Role of sphingosine-1-phosphate (S1P) and the S1P(2) receptor in allergen-induced, mast cell-dependent contraction of rat lung parenchymal strips.

Lung parenchymal strips isolated from ovalbumin-sensitized rats manifest a mast cell-dependent, biphasic contraction when challenged with allergen. The first phase is mediated by the release of preformed 5-HT while the second phase is dependent on de novo synthesis of leukotrienes. Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite which is readily generated in mast cells and has been demonstrated to be an important regulator of allergen-induced mast cell activation. We have used the parenchymal strip to explore the role of sphingosine 1-phosphate and the S1P(2) receptor in the two components of the acute response to allergen. Lung parenchymal strips were prepared from Brown Norway rats actively sensitized to ovalbumin. The strips were set up in organ baths and contractile responses measured isometrically. The inhibitors of sphingosine kinase, D-erythro-NN-dimethylsphingosine (dimethylsphingosine) and 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) inhibited concentration-dependently both phases of the contractile response induced by 0.1 microg ml(-1) ovalbumin. The effects were seen at concentrations similar to those which inhibit the purified enzyme and were selective in that neither the contractile response to adenosine nor that to 5-hydroxytryptamine was affected. JTE-013 (a selective S1P(2) receptor antagonist) also blocked the response to ovalbumin (0.1 microg ml(-1)). However, the concentrations of JTE-013 required (microM) were substantially higher than its affinity for the S1P(2) receptors (nM). However, when tested against a lower concentration of ovalbumin (0.03 microg ml(-1)), JTE-013 inhibited the response with nM potency. These data demonstrate the importance of S1P and the S1P(2) receptor as regulators of allergen-induced activation of mast cells in their natural environment in the rat lung.

In vivo pharmacological evaluation of compound 48/80-induced airways oedema by MRI.

BACKGROUND AND PURPOSE: Allergen-induced airways oedema in actively sensitized rats has been studied earlier by magnetic resonance imaging (MRI). We used MRI to follow the consequences of non-immunological mast cell activation induced by compound 48/80 in the rat lungs in vivo. EXPERIMENTAL APPROACH: Male naïve rats were scanned by MRI prior to and at several time points following intratracheal administration of the mast cell secretagogue, compound 48/80. The effects of a range of drugs on the response induced by compound 48/80 were studied. KEY RESULTS: Strong fluid signals were detected by MRI in the lungs at 24 h after compound 48/80, correlating with increased protein concentration and inflammatory cell infiltration in bronchoalveolar lavage, and with perivascular oedema observed histologically. Pharmacological intervention demonstrated that the increase in MRI signal volume induced by compound 48/80 24 h after challenge was blocked by disodium cromoglycate and the glucocorticoid, budesonide. Pretreatment with wortmannin, capsazepine, DNK333 (a dual neurokinin (NK) 1 and NK2 antagonist) or the anti-allergy drug CGS8515, but not indomethacin, resulted in partial inhibition. CONCLUSIONS AND IMPLICATIONS: Compound 48/80 induced a complex inflammatory reaction which did not solely involve mast cell degranulation but also activation of sensory nerves and was qualitatively similar to allergen challenge. Changes observed by MRI correlated with decreases in protein concentration in BAL fluid. However, the magnitude of the changes detected was greater using MRI. Our results demonstrate that MRI is a sensitive and efficient tool to assess the effects of drugs on lung inflammation.

Capsaicin-induced mucus secretion in rat airways assessed in vivo and non-invasively by magnetic resonance imaging.

