Guinea Pig Lung Slices Research Articles

Allosteric modulator potentiates β2AR agonist-promoted bronchoprotection in asthma models.

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote - with limited efficacy - bronchodilation in asthma. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to guinea pig β2ARs and downstream signaling of β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated β2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

The novel TRPA1 antagonist BI01305834 inhibits ovalbumin-induced bronchoconstriction in guinea pigs

BackgroundAsthma is a chronic respiratory disease in which the nervous system plays a central role. Sensory nerve activation, amongst others via Transient Receptor Potential Ankyrin 1 (TRPA1) channels, contributes to asthma characteristics including cough, bronchoconstriction, mucus secretion, airway hyperresponsiveness (AHR) and inflammation. In the current study, we evaluated the efficacy of the novel TRPA1 antagonist BI01305834 against AHR and inflammation in guinea-pig models of asthma.MethodsFirst, a pilot study was performed in a guinea-pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on (1) AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), (2) magnitude of EAR and LAR and (3) airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating-effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann–Whitney U test or One-way nonparametric Kruskal–Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett’s post-hoc test where appropriate.ResultsA dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study, 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea-pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea-pig trachea strips.ConclusionsTRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

Bronchoconstriction Induces TGF-β Release and Airway Remodelling in Guinea Pig Lung Slices

Airway remodelling, including smooth muscle remodelling, is a primary cause of airflow limitation in asthma. Recent evidence links bronchoconstriction to airway remodelling in asthma. The mechanisms involved are poorly understood. A possible player is the multifunctional cytokine TGF-β, which plays an important role in airway remodelling. Guinea pig lung slices were used as an in vitro model to investigate mechanisms involved in bronchoconstriction-induced airway remodelling. To address this aim, mechanical effects of bronchoconstricting stimuli on contractile protein expression and TGF-β release were investigated. Lung slices were viable for at least 48 h. Both methacholine and TGF-β1 augmented the expression of contractile proteins (sm-α-actin, sm-myosin, calponin) after 48 h. Confocal fluorescence microscopy showed that increased sm-myosin expression was enhanced in the peripheral airways and the central airways. Mechanistic studies demonstrated that methacholine-induced bronchoconstriction mediated the release of biologically active TGF-β, which caused the increased contractile protein expression, as inhibition of actin polymerization (latrunculin A) or TGF-β receptor kinase (SB431542) prevented the methacholine effects, whereas other bronchoconstricting agents (histamine and KCl) mimicked the effects of methacholine. Collectively, bronchoconstriction promotes the release of TGF-β, which induces airway smooth muscle remodelling. This study shows that lung slices are a useful in vitro model to study mechanisms involved in airway remodelling.

Allergen‐induced airway constriction in guinea pig lung slices is attenuated by arginase inhibition via increased nitric oxide production

In a guinea pig model of allergic asthma, arginase inhibition protects against allergen‐induced airway obstruction and greatly (>30‐fold) reduces airway sensitivity towards the inhaled allergen. Since arginase regulates nitric oxide (NO) synthesis via competition with NO synthase (NOS) for the common substrate L‐arginine, we hypothesized that the anti‐allergic effect of arginase inhibition involves increased NO synthesis. Ovalbumin (OVA; 0.0001–100 μg/ml)‐induced airway constriction was measured in lung slices obtained from actively‐sensitized guinea pigs using video‐assisted microscopy, in the absence or presence of inhibitors of arginase (ABH, 1μM or nor‐NOHA, 5 μM) and/or NOS (L‐NAME, 100 μM). OVA induced a dose‐dependent airway constriction with a mean maximal effect (Emax) of 98% and a mean EC50 of 0.039 μg/ml. ABH significantly reduced the Emax of OVA to 50%, whereas the sensitivity towards OVA was reduced by >100‐fold, to 5 μg/ml. nor‐NOHA mimicked the effects of ABH, whereas L‐NAME slightly increased contractile responses towards OVA (Emax: 100%; EC50: 0.007 μg/ml). L‐NAME partially reversed the protective effect of ABH against OVA (Emax: 74%; EC50: 0.125 μg/ml). In conclusion, arginase inhibition reduces allergen‐induced contractile responses of intrapulmonary airways by increasing NO synthesis.Supported by Erasmus Mundus partnership Join EU‐SEE and NIH grant HL‐103405.

