Pharmacologic actions of the second generation leukotriene B4 receptor antagonist LY29311: in vivo pulmonary studies.

We examined the in vivo actions of LY293111 sodium (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]pro poxy]phenoxy] benzoic acid sodium salt). Guinea pigs were used to evaluate the effect of this agent on (1) acute airway obstruction produced by intravenous leukotriene B4, (2) pulmonary granulocyte infiltration and delayed onset airway obstruction resulting from a 4-h leukotriene B4 inhalation and (3) lung inflammation after aerosol challenge with the divalent cationic ionophore A23187 (6S-[6alpha(2S*,3S*),8beta(R*),9beta,11alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)e thyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazole carboxylic acid). Airway obstruction was quantitated using pulmonary gas trapping measurements and lung inflammation was evaluated by bronchoalveolar lavage (BAL) and histology. LY293111 sodium produced a dose-related inhibition of acute leukotriene B4-induced airway obstruction when administered i.v. (ED50=14 microg/kg) or p.o. (ED50=0.4 mg/kg). In contrast, LY293111 sodium did not inhibit the pulmonary gas trapping caused by aerosols of histamine, leukotriene D4, or the thromboxane mimetic U46619 (15 [(S)-hydroxy11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid]). Oral LY293111 sodium inhibited leukotriene B4-induced bronchoalveolar lavage granulocyte infiltration and delayed onset airway obstruction at doses as low as 0.3 mg/kg. In A23187-challenged animals, pulmonary inflammation was markedly inhibited at 1 h, but not 2 h and 4 h post-exposure. We conclude that LY293 11 sodium is a selective leukotriene B4 receptor antagonist with potent pulmonary anti-inflammatory activity.

Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies.

The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.

Differential effects of cyclosporine A after acute antigen challenge in sensitized cats in vivo and ex vivo.

1. We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (R(L)) in vivo, and tracheal smooth muscle (TSM) contraction ex vivo after antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen-induced responses of TSM in vitro. 2. Cats (n=10) were sensitized by i.m. injection of Ascaris suum antigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin-2 secretion from feline peripheral blood mononuclear cells ex vivo. 3. Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542+/-74 pg ml(-1) post AA for CsA+ and from 74+/-19 pg ml(-1) at baseline, to 970+/-180 pg ml(-1) post AA CsA- (P<0.05; P=NS vs CsA+). 4. In excised TSM, active tension elicited by exposure to AA in vitro was 107+/-38% KCl in the CsA+ group vs 144+/-56% KCl in the CsA- group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre-incubated with 1 microM CsA in vitro (8+/-8% KCl, P<0.05 vs CsA+ and CsA-). This was associated with inhibited release of 5-hydroxytryptamine into the organ bath fluid of tissues treated with CsA in vitro only. 5. We demonstrated that CsA treatment in vivo does not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell function ex vivo do not reflect lack of effects of CsA on mast cell function in vivo in this animal model of atopic asthma.

The regulation of CD11b integrin levels on human blood leukocytes and leukotriene B4-stimulated skin by a specific leukotriene B4 receptor antagonist (LY293111).

CD11b is part of the beta2-integrin Mac-1 and plays an important role in neutrophil adhesion. Leukotriene B4 (LTB4) is an active upregulator of neutrophil CD11b-expression, acts as a potent chemoattractant to neutrophils and is also known to upmodulate epidermal proliferation. We performed a placebo-controlled study on LY293111, an oral LTB4 receptor antagonist. Twenty healthy male volunteers were randomised over three treatment groups that received placebo, 48 mg, or 200 mg drug twice daily for 10 days. Before and after treatment, flow cytometrical CD11b assessment was performed on in vitro LTB4-stimulated peripheral blood neutrophils. Additionally, skin biopsies were taken at 24 and 72 h after epicutaneous LTB4 application, before and after treatment. The effects on skin were assessed immunohistochemically using various markers. All observed effects were dose related. CD11b upregulation on blood neutrophils was significantly suppressed in both treatment groups compared to placebo. In skin, a significant suppression of inflammation and hyperproliferation occurred. Pronounced inhibition was observed on neutrophil migration into the epidermis and the inflammatory infiltrate was decreased. A similar but weaker response was seen in the dermis. The number of cycling cells as well as suprabasal keratin-16 expression were decreased in both treatment groups. LY293111 proved to be a potent inhibitor of LTB4-induced cutaneous inflammation and hyperproliferation. The potent antiinflammatory effect in vivo and the fact that in the present study the compound showed no clinically significant side effects make it an interesting drug in the future treatment of inflammatory conditions predominated by neutrophils.