BACKGROUND AND PURPOSE: An up-regulation of the sensory neural pathways in the lung has been implicated in asthma and chronic obstructive pulmonary disease (COPD) and is thought to contribute to mucus hypersecretion, an essential feature of both diseases. The aim of this study was to assess non-invasively the acute effects (up to 60 min) of sensory nerve stimulation by capsaicin in the lung, using magnetic resonance imaging (MRI). EXPERIMENTAL APPROACH: Male Brown Norway rats were imaged prior to and 10, 30 and 60 min after intra-tracheal challenge with capsaicin (30 microg kg(-1)) or vehicle (0.5% ethanol solution). In subsequent studies, pre-treatment with the transient receptor potential vanilloid (TRPV)-1 antagonist, capsazepine; the dual neurokinin (NK) 1 and NK2 receptor antagonist, DNK333 and the mast cell stabilizer, di-sodium cromoglycate (DSCG) was used to modulate the effects of capsaicin. KEY RESULTS: Diffuse fluid signals were detected by MRI in the lung as early as 10 min after capsaicin, remaining constant 30 and 60 min after treatment. Broncho-alveolar lavage (BAL) fluid analysis performed 60 min after capsaicin revealed increased mucin concentration. Capsazepine (3.5 mg kg(-1)), DNK333 (10 mg kg(-1)) but not DSCG (10 mg kg(-1)) administered prophylactically were able to block the effect of capsaicin in the airways. CONCLUSIONS AND IMPLICATIONS: These observations suggest that the fluid signals detected by MRI after capsaicin administration reflected predominantly the release of mucus following activation of sensory nerves. They point to the opportunity of non-invasively assessing with MRI the influence of neuronal mechanisms in animal models of asthma and COPD.

Effects of endotoxin and allergen alone and in combination on the sensitivity of the rat airways to adenosine.

1 The airways of patients with asthma are hyperresponsive to adenosine. The phenomenon can be mimicked in the actively sensitized Brown Norway rat by exposure to allergen or lipopolysaccharide (LPS). We wondered whether combined treatment with allergen and endotoxin would result in additive effects or synergism with respect to increasing the sensitivity of the airways of the Brown Norway rat to adenosine. 2 Animals actively sensitized to ovalbumin and challenged intratracheally with allergen or endotoxin manifested increased bronchoconstrictor responses to adenosine. A combination of ovalbumin and endotoxin also increased the response to adenosine but the effects were at best additive. 3 Changes in the response to adenosine were selective as responses to 5-hydroxytryptamine were unaltered following ovalbumin or LPS either alone or in combination. 4 Thus, endotoxin and allergen acting together could play a role in up-regulating the response of the human asthmatic airway to adenosine. However, our data suggest that the interaction would be additive rather than synergistic.

Suppression of histamine-induced tachypnoea in the rhesus monkey by sibenadet: no role for dopamine D2 receptors.

1. Sibenadet (Viozan), a dual dopamine D(2)/beta(2)-adrenoceptor agonist, suppresses histamine-induced tachypnoea in the dog by activating dopamine D(2) receptors. We here compare the effects of sibenadet and formoterol, a selective beta(2)-adrenoceptor agonist, on histamine-induced tachypnoea in the rhesus monkey. 2. Anaesthetized, spontaneously breathing, rhesus monkeys were set up for measuring airways resistance, respiratory rate, blood pressure and heart rate. 3. Both sibenadet and formoterol administered by aerosol, induced inhibition of the bronchoconstrictor response to aerosolized methacholine accompanied by tachycardia. Sibenadet, but not formoterol, also reduced blood pressure. 4. Administration of histamine by inhalation induced tachypnoea which was accompanied by bronchoconstriction. Tachypnoea to histamine was suppressed by both sibenadet and formoterol at doses which manifest anti-bronchoconstrictor activity. These effects and the accompanying tachycardia but not the hypotension induced by sibenadet were abolished by pretreatment with propranolol. 5. The dopamine D(2) receptor agonist, quinagolide, did not suppress tachypnoea to histamine despite inducing a fall in blood pressure indicating activation of dopamine D(2) receptors. 6. Thus, both sibenadet and formoterol suppress histamine-evoked tachypnoea in the rhesus monkey. The effect arises exclusively through activation of beta(2)-adrenoceptors and probably reflects the anti-bronchoconstrictor effects of these agents. The results reveal a fundamental difference in the role of dopamine receptors in the airways of dog and rhesus monkey.

Airway hyperresponsiveness to bradykinin induced by allergen challenge in actively sensitised Brown Norway rats.

