and late increases in specific lung resistance of 380 +/- 42% and 175 +/- 16% over baseline immediately and 7 h after challenge, respectively, as well as airway hyperresponsiveness continuing for 14 d after antigen challenge. Treatment with HP 1/2 (1 mg/kg, i.v.) 30 min before antigen challenge did not affect the early increase in specific lung resistance but inhibited the late-phase increase at 5-8 h by 75% (P < 0.05) and inhibited the post-antigen-induced airway hyperresponsiveness at 1, 2, 7, and 14 d (P < 0.05, for each time). Intravenous HP 1/2 given 2 h after antigen challenge likewise blocked late-phase airway changes and postchallenge airway hyperresponsiveness. Airway administration of HP 1/2 (16-mg dose) was also effective in blocking these antigen-induced changes. Response to HP 1/2 was specific since an isotypic monoclonal antibody, 1E6, was ineffective by intravenous and aerosol administration. Inhibition of leukocyte recruitment did not totally account for the activity of anti-alpha 4 antibody since HP 1/2 neither diminished the eosinopenia or lymphopenia that followed antigen challenge nor consistently altered the composition of leukocytes recovered by bronchoalveolar lavage. Because airway administration of HP 1/2 was also active, HP 1/2 may have inhibited cell activation. Reduction of platelet-activating factor-induced eosinophil peroxidase release from HP 1/2-treated eosinophils supports such a mechanism. These findings indicate a role for alpha 4-integrins in processes that lead to airway late phase responses and persisting airway hyperresponsiveness after antigen challenge.

L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition.

Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2 nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFalpha formation in whole blood (IC50 = 161 nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNgamma and no detectable inhibition of IL-13 formation up to 1 microM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Scavenging of reactive oxygen species by a glycolipid fraction of Mycobacterium avium serovar 2.

Previous experiments indicated that MIF-A3, a peptidoglycolipid extracted from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18), inhibits the killing of Candida albicans by activated bovine peripheral blood-derived macrophages and murine thioglycollate-elicited peritoneal macrophages in vitro. Subsequent in vitro data from our laboratory indicated that this reduction in killing may be related to the ability of MIF-A3 to scavenge reactive oxygen species (ROS). In this study we examined this hypothesis directly by determining if MIF-A3 reduced exogenous H2O2-induced candidacidal activity. When Candida albicans was incubated with H2O2 (4 mM) alone, colony-forming units/ml x 10(4) (CFU/ml) were 0.4 +/- 0.1 (mean +/- SE, n = 4) as compared to 11.3 +/- 2.0 CFU/ml in control (untreated) cultures (p < .05). The addition of catalase at concentrations > or = 6.8 U/ml, completely blocked the fungicidal effect of H2O2. However, reducing the amount of catalase from 6.8 U/ml to 3.4 U/ml resulted in a loss of scavenging activity, which was associated with a 50% increase in H2O2-mediated killing. Substituting MIF-A3 (400 micrograms/ml) for catalase, also reduced H2O2-induced fungicidal activity. In the absence of MIF-A3, H2O2 reduced Candida albicans to less than 10(3) CFU/ml. However, in the presence of MIF-A3 the CFU/ml of Candida albicans increased 7.5-fold. Based on concentration-response curves of H2O2 inhibition vs. increasing amounts of catalase we determined that the relative inhibitory capacity of the MIF-A3 (400 micrograms/ml) was approximately 1.0 U/ml "catalase equivalents." These findings provide direct evidence that MIF-A3 can scavenge H2O2, and reduce H2O2-induced killing of Candida albicans.

Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses.

Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

Effect of nedocromil sodium on antigen-induced airway responses in allergic sheep.

We studied the effects of nedocromil sodium and sodium cromoglycate on early and late bronchial responses, the airway inflammation associated with the late response to inhaled Ascaris suum antigen in allergic sheep in vivo, and the antigen-induced contractile responses of sheep tracheal smooth muscle in vitro. In addition, we examined the effect of nedocromil sodium on the development of antigen-induced airway hyper-responsiveness in this model. Pretreatment with either nedocromil sodium or sodium cromoglycate was effective in blocking antigen-induced early and late responses in allergic sheep. Both drugs also prevented the influx of eosinophils into the airways as assessed by bronchoalveolar lavage, observed during the late response in this model. No difference in drug potency was observed in vivo, but in vitro nedocromil sodium was 10-fold more potent than sodium cromoglycate against antigen-induced contractions of sheep tracheal smooth muscle. Nedocromil sodium was effective in blocking antigen-induced late responses and the subsequent development of airway hyper-responsiveness irrespective of whether the drug was given before antigen challenge or after the immediate response to antigen but before the late response. These findings indicate that nedocromil sodium is effective in the sheep model of asthma and therefore may be beneficial in the treatment of reversible obstructive airway disease in man.