Effect of a leukotriene B4 receptor antagonist, LY293111, on allergen induced responses in asthma.

BACKGROUND: Leukotriene (LT) B4 is a potent neutrophil chemoattractant and also stimulates eosinophils in vitro, but its role in asthmatic inflammation is unknown. METHODS: The effect of the novel LTB4 receptor antagonist, LY293111, was examined using allergen challenge as a model for asthmatic inflammation in 12 atopic asthmatic subjects in a double blind placebo controlled crossover trial. Subjects with an established early (EAR) and late asthmatic response (LAR) to allergen at screening received oral LY293111 in a dose of 112 mg three times daily for seven days or placebo before further allergen challenge. Each treatment was separated by a washout period of 28 days. Individuals underwent histamine challenge one hour before and three hours after allergen challenge. Bronchoalveolar lavage (BAL) fluid was obtained at bronchoscopy 24 hours after allergen challenge. RESULTS: There was no difference in baseline lung function, EAR, LAR, or in airway responsiveness to histamine before and after allergen between placebo and LY293111. By contrast, treatment with LY293111 significantly reduced the number of neutrophils in BAL fluid expressed as both absolute cell numbers and percentage cell differential counts: absolute cell counts, median (range) 0.04 (0.02-0.15) x 10(6) after LY293111, 0.09 (0.02-0.43) x 10(6) after placebo; percentage differential cell counts 0.35 (0.1-2.0) after LY293111, 0.80 (0.1-3.6) after placebo (p < 0.05). Eosinophils, macrophages, and lymphocytes in BAL fluid did not differ between treatments. There was a significant reduction in the concentration of myeloperoxidase (MPO) with both placebo (16 (6.6) ng/ml) and LY293111 (3.5 (1.8) ng/ml) and of LTB4 (placebo 4.6 (1.2) pg/ml, LY293111 2.2 (0.2) pg/ml). Concentrations of LTC4 and interleukin 8 were reduced, although not significantly, whereas concentrations of interleukin 6, GM-CSF, and TNF-alpha were unchanged by LY293111. CONCLUSIONS: These results demonstrate an influence of LTB4 on neutrophil influx and activation in the airway following allergen challenge. Despite this anti-inflammatory effect, there was no measured physiological benefit and this questions the functional role of the neutrophil in the pathophysiology of allergen induced asthma.

Inhibition of ex vivo neutrophil activation by oral LY293111, a novel leukotriene B4 receptor antagonist.

1. The effects of orally administered LY293111 on ex vivo neutrophil Mac-1 upregulation were determined in a total of 24 healthy male subjects within three study periods. 2. In the first period, eight volunteers received 60 mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average ex vivo Mac-1 response of the LY293111 group was 56% of the predose control (95% confidence interval (CI) 44.3 to 67.9%; P < 0.01). The inhibitory effect was maximum at the end of dosing and had disappeared by day 14. 3. In the second period, eight subjects received 120 mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average response of the LY293111 group was 70% of the pre-dose control (95% CI 59.7 to 81.0%; P < 0.01). The inhibitory effect was maximum the day following the initial dose and continued throughout the dosing period. 4. In the third period, eight subjects received 200 mg LY293111 or placebo twice daily in 15 total doses over 8 days followed by a 1 week follow-up. Mac-1 upregulation was 64% of pre-dose levels (95% CI 53.8 to 75.1%; P < 0.01) over the course of the study period. The inhibition had disappeared 2 days following the final dose. Alternate neutrophil stimulation by fMLP was not inhibited. 5. No statistically significant inhibition was observed for placebo-treated subjects. 6. No statistically significant differences were apparent between the active dose regimens. 7. The results indicate that orally administered LY293111 is pharmacologically active in humans. Results from this study may be useful in determining dose selection for efficacy trials.