The mechanism(s) of bradykinin-induced bronchoconstriction was investigated in the Brown Norway (BN) rat model of allergic asthma. Bronchoconstrictor responses to i.v. bradykinin in BN rats were maximally augmented 24 h following challenge with allergen and declined at later time points. Histological evaluation of the inflammatory status of the lungs after ovalbumin (OA) challenge showed a marked inflammatory response, which was maximal at 24 h and declined thereafter. However, pretreatment with budesonide did not inhibit the augmented bronchoconstrictor response to bradykinin 24 h after allergen challenge. The selective B1 receptor agonist, Lys-[desArg9]-BK had no bronchoconstrictor effects, whereas the selective B2 receptor antagonist, HOE 140, abolished the response to bradykinin in OA-challenged animals. The augmented response to bradykinin was not affected by methysergide, indomethacin, disodium cromoglycate, iralukast, the 5-lipoxygenase inhibitor, CGS8515, or the NK2 receptor antagonist, SR48968. It was, however, partially inhibited by atropine both in saline- and OA-challenged animals. Pretreatment with captopril and thiorphan markedly potentiated responses to bradykinin both in saline- and OA-challenged animals. Thus, augmentation of the bronchoconstrictor response to bradykinin occurs in actively sensitised BN rats 24 h after challenge with OA and is associated with marked pulmonary inflammation. The response is entirely B2 receptor mediated and approximately 50% of the response is cholinergic. However, mast cell activation, the products of the cyclooxygenase or 5-lipoxygenase pathways and tachykinins are not involved. Peptidase inhibition mimics the effect of allergen challenge on the bronchoconstrictor response to bradykinin and it remains possible that the mechanism of the augmented response to bradykinin following allergen challenge involves downregulation of peptidase activity as a consequence of the inflammatory response.

Role of endogenous adenosine in the acute and late response to allergen challenge in actively sensitized Brown Norway rats.

1. Endogenous adenosine has been suggested to amplify the response of airway mast cells to allergen in vivo. We have sought evidence for this by monitoring the acute and late-phase response to allergen in Brown Norway (BN) rats actively sensitised to ovalbumin (OA) and treated either with adenosine deaminase (ADA) linked covalently to polyethylene glycol (PEG-ADA; Adagen) to decrease adenosine availability or with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an inhibitor of ADA, plus S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of facilitated adenosine transport, to increase adenosine availability. 2. The cardiovascular effects of adenosine (0.01-3 mg kg(-1) i.v.) were significantly reduced in PEG-ADA-treated animals and augmented in EHNA/NBTI-treated animals. The difference in sensitivity to adenosine in the treated groups was 33- and 15-fold, at the level of 30% reduction in blood pressure and heart rate, respectively. 3. The acute response to allergen, given either intravenously or intratracheally, was quantified as bronchoconstriction. The late phase to allergen was measured as the influx and activation of immunoinflammatory cells into the bronchoalveolar lavage fluid 24 h after challenge. 4. Despite evidence of a substantial difference in adenosine availability following pretreatment with PEG-ADA or EHNA/NBTI, there were no differences in either the acute or late response to allergen in the actively sensitised BN rat. 5. Our data suggest no role for endogenous adenosine in determining the response to allergen under our experimental conditions.

Species differences in bradykinin receptor-mediated responses of the airways.

1. Bradykinin (BK) is a nine amino acid peptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) formed from the plasma precursor kininogen during inflammation and tissue injury. The actions of BK are mediated by G protein-coupled cell surface receptors, designated B1 and B2. 2. BK has a plethora of effects in the airways including bronchoconstriction, bronchodilation, stimulation of cholinergic and sensory nerves, mucus secretion, cough and oedema resulting from promotion of microvascular leakage. These airway effects are mediated in the main by the B2 receptor subtype. 3. BK acts mainly indirectly, primarily through airway nerve activation, but also by the release of prostanoids, thromboxanes and nitric oxide (NO). 4. Airway responses to BK have been studied in detail in guinea-pigs, mice, sheep and rats. This review describes the effects of BK in these species and draws comparison with its effects in normal humans and patients with respiratory diseases. 5. Despite its many and varied effects in the airways of animals and man, the exact contribution of BK to airways disease remains unclear.