Inhibition of allergen-induced pulmonary responses by the selective tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-phenoxy]-pen tane (AMG-126737).

Emerging evidence suggests that mast cell tryptase is a therapeutic target for the treatment of asthma. The effects of this serine protease are associated with both pathophysiologic pulmonary responses and pathologic changes of the asthmatic airway. In this study, the tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-p henoxy]-pentane (AMG-126737) was evaluated for its pharmacologic effects against allergen-induced airway responses. AMG-126737 is a potent inhibitor of human lung mast cell tryptase (Ki = 90 nM), with greater than 10- to 200-fold selectivity versus other serine proteases. Intratracheal administration of AMG-126737 inhibited the development of airway hyperresponsiveness in allergen-challenged guinea pigs with an ED50 of 0.015 mg/kg. In addition, the compound exhibited oral activity in the guinea pig model. The in vivo activity of AMG-126737 was confirmed in a sheep model of allergen-induced airway responses, where the compound inhibited early and late phase bronchoconstriction responses and the development of airway hyperresponsiveness. These results support the proposed role of tryptase in the pathology of asthma and suggest that AMG-126737 has potential therapeutic utility in this pulmonary disorder.

Plasma thrombospondin levels in sheep with allergic asthma.

STUDY OBJECTIVE: In search of a better universal marker of tissue inflammation in allergic reactions, we studied platelet activation and its relation to allergic asthma in sheep. SETTING: University research laboratory. SUBJECTS: Twenty-three allergic sheep. DESIGN AND INTERVENTIONS: We serially measured plasma thrombospondin (TSP), a glycoprotein secreted from activated platelets, using enzyme-linked immunosorbent assay with a monoclonal antibody. We validated the assay by establishing a standard curve that closely matches that of humans. MEASUREMENTS AND RESULTS: Of the 23 allergic sheep tested, 10 demonstrated early airway response (ER group) and 13 had early and late dual airway responses (DR group) to inhaled Ascaris suum antigen. TSP was measured before, and at 0, 1, 2, 3, 4, 6, 8, and 24 h postantigen challenge. Both groups showed an increase in plasma TSP level after antigen challenge, but only the DR group showed a significant increase in TSP at I h (threefold) and 8 h (sixfold) postchallenge. The two sheep that received carbachol challenge showed a slight rise in TSP at 8 h postchallenge. There was a significant correlation between the TSP level at I h and the peak early airway response in the DR group. CONCLUSIONS: These results suggest that the two groups of allergic sheep may have two distinct induction processes leading to airway obstruction. The dual peak rise in TSP level that coincided with the airway response also suggests that an early priming effect exists in the DR group, which might have led to the late response. We conclude that intravascular platelet activation often occurs in mast cell-mediated allergic disorders. Therefore, plasma TSP may be useful for monitoring the course of allergic disorders.

The effect of nedocromil sodium and cromolyn sodium on antigen-induced responses in allergic sheep in vivo and in vitro.

We studied the effects of nedocromil sodium and cromolyn sodium on early and late bronchial responses to inhaled Ascaris suum antigen in allergic sheep in vivo, and the antigen-induced contractile responses of sheep tracheal smooth muscle in vitro. For the in vivo studies the sheep were pretreated with aerosols of placebo (buffered saline solution), 20 mg of nedocromil sodium, or 20 mg of cromolyn sodium (both dissolved in 3 ml of buffered saline solution) and then challenged with aerosol antigen. Specific pulmonary resistance (SRL) was measured before and after challenge to document the responses of the airways. In the trial with placebo, challenge with antigen resulted in significant early and late increases in SRL. Treatment with nedocromil sodium significantly reduced the early response to antigen and blocked the late response. Cromolyn sodium gave the same results; there were no statistical differences between the responses of the airways for the two dogs. In vitro nedocromil sodium at doses of 10(-6)M and 10(-5)M inhibited significantly the contractile responses of sheep tracheal smooth muscle to A suum. Cromolyn sodium only showed efficacy at 10(-5)M. These results suggest that nedocromil sodium may be potentially useful in the treatment of reversible allergic disease of the airways.

The role of leukotrienes in allergen-induced late responses in allergic sheep.