Recombinant human secretory phospholipase A2 released thromboxane from guinea pig bronchoalveolar lavage cells: in vitro and ex vivo evaluation of a novel secretory phospholipase A2 inhibitor.

The primary objective of this study was to develop a functional assay that could provide rapid and reliable information on some pharmacologic characteristics of a novel inhibitor of human secretory phospholipase A2 (sPLA2). Guinea pig bronchoalveolar lavage (BAL) fluid, containing predominantly macrophages, eosinophils and epithelial cells, released thromboxane A2, as measured by thromboxane B2, in a concentration-dependent manner on exposure to recombinant human sPLA2 (rh-sPLA2). Similarly, n-formyl-L-methionyl-L-leucyl-L-phenylalanine (n-F-Met-Leu-Phe) or arachidonic acid also released this lipid mediator. Indomethacin, a cyclooxygenase inhibitor, blocked synthesis of thromboxane in response to these agents. p-Bromophenacylbromide-inactivated rh-sPLA2 was substantially less effective than the untreated enzyme in causing release of thromboxane. LY311727 is a potent indole-derived inhibitor of the isolated enzyme (IC50 = 23 nM). Incubation of this agent with BAL cells, just before addition of rh-sPLA2, reduced release of thromboxane with an IC50 = 1.8 x 10(-6) M. Specificity for sPLA2 was demonstrated in that LY311727, unlike indomethacin, did not reduce synthesis and subsequent release of thromboxane A2 in response to arachidonic acid. Using this technique as a basis, we determined whether LY311727 could sufficiently accumulate in lung after i.v. administration to inhibit rh-sPLA2-induced thromboxane A2 release from BAL cells. The compound, given i.v. to guinea pigs 5 min before collecting BAL fluid, produced a dose-dependent inhibition of rh-sPLA2 with an ED50 = 50 mg/kg. Thus, new in vitro and ex vivo assays were developed that permit functional evaluation of novel sPLA2 inhibitors. These techniques should serve as secondary assays for evaluation of human sPLA2 inhibitory activity from a chemical series and in addition provide initial data related to metabolic stability and distribution to the lung.

Blockade of human neutrophil activation by 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5- hydroxyphenoxy]propoxy]phenoxy]benzoic acid (LY293111), a novel leukotriene B4 receptor antagonist.

Leukotriene B4 (LTB4), a naturally occurring pro-inflammatory product of arachidonic acid metabolism, has been associated with human inflammatory disease. This study compares the abilities of two LTB4 receptor antagonists, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]- propoxy]phenoxy]benzoic acid (LY293111) and 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), to displace LTB4 binding and their functional blockade of human neutrophil activation. LY293111 inhibited the binding of [3H]LTB4 with a Ki of 25 nM; SC-41930 displayed a similar potency (Ki = 17 nM). In contrast, LY293111 prevented LTB4-induced calcium mobilization with an IC50 = 20 nM, or 40 times more effectively than SC-41930 (IC50 = 808 nM). LY293111 was 300 times more potent than SC-41930 in blocking LTB4-induced CD11b up-regulation on isolated neutrophils. LY293111 also arrested LTB4-induced up-regulation of CD11b on neutrophils in whole human blood. LY293111 was not effective in blocking human neutrophil activation responses induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), platelet-activating factor (PAF), human recombinant endothelial interleukin-8 (IL-8) or human recombinant complement component 5a (C5a).

Cyproheptadine-induced attenuation of type-I immediate-hypersensitivity reactions of airway smooth muscle from immune-sensitized cats.