Airway hyperresponsiveness to adenosine induced by lipopolysaccharide in Brown Norway rats.

We have explored the effects of bacterial endotoxin (lipopolysaccharide; LPS) on the response of the airways of Brown Norway (BN) rats to adenosine. Comparisons have been drawn with the effects on responses to methacholine and 5-hydroxytryptamine. In vehicle-challenged animals, adenosine, given i.v. was only a weak bronchoconstrictor. In contrast, 1 h following intratracheal administration of LPS, 0.3 mg kg-1, bronchoconstrictor responses to adenosine were markedly and selectively enhanced. At this time point, there were no significant changes in leukocyte numbers, eosinophil peroxidase and myeloperoxidase activities or protein concentrations in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, the sensitivity of the airways to both adenosine and methacholine was reduced relative to the earlier time point and there were substantial increases in each marker of inflammation in BAL fluid. The bronchoconstrictor response to adenosine was blocked selectively by methysergide, disodium cromoglycate and the broad-spectrum adenosine receptor antagonist, 8-SPT, but not by DPCPX or ZM 243185, selective antagonists for the A1 and A2A receptors, respectively. Thus, the response to adenosine augmented following LPS is mast cell mediated and involves a receptor which can be blocked by 8-SPT but not by selective A1 or A2A receptor antagonists. It thus bears similarity to the augmented response to adenosine induced by allergen challenge in actively sensitized BN rats. Exposure to LPS could be a factor along with allergen in determining the increased sensitivity of the airways of asthmatics to adenosine.

Evidence for an atypical receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitized Brown Norway rats.

The bronchoconstrictor response to adenosine is markedly and selectively increased following ovalbumin (OA) challenge in actively sensitized, Brown Norway rats. We present a pharmacological analysis of the receptor mediating this response. Like adenosine, the broad-spectrum adenosine receptor agonist, NECA, induced dose-related bronchoconstriction in actively sensitized, OA-challenged animals. In contrast, CPA, CGS 21680 and 2-Cl-IB-MECA, agonists selective for A(1) A(2A) and A(3) receptors, respectively, induced no, or minimal, bronchoconstriction. Neither the selective A(1) receptor antagonist, DPCPX, nor the selective A(2A) receptor antagonist, ZM 241385, blocked the bronchoconstrictor response to adenosine. MRS 1754, which has similar affinity for rat A(2B) and A(1) receptors, failed to block the bronchoconstrictor response to adenosine despite blockade of the A(1) receptor-mediated bradycardia induced by NECA. 8-SPT and CGS 15943, antagonists at A(1), A(2A), and A(2B) but not A(3) receptors, inhibited the bronchoconstrictor response to adenosine. However, the degree of blockade (approximately 3 fold) did not reflect the plasma concentrations, which were 139 and 21 times greater than the K(B) value at the rat A(2B) receptor, respectively. Adenosine and NECA, but not CPA, CGS 21680 or 2-Cl-IB-MECA, induced contraction of parenchymal strip preparations from actively sensitized OA-challenged animals. Responses to adenosine could not be antagonized by 8-SPT or MRS 1754 at concentrations >50 times their affinities at the rat A(2B) receptor. The receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in actively sensitized BN rats cannot be categorized as one of the four recognized adenosine receptor subtypes.

Comparison of the anti-bronchoconstrictor activities of inhaled formoterol, its (R,R)- and (S,S)-enantiomers and salmeterol in the rhesus monkey.