Evidence has been presented that lipoxygenase products of arachidonic acid metabolism released during the early stages of allergic reactions play an important role in the subsequent development of late bronchial responses. Moreover, because of this, the activity of the lipoxygenase pathway and sensitivity to the products generated may be important factors that distinguish between dual and acute responders. We recognize that these studies were performed in an experimental animal model of allergic airway disease. If the same mechanisms that are active in sheep are proven active in humans, then pharmacologic modification of the lipoxygenase pathway or its products (or both) may be important for the treatment of some forms of asthma.

Pathophysiologic role of alpha 4 integrins in the lung.

Evidence for a central role for the integrins alpha 4 beta 1 and alpha 4 beta 7 in leukocyte pathophysiology is rapidly accumulating. Five distinct alpha 4 mAbs, each able to block alpha 4-dependent adhesion in vitro, show beneficial effects in vivo in six different species, and in a wide variety of organ systems, including colon, lung, skin, neural tissue, pancreas, peritoneum, and the vessel wall. In particular, a clear role for these integrins in lung pathophysiology is implied on the basis of in vivo studies in four different species. Although several issues remain to be resolved, including the relative importance of alpha 4 beta 1 and alpha 4 beta 7, and the relative roles of their counterligands, VCAM1, fibronectin, and MAdCAM, the data argue that alpha 4 integrins will likely be critical to both the normal physiology and pathology of the lung in man. To this end, we (Adams, Lin, Lobb, and Gill, unpublished data) and others have generated peptidomimetic small molecule antagonists of VLA4 based on the connecting segment 1 (CS1) peptide sequence of fibronectin that are potent blockers of integrin adhesive function in vitro and show efficacy in vivo. We have found that our inhibitors are excellent blockers of both murine contact hypersensitivity, and of the LPR and AHR in the sheep allergic airways model (Abraham, Lobb, Adams, and Gill, unpublished data), and are therefore possible candidates for clinical intervention in human asthma. The use of the VCAM-Ig fusion protein as a probe for high-affinity alpha 4 integrins has further enhanced our understanding of alpha 4 integrin function in the lung. While integrin upregulation in vitro has been observed many times, and high affinity (as opposed to avidity) of integrins seen in vitro in several systems, in vivo proof of integrin upregulation to a high-affinity state has been difficult to obtain in the absence of selective probes. Our data provide key information in this regard and strongly argue not only that integrin upregulation does indeed occur in vivo, but also that it is in fact obligatory for the leukocyte pathologies we have examined to date. Further studies are clearly warranted to further examine mechanisms of action, and to confirm and extend these studies, both with the alpha 4 integrins and with other integrin families. In summary, our studies of alpha 4 integrins continue to provide novel insights into the pathophysiology of integrin function and into future directions for drug discovery.

Separation of late bronchial responses from airway hyperresponsiveness in allergic sheep.

Allergic sheep with antigen-induced early and late responses were used to determine whether airway hyperresponsiveness (AHR) to carbachol is present during the late response and whether blocking the late response with the leukotriene D4 (LTD4) antagonist MK-571 also blocks this AHR. To do this, we first showed that MK-571 blocked the antigen-induced late response, and then, in a separate study, we determined the effect of MK-571 treatment on airway responsiveness 6 h after antigen challenge (at the start of the late response). MK-571 (5 mg, by metered dose inhaler) given 30 min before and 4 h after Ascaris suum challenge had no effect on the acute response to antigen but blocked (P less than 0.05) the late response compared with placebo (n = 7). In the second study (n = 6), the antigen-induced acute increases in mean specific lung resistance (sRL) were also similar in the placebo (249%) and drug trials (247%). By 6 h postchallenge, however, mean sRL in the placebo trial began to increase (54%, P less than 0.05), whereas in the drug trial mean sRL was baseline. Nevertheless, AHR was apparent in both trials as indicated by a mean twofold leftward shift in the dose-response curves to inhaled carbachol (P less than 0.05 vs. prechallenge). Bronchoalveolar lavage at 6 h showed that MK-571 did not prevent the inflammatory cell influx into the lung. These observations suggest that although LTD4 may be a mediator of the late response in sheep, it is not a primary mediator affecting cholinergic AHR during this period.

A PAF antagonist blocks antigen-induced airway hyperresponsiveness and inflammation in sheep.