We assessed the effect of serotonergic inhibition by cyproheptadine on the responsiveness of tracheal smooth muscle (TSM) strips and epithelium-intact third-generation bronchial rings from immune-sensitized (Ascaris suum) cats after exposure to antigen. Cats were sensitized by IM administration of antigen and adjuvant twice over a 4-week period. Sensitization was confirmed in vivo by skin testing with antigen and by physiologic airway responses after exposure to nebulized antigen. Tissues were tethered isometrically to force transducers and were actively equilibrated by exposures to KCl-substituted perfusate. Maximal response after exposure to antigen (expressed as percentage of maximal contraction of each tissue to 63 mM KCl (%KCl) was 169 +/- 18% KCl for sensitized TSM and 43 +/- 18% KCl for sensitized TSM pretreated with cyproheptadine (P < 0.001). Similarly, maximal response to antigen was 81 +/- 27% KCl for sensitized bronchial rings, compared with 16 +/- 14% KCl for sensitized bronchial rings pretreated with cyproheptadine (P = 0.05 vs control). Blockade of leukotriene synthesis by 10(-6) to 10(-4) M A-64077, a selective 5-lipoxygenase inhibitor, had no significant effect on peak response for either TSM (193 +/- 13% KCl vs 169 +/- 18% KCl for sensitized untreated TSM) or bronchial rings (79 +/- 20% KCl vs 81 +/- 27% KCl for sensitized untreated bronchial rings). Release of serotonin from airway tissues was confirmed by the presence of serotonin in the perfusate of 8 sensitized TSM preparations after, but not before, antigen challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE.

In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC4.

Beta adrenergic modulation of formyl-methionine-leucine-phenylalanine-stimulated secretion of eosinophil peroxidase and leukotriene C4.

The inhibitory effect of beta-2 adrenergic receptor stimulation on leukotriene C4 (LTC4) secretion and eosinophil peroxidase (EPO) release caused by exogenous activation with 10(-8) to 10(-6) M formyl-met-leu-phe (fMLP) + 5 micrograms/ml of cytochalasin B (Cyto B) in purified human peripheral blood eosinophils was studied. Cells from normal subjects were isolated by negative immunoselection and remained > or = 98% viable as determined by trypan blue exclusion. Duplicate aliquots of eosinophils (10(5) cells/intervention) were activated with 1) fMLP + Cyto B alone, 2) fMLP + Cyto B after pretreatment with 10(-8) M albuterol, 3) 10(-8) M albuterol + fMLP + Cyto B after pretreatment with 10(-8) M propranolol or 4) vehicle control. After incubation, the supernatants were tested for concentration of LTC4 and EPO. Concentration-related release of EPO was demonstrated for 10(-8) M fMLP + 5 micrograms/ml of Cyto B to 10(-6) M fMLP + 5 micrograms/ml of Cyto B, and the greatest concentration of fMLP was used in all subsequent studies. FMLP + Cyto B caused substantial LTC4 secretion in eosinophils (300 +/- 83.0 pg/ml) as compared to sham-activated eosinophils (3.3 +/- 1.9 pg/ml; P < .02). Similarly, maximum EPO release increased from 277 +/- 17.8 to 3956 +/- 1230 ng/10(6) cells (P < .02) after activation with fMLP + Cyto B. Treatment with albuterol decreased markedly both LTC4 secretion to 144 +/- 54.0 pg/ml (P < .05 vs. fMLP + Cyto B-activated eosinophils) and EPO release to 1993 +/- 368 ng/10(6) cells (P < .05 vs. fMLP + Cyto B-activated eosinophils).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of eicosanoids in hyperpnea-induced airway responses in guinea pigs.

Guinea pigs mechanically hyperventilated with dry gas exhibit hyperpnea-induced bronchoconstriction (HIB) and hyperpnea-induced bronchovascular hyperpermeability (HIBVH). Tachykinins released from airway C-fiber neurons are the central mediators of guinea pig HIB but play only a contributory role in HIBVH. Recent studies suggest that eicosanoid mediators can provoke bronchoconstriction and bronchovascular hyperpermeability, are released by dry gas hyperpnea, and can themselves elicit or modulate tachykinin release. We therefore hypothesized that eicosanoids may participate in HIB and/or HIBVH. To test these hypotheses, we analyzed respiratory system resistance changes and Evans blue-labeled albumin extravasation into the airways of 60 tracheostomized and mechanically ventilated guinea pigs. Animals were subjected to 10 min of isocapnic dry gas hyperpnea or to quiet breathing of humidified gas and received as pretreatment either piroxicam, a cyclooxygenase (CO) inhibitor; A-63162, a 5-lipoxygenase (5-LO) inhibitor; BW-755c, a combined CO and 5-LO inhibitor; ICI-198,615, a leukotriene D4 receptor antagonist; or no drug. HIB was substantially (50-80%) reduced by each of the four eicosanoid-modulating drugs. In contrast, HIBVH was reduced only by BW-755c, and this effect occurred only within the extrapulmonary airways (42% reduction). These data indicate that both CO and 5-LO products, including leukotriene D4, participate in the pathogenesis of HIB but that, like tachykinins, they play only a small contributory role in HIBVH. Together with our previous demonstration that sensory neuropeptide release is critical for the occurrence of HIB, we conclude that the roles of eicosanoids and tachykinins in guinea pig HIB are interdependent.