Inhibition by ibudilast of leukotriene D4‐induced formation of inositol phosphates in guinea‐pig lung

1. The effects of a novel anti-asthmatic drug, 3-isobutyryl-2-isopropylpyrazolo [1,5-a]pyridine (ibudilast, KC-404) on leukotriene D4 (LTD4)-induced formation of inositol phosphates were studied in slices of guinea-pig lung and hippocampus. 2. In guinea-pig lung, ibudilast inhibited LTD4 (0.01-1 microM)-induced formation of inositol monophosphate (IP1) in a concentration-dependent manner (IC50 = 10 microM) without affecting LTD4 receptor binding. 3. Ibudilast (10 microM) inhibited histamine (0.1-1 mM)-induced formation of IP1 in guinea-pig lung slices but not in hippocampal slices. 4. Inhibition of agonist-induced formation of IP1 by ibudilast was non-competitive. 5. Ibudilast had no effect on either GTP- or calcium-stimulated phosphatidylinositol specific-phospholipase C activity of lung membranes. 6. These results suggest that ibudilast has no direct effect on LTD4 receptors, GTP binding proteins (G proteins) or phospholipase C, but inhibits inositol phosphate formation, possibly by interfering with the coupling between receptors and G proteins.

REV 2871 (CHBZ): A potent antiallergic agent with a novel mechanism of action: I. Activity profile as an inhibitor of mediator release

REV 2871 (CHBZ) and its putative metabolite REV 3579-Z (also designated in the literature as RHC 3579-Z) were shown to be potent and orally effective inhibitors of passive cutaneous anaphylaxis (PCA) in the rat (ED 50 = 12 mg/kg). The activity profiles of CHBZ, REV 3579-Z and disodium cromoglycate (DSCG) were compared as inhibitors of histamine release (HR) in vitro from rat mast cells, human basophils, and guinea pig lung slices. CHBZ was a potent inhibitor of both immunologie and non-immunologic HR (I 50 2–20 μM from rat mast cells). The activity profile of CHBZ as an inhibitor of HR from rat mast cells differed from that of DSCG and REV 3579-Z in the following respects: (a) increasing inhibition of HR with increasing preincubation time; (b) irreversibility of the inhibition; (c) lack of tachyphylaxis and cross-tachyphylaxis to DSCG; (d) potentiation of the inhibition of antigen-induced release of histamine (AIR) by DSCG; and (e) inhibition of HR induced by dextran + phosphatidyl serine, compound 48 80 , ionophore A23187 and platelet activating factor (PAF). In the human basophil model, CHBZ was: (a) a potent inhibitor (I 50 = 25 μM) of anti-IgE-induced release (AbIR), whereas DSCG and REV 3579-Z had no effect on AbIR; (b) more potent as an inhibitor of AbIR than ionophore-induced release, whereas the reverse was true for proxicromil; (c) an inhibitor of PAF-induced release, whereas proxicromil stimulated it; and (d) potentiative with proxicromil for inhibition of AbIR. In the guinea pig lung slice model, CHBZ inhibited AIR (I 50 = 800 μM) whereas DSCG and REV 3579-Z did not (I 50 > 300 μM). We conclude that CHBZ is an orally effective antiallergic agent whose mechanism of action as an inhibitor of mediator release is different from DSCG and proxicromil.

P4H0117) - The desensitization during antigen-antibody reaction of guinea-pig lung slices at various temperature

P4H0117) - The desensitization during antigen-antibody reaction of guinea-pig lung slices at various temperature

Antiallergic activity profiles in vitro of RHC 3164 and related compounds. I. A lack of correlation between inhibition of cyclic nucleotide phosphodiesterases and antigen-induced release of histamine from rat mast cells.

A series of 26 compounds belonging to the chemical class of (1,2,4)triazolo(4,3-a)-quinoxaline-1,4-diones have been investigated for their antiallergic activities in 3 in vitro models of anaphylaxis. Effects of these and other known antiallergic agents on cyclic nucleotide phosphodiesterases (cNUC-PDE) from purified rat mast cells have also been investigated. 18 compounds were potent (I50 less than or equal to 45 microM) inhibitors of antigen-induced release of histamine (AIR) from rat mast cells (RMC), 3 compounds inhibited anti-IgE-induced release of histamine from human basophils (I50 less than or equal to 25 microM) and none of the compounds significantly affected AIR from guinea pig lung slices. 13 of the compounds were more potent than theophylline as inhibitors of cyclic AMP-PDE and/or cyclic GMP-PDE from RMC. Parallel concentration-response curves for the inhibition of cyclic AMP-PDE and cyclic GMP-PDE indicated that these compounds probably interact with enzyme in the same manner. Paired regression analysis of the I50 values for inhibition of AIR and cNUC-PDE from RMC by these compounds revealed no statistically significant correlation between the inhibition of AIR and inhibition of cyclic AMP-PDE or cyclic GMP-PDE. We conclude: (1) some of these compounds are potent inhibitors of immunologic release of histamine from RMC with an in vitro activity profile similar to that of DSCG, and (2) inhibition of cyclic AMP or cyclic GMP hydrolysis by cNUD-PDE by these compounds, DSCG, and 6 known antiallergic agents is not the biochemical mechanism by which they inhibit AIR from RMC.