The principle objective of this study was to define the anti-bronchoconstrictor effects of inhaled racemic formoterol and its (R,R)- and (S,S)-enantiomers in a new model of methacholine-induced bronchoconstriction in the rhesus monkey. A second long-acting beta(2)agonist, salmeterol, was included for comparison. Anaesthetized, spontaneously breathing rhesus monkeys were set up for measuring airway resistance. Blood pressure, heart rate and serum potassium concentrations were measured concomitantly to gauge systemic exposure and the potential for side effects. Formoterol, 0.14, 0.34 and 1.15 microg/kg, administered by aerosol, induced rapidly developing, sustained, dose-related inhibition of the bronchoconstrictor responses to aerosolised methacholine (maximum 76%) accompanied by sustained, dose-related tachycardia. (R,R)-formoterol, 0.56 microg/kg, induced anti-bronconstrictor effects and an associated tachycardia which corresponded closely to the effects seen following twice the dose of the racemate. (S,S)-formoterol, 0.54 microg/kg, was inactive. Salmeterol, 1.4 microg/kg, had no significant anti-bronchoconstrictor effect whereas doses of 5.5 and 30 microg/kg produced quantitatively similar but submaximal anti-bronchoconstrictor effects (maximum 47%). Sustained dose-dependent tachycardia was seen with salmeterol over the full dose range. Thus, the anti-bronchoconstrictor activity of formoterol resides in the (R,R) enantiomer and the (S,S) enantiomer does not interfere with the activity when present in the racemic form. Furthermore, the data indicate that the present model of methacholine-induced bronchospasm in the rhesus monkey could be useful in defining the key properties of beta(2)agonist bronchodilators such as relative potency, efficacy, duration of action and potential for systemic side effects.

Adenosine-mediated mast cell degranulation in adenosine deaminase-deficient mice.

Adenosine is a signaling nucleoside that has been suggested to play a role in asthma in part through its ability to influence mediator release from mast cells. Adenosine levels are elevated in the lungs of asthmatics, further implicating this molecule in the regulation of lung inflammation and suggesting that animal models exhibiting endogenous increases in adenosine will be useful for the analysis of adenosine function. Adenosine deaminase (ADA) is a purine catabolic enzyme responsible for regulating the levels of adenosine in tissues and cells. ADA-deficient mice develop lung inflammation and damage reminiscent of that seen in asthma in association with elevated adenosine levels. In the current study, we investigated the status of mast cells in ADA-deficient lungs. ADA-deficient mice exhibited extensive lung mast cell degranulation concurrent with elevated adenosine levels. ADA enzyme therapy prevented the accumulation of lung adenosine as well as mast cell degranulation, suggesting that this process was dependent on elevated lung adenosine levels. Consistent with this, treatment of ADA-deficient mice with broad spectrum adenosine receptor antagonists attenuated degranulation by 30 to 40%, supporting the involvement of adenosine receptor signaling. Moreover, these studies demonstrate the ability of endogenously generated adenosine to influence lung mast cell degranulation in a receptor-mediated manner and establish ADA-deficient mice as a model system to investigate the specific adenosine receptor responses involved in the degranulation of lung mast cells.

MRI of lung parenchyma in rats and mice using a gradient-echo sequence.

Signal of lung parenchymal tissue from the living rat and mouse lung was detected at 4.7 T with a good signal-to-noise ratio and motion-suppressed artifacts using a short TE gradient-echo sequence. Neither cardiac nor respiratory gating were applied, and animals respired freely during data collection. Mean T(2)* relaxation times of parenchyma in the anterior, middle and posterior regions of both lungs ranged between 403 and 657 micros and 397 and 751 micros, respectively for the rat and mouse. For the rat in the prone position, there was a gradient in T(2)* values, from the posterior to the anterior regions of both lungs. In the supine position, however, T(2)* values were larger in the posterior and in the anterior portions. For the mouse in both prone and supine positions, there was a tendential gradient in T(2)* from the anterior to the posterior portions. The robustness of the approach renders it well suited for routine applications, e.g. in pharmacological studies concerning asthma models in small rodents. The method was applied to lung inflammation models involving challenge with ovalbumin or lipopolysaccharide.

Mechanism of airway hyperresponsiveness to adenosine induced by allergen challenge in actively sensitized Brown Norway rats.