We studied the effects of WEB-2086, a specific antagonist of platelet-activating factor (PAF), on the development of antigen-induced airway hyperresponsiveness and inflammation in sheep (n = 8). For these studies, airway responsiveness was determined from slopes of carbachol dose-response curves (DRC) performed at base line (prechallenge) and 2 h after Ascaris suum antigen challenges in the following three protocols: 1) antigen challenge alone (control trial), 2) WEB-2086 (1 mg/kg iv) given 30 min before antigen challenge (WEB pretreatment), and 3) WEB-2086 given 2 h after antigen challenge, immediately before the postchallenge DRC (WEB posttreatment). Airway inflammation was assessed by bronchoalveolar lavage (BAL) before antigen challenge and after the postchallenge DRC for each trial. A. suum challenge resulted in acute increases in specific lung resistance that were not different among the three trials. Antigen challenge (control trial) caused a 93% increase (P less than 0.05) in the slope of the carbachol DRC when compared with the prechallenge value. WEB pretreatment (1 mg/kg) reduced (P less than 0.05) this antigen-induced hyperresponsiveness, whereas pretreatment with a 3-mg/kg dose completely prevented it. WEB posttreatment was ineffective in blocking this hyperresponsiveness. BAL neutrophils increased after antigen challenge in the control trial and when WEB-2086 was given after antigen challenge (P less than 0.05). Pretreatment with WEB-2086 (1 or 3 mg/kg) prevented this neutrophilia. This study provides indirect evidence for antigen-induced PAF release in vivo and for a role of endogenous PAF in the modulation of airway responsiveness and airway inflammation after antigen-induced bronchoconstriction in sheep.

A possible role for PAF in allergen-induced late responses: modification by a selective antagonist.

We determined whether platelet-activating factor (PAF) plays a role in allergen-induced airway responses by studying the effects of a selective PAF antagonist WEB-2086 on antigen-induced early and late airway responses in allergic sheep. In seven sheep, inhaled Ascaris suum produced significant early (282%) and late (176%) increases in specific lung resistance (sRL). WEB-2086 (1 mg/kg iv) given 20 min before antigen challenge did not affect the early response, but the peak late increase in sRL was only 37% over base line (P less than 0.05 vs. control). To study the mechanism by which PAF contributes to antigen-induced responses, we evaluated the effects of pharmacological probes on PAF-induced bronchoconstriction. Inhaled PAF (dose range 75-700 micrograms) caused reproducible (r = 0.781, P less than 0.05) increases in sRL in eight sheep. The PAF-induced bronchoconstriction was blocked by WEB-2086 (1 mg/kg iv) and by the leukotriene antagonist FPL-55712 (30 mg by aerosol); however, neither the cyclooxygenase blocker indomethacin (2 mg/kg iv) nor the histamine H1-antagonist chlorpheniramine (2 mg/kg iv) blocked the PAF response. WEB-2086, however, did not block bronchoconstriction induced by aerosol leukotriene D4, indicating that PAF acts indirectly through leukotrienes. Finally, we determined whether PAF could induce late airway responses. Inhaled PAF produced an immediate increase in sRL in all seven sheep tested, but late airway responses were observed in only three of the seven sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuation of platelet-activating factor-induced airway responses with methylprednisolone succinate in sheep.

Intratracheal instillation of platelet-activating factor (PAF) in sheep produces bronchoconstriction and airway hyperresponsiveness (AHR) by a two-stage process that involves the initial stimulation of PAF receptors followed by the secondary generation of proinflammatory mediators. Because the biological effects of PAF may be mediated by these proinflammatory metabolites, it is possible that a steroidal anti-inflammatory agent would modify the airway responses of PAF. We measured specific lung resistance (sRL) in sheep (n = 11) before, immediately after, and serially for up to 2 h after intratracheal instillation of PAF (30 micrograms/kg). Airway responsiveness was measured 2 h post-PAF when sRL had returned to baseline and was expressed as the cumulative provocating dose of carbachol that increased sRL to 4 l.cmH2O.l-1.s (PD4). PD4 was determined on a control day and on different experiment days without or after treatment with intravenous methylprednisolone (MPS; 1 mg/kg) administered 3 h before (n = 6), 20 min before PAF (n = 7), or 20 min after PAF challenge (n = 7). PAF increased sRL by 222 +/- 44% (SE) above baseline and decreased PD4 of carbachol by 44 +/- 5% (P less than 0.05). Pretreatment (both 3 h and 20 min) with MPS attenuated the PAF-induced increases in sRL, whereas its administration 20 min post-PAF had no effect. Irrespective of the effects on sRL, MPS administration inhibited the PAF-induced AHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciliary responsiveness in allergic and nonallergic airways.