Antiallergic activity profile in vitro of RHC 2963 and related compounds

RHC 2963 (7-methyl-pyrido (3′, 2′:4,5)-thieno (3,2-d)-1,2,3 triazine-4(3H)-one and 20 related compounds have been investigated for their antiallergic activities in 3 in vitro models of anaphylaxis and for their effects on cyclic nucleoticle phosphosphodiesterases (cNUC-PDE) from purified rat mast cells (RMC). Nine compounds were potent (I 50⩽80 μM) inhibitors of antigen-induced release of histamine (AIR) from RMC, 2 compounds inhibited anti-IgE-induced release of histamine from human basophils (I 50⩽60 μM) and one compound inhibited AIR from guinea pig lung slices (I 50 = 55 μM). RHC 2963 was 18 times more potent than disodium cromoglycate (DSCG) as inhibitor of AIR from RMC and had an activity profile identical to that of DSCG in the following respects: loss of inhibitory activity with increasing preincubation time, tachyphylactic properties and inability to inhibit non-immunologic release of histamine induced by compound 48/80. Neither RHC 2963 nor DSCG had any effect on anti-IgE-induced release of histamine from human basophils or IgG 1-mediated release of histamine from guinea pig lung. Twelve of the compounds in this chemical series were more potent than theophylline as inhibitors of cyclic AMP and/or cyclic GMP phosphodiesterase (PDE) from RMC. Paired regression analysis of the I 50 values for inhibition of AIR and cNUC-PDE from RMC revealed no statistically significant correlation between the inhibition of AIR and inhibition of cAMP- or cGMP-PDE. We conclude: (1) RHC 2963 and some of the related compounds are potent inhibitors of immunologic release of histamine from RMC with a mechanism of action similar to that of DSCG, and (2) inhibition of cAMP- or cGMP-PDE by these compounds is not the biochemical mechanism by which they inhibit AIR from RMC.

Antiallergic Activity of Nylidrin Hydrochloride (RHC 3432-A)

Nylidrin (RHC 3432-A) has been investigated for its antiallergic activity in three in vitro models. Nylidrin was an effective inhibitor of IgE-mediated release of histamine from passively sensitized rat peritoneal mast cells and human basophils, and of IgG1-mediated release of histamine from passively sensitized guinea pig lung slices. The inhibition of the release of histamine by nylidrin in all three models was not antagonized by propranolol, indicating that nylidrin does not inhibit histamine release via stimulation of beta-adrenergic receptors. Isoproterenol and epinephrine were effective as inhibitors of the release of histamine only from guinea pig lung while salbutamol and terbutaline had no effect on immunologic release of histamine in all three models. Detailed comparative studies with disodium cromoglycate (DSCG) indicated that the mechanism of action of nylidrin in the rat mast cell model is different from that of DSCG.

The effect of captopril on plasma histamine and histamine release in vitro.

Plasma histamine and the release of histamine from blood in vitro has been determined in patients being treated for hypertension or heart failure with captopril. This drug does not alter plasma histamine levels in either group of patients or release histamine from human basophils, rat peritoneal mast cells or guinea pig lung slices upon challenge in vitro. We conclude that histamine release does not contribute to the cardiovascular or side effects of captopril.

Drug-induced modifications of the immune response. 1. Substituted 1-phenylisoquinolines.

A series of 1-phenylisoquinolines and related compounds was prepared and tested for potential antiallergic activity. Several compounds of this series inhibited the antigen-induced wheal formation in rat passive cutaneous anaphylaxis (PCA) assay, a commonly used test for antiallergic activity. Many of these compounds also inhibited the antigen-induced histamine release from passively sensitized guinea pig lung slices. Furthermore, almost all of these derivatives inhibited the cyclic nucleotide phosphodiesterase, suggesting this as one of several possible mechanisms of action.