Contraction of guinea pig inferior vena cava by eicosanoids.

Guinea pig inferior vena cava contracted in response to leukotriene (LT)C4, LTD4, LTE4 U46619, phenylephrine, histamine, and KCl. Although LTC4, LTD4, and U46619 were the most potent agonists, active tension generated by these eicosanoids was only about half that of histamine or KCl. LTE4 and phenylephrine were marginally active. Biochemical analysis showed vena cava able to convert about 23% LTC4 to LTD4 and LTE4 in 45 min. Pretreatment with acivicin prevented this by abrogating conversion of LTC4 to LTD4. A subthreshold concentration of LTE4 reduced responses to LTC4 and LTD4. LY171883 and WY-48252 competitively antagonized LTD4-induced contractions of vena cava. In contrast, these antagonists blocked contractions to LTC4 in a biphasic manner. Lower segments of the LTC4 concentration-response curves were less affected than the upper portion suggesting the possibility of 2 LTC4 receptor subtypes. Our results indicate that LTE4 is a weak or partial agonist in this tissue and furthermore they suggest a lack of high affinity receptors for LTE4 favoring LTC4 and LTD4. Indomethacin did not influence contractions to the leukotrienes or histamine. However, the response to U46619 was greatly enhanced suggesting release of a vasodilator prostaglandin as part of the overall response of the vena cava to the thromboxane A2 mimetic.

Bronchopulmonary actions of 1-(1,2,3,4-tetrahydro-1-naphthylenyl)-1H-imidazole, nitric acid salt (LY150310), a substituted imidazole, in the guinea pig.

1-(1,2,3,4-tetrahydro-1-naphthylenyl)-1H-imidazole, nitric acid salt (LY150310), was examined for bronchodilator activity in the guinea pig. In guinea pig tracheal preparations, LY150310 competitively antagonized the contractile effects of exogenous histamine and blocked the histamine-mediated component of contractions produced by ovalbumin challenge. LY150310 had little effect on the nonhistamine component of ovalbumin-induced contractions of lung parenchymal strips, but it enhanced the production of prostaglandin (PG) E2 and PGF2 alpha although it partially inhibited thromboxane B2 formation. In other studies, in which postmortem pulmonary gas trapping was used as an index of in vivo airway obstruction, i.v. LY150310 dose-dependently inhibited the bronchospasm produced by aerosols of the divalent cationic ionophore A23187, histamine, 5-hydroxytryptamine, leukotriene D4, methacholine, ovalbumin or platelet activating factor. LY150310 was equal to or more potent than aminophylline in all test systems. Also, orally administered LY150310 inhibited the airway obstruction produced by selected challenge aerosols. In ex vivo studies, LY150310 elevated PGE2 and tended to decrease thromboxane B2 in sodium arachidonate-stimulated whole blood. However, PGE2 and other cyclooxygenase products did not appear to account for in vivo bronchodilation, because combining LY150310 and piroxicam did not alter inhibition of A23187-induced airway obstruction. Our results demonstrate that LY150310 reduces airway obstruction caused by a variety of bronchoconstrictive agents, including A23187 and ovalbumin. Although this substituted imidazole appears to have activity as a histamine H1-receptor antagonist and can alter prostanoid concentrations in vitro and in vivo, its mode of bronchodilation is unclear.

Prostaglandin secretion by guinea pig tracheal epithelial cells caused by eosinophil major basic protein.