Allergic asthma is associated with airway (smooth muscle) hyperresponsiveness to several chemical mediators of anaphylaxis; however, it is not known whether this is accompanied by mucociliary hyperresponsiveness. The purpose of this study was therefore to determine if airway ciliary activity, a component function of mucociliary clearance, exhibits exaggerated responses to prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), and leukotriene D4 (LTD4) in allergic sheep when compared with nonallergic sheep, and the effects of LTD4 are direct or involve the generation of cyclooxygenase products of arachidonate metabolism. Ciliary beat frequency (CBF) was measured in a perfusion chamber with a microscopic technique using tracheal epithelial cells obtained from brushing of "allergic" (positive cutaneous reaction and previous bronchospastic response to inhaled specific antigen) and "nonallergic" (negative cutaneous reaction, no previous inhalation challenge with antigen) sheep. Mean base-line CBF was not different among the groups; PGE1, PGE2, and LTD4 induced dose-dependent increases in CBF, and these increases were not different in allergic and nonallergic sheep. At the highest agonist concentration the mean increase in CBF from base line varied between 13 and 16% (P less than 0.05). The ciliostimulatory effect of LTD4 was significantly blunted by both the sulfidopeptide leukotriene antagonist FPL-55712 and the cyclooxygenase inhibitor indomethacin. These results suggest that allergic sheep fail to exhibit ciliary hyperresponsiveness to selected chemical mediators of anaphylaxis and the ciliostimulatory effect of LTD4 depends on the activation of cyclooxygenase and possibly the generation of prostaglandins.

Late-phase bronchial vascular responses in allergic sheep.

Sheep were classified on the basis of their airway response to Ascaris suum antigen aerosols as allergic or nonsensitive. Allergic sheep were classed as acute or dual responders. Acute responders had only an immediate increase in mean airflow resistance after antigen, whereas dual responders had an immediate and late-phase (6-8 h after antigen challenge) increase in mean airflow resistance; nonsensitive sheep had minimal airway responses to antigen (less than 30% increase from base line). The sheep were anesthetized 2 wk later and, after a left thoracotomy, were challenged with antigen to determine bronchial vascular responses; bronchial artery blood flow was measured with an electromagnetic flow probe. Airway responses to antigen aerosol challenge were similar in the anesthetized and conscious animals. The mean fall in bronchial vascular resistance (BVR) immediately after antigen challenge was similar in acute and dual responders (41 +/- 7 and 47 +/- 9% of base line, respectively). In dual responders, late-phase airway responses were preceded by a significant increase from base line in Qbr and a fall in bronchovascular resistance (BVR). The mean fall in BVR 6-8 h after antigen challenge in documented dual responders was significantly different from bronchial vascular responses in acute responders (59 +/- 3 vs. 89 +/- 10%, respectively). Sheep without airway responses to A. suum had no significant changes in bronchial hemodynamics or airways mechanics. Late-phase-associated changes in BVR are a specific response to antigen challenge and may be a sensitive index of mediators being released.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific endothelin-1 antagonist blocks inhaled endothelin-1-induced bronchoconstriction in sheep.

In this study we used conscious sheep to compare the relative potencies of inhaled endothelin- (ET) 1 and ET-3 and to determine whether a newly described selective ET-1 receptor antagonist, [diaminopropionic acid1-Asp15]ET-1 ([Dpr1-Asp15]ET-1), when given as an aerosol blocks ET-1-induced bronchoconstriction. Partial concentration-response curves to ET-1 and ET-3 were obtained by measuring the change in pulmonary airflow resistance (RL) after aerosol challenge. Both ET-1 (n = 6) and ET-3 (n = 4) caused concentration-dependent (10(-10)-10(-7) bronchoconstriction, but ET-3 was 400-fold less potent than ET-1. Pretreatment (30 min) with 25 breaths of 5 x 10(-8) M [Dpr1-Asp15]ET-1 caused a 100-fold rightward shift of the ET-1 concentration-response curve. The activity of the ET-1 antagonist (25 breaths of 5 x 10(-8) M) was compared with that of various control peptides (25 breaths of 10(-8) M). ET-1 (50 breaths of 10(-7) M) caused an increase in RL of 133 +/- 33% (SE) over baseline. [Dpr1-Asp15]ET-1 significantly inhibited this response by 54%. No protection was seen with a monocyclic control peptide or a thrombin receptor peptide. There was, however, a small protective effect (38%, P < 0.05) seen with [Dpr1-Asp15]ET-3, a structurally homologous ET-3 antagonist. [Dpr1-Asp15]ET-1 had no effect on carbachol (n = 3) or leukotriene D4-induced bronchoconstriction. Thus inhaled ET-1 and, to a lesser extent, ET-3 cause concentration-dependent bronchoconstriction in conscious sheep.(ABSTRACT TRUNCATED AT 250 WORDS)