1. We have explored the role of allergen sensitization and challenge in defining the response of the airways of the Brown Norway (BN) rat to adenosine. 2. In naïve animals or in rats sensitized to ovalbumin (OA) adenosine induced only weak bronchoconstrictor responses. Challenge of sensitized animals with OA induced a marked airway hyperresponsiveness to adenosine which was not seen with methacholine or bradykinin. 3. The augmented bronchoconstrictor response to adenosine was not affected by acute bivagotomy or atropine nor mimicked by an i.v. injection of capsaicin. It was, however, blocked selectively by disodium cromoglycate methysergide or ketanserin and reduced in animals treated sub-chronically with compound 48/80. 4. The augmented response to adenosine was associated with increases in the plasma concentrations of both histamine and 5-hydroxytryptamine (5-HT), which were attenuated by pretreatment with disodium cromoglycate, and degranulation of mast cells in the lung. 5. Parenchymal strips from lungs removed from sensitized rats challenged with OA gave augmented bronchoconstrictor responses to adenosine relative to strips from sensitized animals challenged with saline. Responses were inhibited by methysergide and disodium cromoglycate. 6. These data demonstrate a marked augmentation of the bronchoconstrictor response to adenosine in actively sensitized BN rats challenged with OA. The augmented response is primarily a consequence of mast cell activation, leading to the release of 5-HT, which in turn induces bronchoconstriction. Our data further suggest the involvement of a discrete lung-based population of mast cells containing and releasing mainly 5-HT and brought into play by prior exposure to allergen.

Pulmonary edema induced by allergen challenge in the rat: noninvasive assessment by magnetic resonance imaging.

The course of pulmonary edema formation after an intratracheal (i.t.) instillation of ovalbumin was followed noninvasively by magnetic resonance imaging (MRI) in actively sensitized Brown Norway (BN) rats. Changes in edema volume assessed by MRI mimicked the results from the analysis of the number and activation of inflammatory cells recovered from the broncho-alveolar lavage (BAL) fluid. Rats treated with budesonide did not develop edema following challenge with ovalbumin, and these animals showed a significant decrease in BAL fluid inflammatory cell numbers and eosinophil peroxidase and myeloperoxidase activities. Thus, following lung edema formation by MRI provides a reliable means of assessing pulmonary inflammation after allergen challenge. Unlike BAL fluid analysis, which requires killing animals at each time point, this method is noninvasive. MRI could be of importance for the noninvasive profiling of anti-inflammatory drugs in animal models of asthma and in the clinic. Magn Reson Med 45:88-95, 2001.

Inhibition by viozan of extravasation induced in rat trachea by capsaicin is mediated exclusively by beta 2-adrenoceptors.

The mechanism by which 2(3H)-benzothiazolone, 4-hydroxy-7-[2-[[2-[[3-(2-phenylethoxy)propyl]-sulphonyl]ethyl]amino]ethyl]-monohydrochloride (AR-C68397AA; viozan), a dual dopamine D2/beta2-adrenoceptor agonist which has shown promise in the treatment of chronic obstructive pulmonary disease (COPD), inhibits the extravasation of plasma protein induced by capsaicin in the tracheas of Brown Norway rats has been re-evaluated. Viozan (10-30 microg/kg given intratracheally; i.t.) inhibited dose-dependently the extravasation of plasma protein tagged with Evans Blue into rat trachea induced by capsaicin (10 microg/kg i.t.). Similar effects were seen with the selective beta2-adrenoceptor agonist, salbutamol (3-10 microg/kg i.t.), but the selective dopamine D2 receptor agonist, quinagolide (10-30 microg/kg i.t.), was inactive. The effects of viozan and salbutamol were abolished by propranolol (3 mg/kg) given intraperitoneally (i.p.) but unaffected by sulpiride (3 mg/kg i.p.). Thus, in c,ontrast to claims in the literature, a functional response to dopamine D2 receptor activation in a preclinical model of oedema arising from sensory nerve fibre activation in the rat lung could not be demonstrated. Moreover, no qualitative difference could be demonstrated between the response to a dual D2/beta2-adrenoceptor agonist and a selective beta2-adrenoceptor agonist. The observations call into question whether a dual D2/beta2-adrenoceptor agonist such as viozan would bring added benefit over established selective beta2-adrenoceptor agonists in the therapy

Effects of wortmannin on airways inflammation induced by allergen in actively sensitised Brown Norway rats.