We examined the effect of eosinophil major basic protein (MBP) on prostaglandin (PG) secretion from guinea pig tracheal epithelial (GPTE) cells. Primary cultures of GPTE cells were incubated with 10(-6) M MBP for up to 6 h and then stimulated with 10(-6) M bradykinin (BK). PGE2, 6-ketoprostaglandin F1 alpha (PGF1 alpha), PGF2 alpha, and thromboxane B2 (TxB2) concentrations in media were determined by enzyme-linked immunoabsorbent assay (EIA). Incubation with MBP for 6 h caused secretion of both PGE2 (17,614 +/- 4,416 vs. 1,426 +/- 555 pg/10(6) cells at baseline, P < 0.001, n = 7) and PGF2 alpha (20,303 +/- 5,724 vs. 3,790 +/- 1.075 pg/10(6) cells at baseline, P < 0.002, n = 7). Secretion of PGE2 and PGF2 alpha stimulated by MBP required at least 2 h. Incubation with MBP for 6 h also augmented the subsequent response to BK: PGE2 secretion was 29,215 +/- 6,853 vs. 3,445 +/- 1,041 pg/10(6) cells for BK alone (P < 0.0001), and PGF2 alpha secretion was 25,407 +/- 6,237 vs. 5,213 +/- 1,535 pg/10(6) cells for BK alone (P < 0.0001). MBP did not change 6-keto-PGF1 alpha and TxB2 secretion. Incubation of GPTE cells from seven animals with polylysine, a protein with mass and ion charge similar to MBP, for 2 h, both caused secretion of PGE2 (8,579 +/- 3,244 vs. 788 +/- 419 pg/10(6) cells at baseline, P < 0.01) and augmented the response to BK (12,732 +/- 4,788 vs. 1,653 +/- 680 pg/10(6) cells after BK alone, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Augmented muscarinic responsiveness caused by 5-lipoxygenase products secreted from alveolar macrophages in isolated-perfused rat lung.

We examined the effect of activated alveolar macrophages (AM) on airway responsiveness to muscarinic stimulation in 33 adult Sprague-Dawley rats. An isolated-perfused lung preparation was used to ensure precise and uniform delivery of cells into peripheral airways. The bronchoconstrictor response to acetylcholine (ACh) delivered into the pulmonary arterial circulation was augmented in 8 rats after infusion of 3 x 10(6) AM activated with 10(-6) M f-met-leu-phe and 5 micrograms/ml of cytochalasin B. Lung resistance (RL) caused by 10(-6) mol ACh increased 2.5-fold from 0.10 +/- 0.004 cm H2O/ml/s before infusion of activated AM to 0.35 +/- 0.05 cm H2O/ml/s after infusion of activated AM (N = 8; p < 0.05); the response to ACh was not augmented after infusion of nonactivated AM (N = 7) or vehicle control (N = 6). Baseline RL before ACh was similar in all three groups (p NS). Perfusion with activated AM also significantly increased the wet/dry (W/D) lung weight ratios (7.1 +/- 0.5) compared with nonactivated AM (5.2 +/- 0.1) or vehicle control (5.5 +/- 0.3) (p < 0.05 versus either nonactivated AM or vehicle control). A63162, a 5-lipoxygenase inhibitor, but not indomethacin, a cyclooxygenase inhibitor, completely inhibited augmentation of bronchoconstrictor responses to ACh caused by activated AM and also completely attenuated the increase in W/D lung weight ratios. A highly significant (p < 0.01) correlation (R = 0.76) between W/D lung weight ratios and RL was observed after 10(-6) mol ACh (the greatest dose of ACh administered). Baseline RL was equivalent for all groups before and after infusion of cells or vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2.