Antigen-induced release of histamine from passively sensitized guinea pig lung slices. I. Optimum conditions for in vitro passive sensitization and challenge with antigen.

The effect of several parameters (pH, Ca++ concentration, time, temperature, lung slice size) on in vitro antigen-induced histamine release from passively sensitized guinea pig lung slices was investigated. With respect to pH, Ca++ concentration and time, the optimal conditions for the passive sensitization step (pH 7.8, Ca++ 1.5 mM, 2 h) were distinctly different from those for the antigen challenge step (pH 8.2, Ca++ 10mM, 15 min). Maximum antigen-induced release was obtained with 3 X 0.25 X 0.25 mm lung slices at 37 degrees C using air as gas phase.

Catabolism of epinephrine in control, sensitized and anaphylactic guinea pig lung slices

Catabolism of epinephrine in control, sensitized and anaphylactic guinea pig lung slices

Mechanisms of immunologic injury of rat peritoneal mast cells. I. The effect of phosphonate inhibitors on the homocytotropic antibody-mediated histamine release and the first component of rat complement.

The ability of a number of p-nitrophenylethyl, alkyl phenylalkyl, chloroalkyl, and aminoalkyl phosphonates to inhibit the homocytotropic antibody-mediated release of histamine from rat peritoneal mast cells has been tested. The effectiveness of these same phosphonates against the activated first component of rat complement (C'1a) has also been investigated. The rat mast cell esterase activated by the reaction of antigen and homocytotropic antibody resembles chymotrypsin in its reactivity with the phenylalkyl and chloroalkyl phosphonate, but is unlike this protease in its greater responsiveness to the 5-aminopentyl phosphonate relative to the pentyl phosphonate. The antigen-homocytotropic antibody-activated mast cell esterase and chymotrypsin, thus, appear to be similar, but different enzymes; i.e., they are parazymes (see reference 4, p. 501). There are distinct differences in the pattern of inhibition given by the phenylalkyl and aminoalkyl and alkyl phosphonates of the homocytotropic antibody-mediated histamine release from rat peritoneal mast cells and from guinea pig lung slices. On the basis of these differences it is concluded that the esterases activated by the combination of antigen and homocytotropic antibody on the mast cells of the two species are not the same. The arithmetic dose response curve found for the action of the phosphonates on the antigen-induced histamine release from rat peritoneal mast cells contrasted sharply with the logarithmic relationship found when these same inhibitors acted on the guinea pig lung system. This suggests that in addition to the antigen-antibody-activated esterases being unlike, the detailed mode of histamine release from the mast cells of the guinea pig lung differs from that of the mast cells of the rat peritoneum. Distinct and large differences were found in the pattern of inhibition of histamine release from rat peritoneal mast cells and of rat C'1a given by the phenylalkyl, and chloroalkyl and alkyl phosphonates implying that esterase activated by the combination of antigen with the sensitized rat peritoneal mast cells is not C'1a. Thus, the results with the peritoneal mast cells lead to the same conclusion as the previous work with guinea pig lung slices; i.e., the antigen-antibody-activated esterase involved in the homocytotropic antibody-mediated release of histamine is not part of the complement system.

A slow and progressive action of cyanide on the anaphylactic reaction of guinea pig lung slices

The effect of pretreatment of sensitized guinea pig lung slices with various concentrations of cyanide on the anaphylactic histamine release and the oxygen uptake was studied. Pretreatment with 1–10 mM cyanide inhibits both histamine release and oxygen uptake, while pretreatment with 0.01–0.1 mM inhibits histamine release without affecting oxygen uptake. The effect of cyanide on histamine release increases with time of contact and is not reversible by washing or by further incubation in cyanide-free medium. The addition of succinate has no effect on the histamine release of cyanide-treated slices, although it increases the oxygen uptake. Glucose, adenosine, and ribose remove the inhibiting effect of cyanide pretreatment on histamine release. Uncoupling of oxidative phosphorylation is suggested as an explanation for the mechanism of the effect of cyanide on the anaphylactic reaction.

Influence of sodium succinate and malonate on the histamine release in the anaphylactic reaction in vitro

Succinate increases and malonate decreases the amount of histamine liberated by sensitized guinea-pig lung slices submitted to the anaphylactic reaction. The results indicate a linkage between cellular metabolism and the mechanism of histamine release by antigen-antibody reaction.

The effect of succinate and malonate on the chemical release of histamine from Guinea-pig lung slices

The effect of succinate and malonate on the chemical release of histamine from Guinea-pig lung slices