We have investigated the effect of wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase, on the immediate-type allergic response and the late phase pulmonary inflammation induced by allergen challenge in the ovalbumin-sensitised Brown Norway rat. Intratracheal (i.t.) instillation of ovalbumin induced dose-related bronchoconstrictor responses. Administration of wortmannin (1, 10 or 100 microg kg(-1) i.t., 1 h prior to challenge) induced a marked and dose-dependent inhibition of ovalbumin-induced bronchospasm (ED(50) ca. 5 microg kg(-1) i.t.). At similar doses, wortmannin also suppressed the bronchoconstrictor responses to 5-hydroxytryptamine and methacholine but the degree of blockade of these spasmogens (1.4-1.9-fold) was less than that of ovalbumin (>20-fold). Wortmannin, given intratracheally 1 h prior to allergen challenge, also suppressed the increases in bronchoalveolar lavage fluid leukocyte numbers and eosinophil peroxidase activity measured 24 h post challenge. However, relatively high doses were necessary (ED(50) ca. 100 microg kg(-1) i.t.). The potency of wortmannin was increased when dosed 1 h prior to and 24 h after allergen challenge and the readout was 48 h after challenge (ED(50) 3-5 microg kg(-1) i.t.). Thus, wortmannin is a potent inhibitor of the bronchoconstrictor response induced by allergen in the airways of actively sensitised Brown Norway rats. Inhibition of phosphatidylinositol-3-kinase, an obligatory step in mast cell activation in response to allergen, is the presumed mechanism of action. The fact that similar doses of wortmannin do not suppress the late response to allergen suggests a minimal role for the mast cell in generating the late response to allergen in this model. The striking increase in potency to inhibit the late response when dosed 1 h prior to and 24 h after allergen challenge with the readout taken at 48 h may represent an effect of wortmannin to suppress the migration of leukocytes.

Effects of wortmannin on bronchoconstrictor responses to adenosine in actively sensitised Brown Norway rats.

The bronchoconstrictor response to adenosine in the actively sensitised Brown Norway rat is markedly augmented following low level allergen (ovalbumin) challenge. The response reflects activation of the A(2B) receptor subtype and is mediated by 5-hydroxytryptamine (5-HT) released as a consequence of mast cell activation. We describe here the effects of wortmannin, a potent inhibitor of phosphatidylinositol-3-kinase and mast cell exocytosis, on the response to adenosine. Bronchoconstrictor responses to adenosine elicited 3 h following ovalbumin challenge were markedly and dose-dependently reduced by wortmannin given intratracheally (i. t.), 1 h prior to or 2 h post-allergen challenge. Responses to methacholine, which activates bronchial smooth muscle directly, and 5-HT were also reduced following wortmannin but to a lesser extent than those to adenosine. Bronchoconstrictor responses to adenosine 3 h post-challenge with vehicle were also markedly reduced by wortmannin given intratracheally (i.t.), 1 h prior to the "sham" challenge. Plasma histamine and 5-HT levels increased in response to adenosine given 3 h following ovalbumin challenge. The increases were suppressed by wortmannin given i.t., 2 h post-ovalbumin challenge. A reduction in the sensitivity of the airways to 5-HT explains in part the reduced bronchoconstrictor response to adenosine induced by wortmannin. A direct action to suppress 5-HT release from airway mast cells induced by adenosine also contributes to the reduction in the response. Inhibition of phosphatidylinositol-3-kinase is the presumed mechanistic basis for the observed effects.

BP-294 (Ste Civile Bioprojet).

BP-294 (a prodrug of R-alpha-methylhistamine) is a histamine-H(3) agonist under development by Bioprojet, which is currently in phase II clinical trials as a potential treatment for asthma [219798].