The unique granular proteins of eosinophils may have a pathogenetic role in asthma and in the defense against parasitic infestations. However, the mechanisms regulating eosinophil degranulation are largely unknown. We examined the hypothesis that release of these proteins is regulated by endogenous activation of phospholipase A2. Human eosinophils (HE) were isolated from the peripheral blood of 42 subjects either by Percoll density separation or by negative-selection immunomagnetic fractionation. Eosinophil activation was initiated in vitro with 10(-6) M FMLP and 5 micrograms/ml cytochalasin B and was assessed by measurement of eosinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radical (.O2-) secretion. Treatment of HE with 100 microM mepacrine before activation blocked EPO release (2.0 +/- 0.2 vs 10.2 +/- 2.1% cell content for activated HE, P < 0.004, n = 9), .O2- generation (2.6 +/- 0.9 vs 44.2 +/- 10.8 nmol/ml per 10(6) HE, P < 0.002, n = 5), and LTC4 secretion (68.2 +/- 32.2 vs 1,125.2 +/- 526.8 pg/ml per 10(6) HE, P < 0.04, n = 8). Pretreatment of HE with 100 microM 4-bromophenacyl bromide before activation similarly blocked EPO release, .O2- generation and LTC4 secretion. Addition of AA to HE after treatment with 100 microM mepacrine and before subsequent activation reversed the inhibition of both EPO (10.4 +/- 2.2% with 1 microM AA vs 2.0 +/- 0.2% for mepacrine, n = 5, P < 0.02) and LTC4 secretion (695.1 +/- 412.9 with 10 microM AA vs 68.2 +/- 32.2 pg/ml per 10(6) HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of .O2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and .O2- by eosinophils is regulated endogenously by phospholipase A2.

Pulmonary actions of LY255283, a leukotriene B4 receptor antagonist.

The actions of LY255283, a leukotriene (LT) B4 receptor antagonist, were examined on guinea pig lung. LTB4 and LY255283 displaced [3H]LTB4 from its binding site on lung membranes with pKi values of 9.9 and 7.0, respectively. In the functional correlate of the binding studies, LY255283 competitively reduced contractile responses of lung parenchyma to LTB4 (pA2 = 7.2). LTB4 produced airway obstruction which was reduced by LY255283 administered i.v. (ED50 = 2.8 mg/kg) or orally (ED50 = 11.0 mg/kg). Contractile responses to histamine, LTD4 and the thromboxane mimetic, U46619, were not reduced by LY255283. The compound also did not inhibit cyclooxygenase or 5-lipoxygenase enzymes. We conclude that LY255283 selectively antagonized pharmacologic responses to LTB4 on lung tissue and appears to be a useful tool to investigate the role of LTB4 in pulmonary disease.

Contributory role of lung pleura to release of anaphylactic mediators from guinea pig lung in response to ovalbumin or A23187.

Previous findings revealed greater contractile responses of guinea pig lung pleural surface strips to antigen or A23187 challenge than denuded lung parenchymal strips (lung strip devoid of any pleura). Moreover, we have identified a high density of mast cells distributed throughout the lung pleura. The present study examined mediators released from guinea pig lung pleural surface and denuded lung parenchyma fragments in response to immunologic challenge with ovalbumin (OA) or non-immunologic challenge with the ionophore A23187. Histamine levels were measured radioenzymatically; leukotrienes (LTs), prostaglandins (PGs) and thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were quantitated using an enzyme immunoassay. Histamine release reached a maximal level 3-5 min after OA challenge, whereas A23187-induced histamine release increased gradually in a time-dependent manner. Similar kinetics were observed in the release of LTs, PGs and TXA2. Pleural surface released a substantially (P < 0.05) greater amount of histamine to both challenges than denuded parenchyma. Moreover, histamine content in pleural surface was significantly (P < 0.05) higher than in denuded parenchyma. Pleural surface also released considerably (P < 0.05) more LTB4, LTC4, and LTE4 in response to OA and A23187 than denuded parenchyma. In contrast, pleural surface and denuded parenchyma released equivalent amounts of PGD2, PGE2, PGF2 alpha, and TXA2 in response to both challenges. The rank order of leukotriene release was LTC4 > LTE4 > LTB4, whereas that of prostanoid release was TXA2 >> PGD2 > or = PGF2 alpha >> PGE2. We conclude that pleural surface is the major source of histamine and leukotrienes released from guinea pig lung in vitro in response to OA and A23187, whereas both pleural surface and denuded parenchyma participate to the same extent in prostaglandin and TXA2 production after such challenges.