Guinea Pig Lung Parenchymal Strips Research Articles

Anti-allergic, anti-asthmatic and anti-inflammatory effects of an oxazolidinone hydroxamic acid derivative (PH-251) – A novel dual inhibitor of 5-lipoxygenase and mast cell degranulation

We have recently reported the discovery of a series of oxazolidinone hydroxamic acid derivatives that are potent inhibitors of 5-lipoxygenase (5-LO) [arachidonate 5-lipoxygenase; EC 1.13.11.34]. We now report that one of the most active members of this series, compound PH-251, [(R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl) methyl)-N-hydroxyoctanamide], also possesses a unique and strong ability to concurrently inhibit mast cell degranulation. PH-251 inhibited the biosynthesis of leukotriene C4 (LTC4), as well as degranulation of IgE/allergen-activated bone marrow-derived mouse mast cells (BMMC) in vitro. In contrast, zileuton (the prototype 5-LO inhibitor) inhibited leukotriene generation, but not degranulation. Consistent with its dual activity, compound PH-251 also significantly inhibited both the early and the late anaphylactic contractions of guinea pig lung parenchymal strip, whereas zileuton inhibited only the late (leukotriene-dependent) contractions. Comparative structure–activity analysis of PH-251 and its structural analogues showed that the anti-degranulation effect appeared to be dependent on the length of the straight-chain hydrocarbon substitution on the hydroxamic acid moiety. In the in vivo studies, PH-251 (3–30 mg/kg s.c.) strongly inhibited various components of zymosan-induced peritonitis - a typical non-allergic LT-dependent animal model of inflammation. In the mouse allergic asthma model, the compound significantly inhibited allergen-induced bronchial eosinophilic inflammation and airway hyper-responsiveness to inhaled methacholine. These results show that PH-251 is a unique dual inhibitor of 5-LO and mast cell degranulation, with in vivo activity in animal models of disease and may therefore offer potential advantages over single-target drugs in the treatment of asthma and other allergic and inflammatory diseases.

Gossypol: a potent inhibitor of PAF-acether- and leukotriene-induced contractions of guinea-pig lung parenchyma strips.

Gossypol, a substance extracted from cotton plants, markedly inhibited the contractile responses of guinea-pig lung parenchyma strips stimulated with leukotriene B4 (LTB4), leukotriene D4 (LTD4) and PAF-acether but not the responses to histamine (except at high concentration). It was slightly less potent than nordihydroguaiaretic acid (NDGA) and a PAF-antagonist (BN 52021). From previously reported effects of gossypol on the cyclooxygenase and lipoxygenases, and on the similarity of the inhibition produced by NDGA and gossypol on the lung parenchyma, it is suggested that the inhibition of the myotropic activity of the lung parenchyma by gossypol is due to interactions with the formation of cyclooxygenase products within the guinea-pig lung.

Comparison of early and late responses to antigen of sensitized guinea pig parenchymal lung strips

The peripheral lung parenchyma has been studied as a component of the asthmatic inflammatory response. During induced constriction, tissue resistance increases in different asthma models. Approximately 60% of the asthmatic patients show early and late responses. The late response is characterized by more severe airway obstruction. In the present study, we evaluated lung parenchymal strips mechanics in ovalbumin-sensitized guinea pigs, trying to reproduce both early and late inflammatory responses. Oscillatory mechanics of lung strips were performed in a control group (C), in an early response group (ER), and in two late response groups: 17 h (L1) and 72 h (L2) after the last ovalbumin challenge. Measurements of resistance and elastance were obtained before and after ovalbumin challenge in C and ER groups and before and after acetylcholine challenge in all groups. Using morphometry, we assessed the density of eosinophils and smooth muscle cells, as well as collagen and elastin content in lung strips. The baseline and postagonist values of resistance and elastance were increased in ER, L1, and L2 groups compared with C (P < or = 0.001). The morphometric analysis showed an increase in alveolar eosinophil density in ER and L2 groups compared with C (P < 0.05). There was a significant correlation between eosinophil density in parenchymal strips of C, L1, and L2 groups and values of resistance and elastance postacetylcholine (r = 0.71, P = 0.001 and r = 0.74, P < 0.001, respectively). The results show that the lung parenchyma is involved in the late response, and the constriction response in this phase is related to the eosinophilic inflammation.

Development of agonists of endothelin-1 exhibiting selectivity towards ETA receptors.

(1) Endothelin-1 (ET-1) is a bicyclic 21-amino-acid peptide causing a potent and sustained vasoconstriction, mainly through the ET(A) receptor subtype. So far, no selective ET(A) agonists are described in the literature. (2) A series of truncated and chemically modified ET-1 analogues were obtained through solid-phase peptide synthesis and their biological activity was assessed on rat thoracic aorta rings (ET(A) receptors) and guinea-pig lung parenchyma strips (ET(B) receptors). (3) Structure-activity studies led to the identification of ET-1 fragments exhibiting an ET(A) selective agonistic activity. (4) In particular, [D-Lys(9)]cyclo(11-15) ET-1(9-21) was the most potent peptide. It appeared as a full agonist of ET(A) receptors, being under two orders of magnitude less potent than ET-1 (EC(50): 2.3 x 10(-7) vs 6.8 x 10(-9) M). Interestingly, even a linear formylated analogue, [Ala(11,15), Trp(For)(21)]ET-1(9-21), showed a selective ET(A) activity (EC(50): 3.0 x 10(-6) M). None of the numerous analogues of the series exhibited substantial effects in the guinea-pig lung parenchyma bioassay. (5) Thus, this study describes the first compounds showing a significant bioactivity in an ET(A) pharmacological preparation while being inactive in an ET(B) paradigm. They show that the ET-1 pharmacophores, responsible for the ET(A)-mediated actions, are located within the 9-21 segment of the molecule. Moreover, the bicyclic structure of ET-1 does not appear as essential for the ET(A)-related vasoconstriction. Results also suggest that the positive charge of the Lys(9) side chain participates in an intramolecular ionic bond with the carboxylate function of Asp(18).

L-CARNITINE DOES NOT EXERT ANY IN VITRO RELAXANT EFFECT IN GUINEA PIG TRACHEA, LUNG PARENCHYMA AND HUMAN BRONCHIAL TISSUE

The aim of the present study was to investigate the probable in vitro relaxant effect of carnitine in guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial tissue. It was suggested by an in vivo study that carnitine pretreatment prevented the subclinic bronchospasm in children who underwent chronic hemodialysis. Tracheal and lung parenchymal preparations of 10 guinea pigs and 5 human bronchial tissues were prepared and mounted in 20-mL organ baths. In the first series of experiments, contractions to carbachol and histamine (10 -9 to 10 -3 M) were compared after the tissues were incubated with different concentrations of L-carnitine (10 -6 to 10 -4 M). pD 2 values were compared with analysis of variance (ANOVA) and P <. 05 was considered as significant. In the second part of experiments, the inhibitory effect of L-carnitine (10 -9 to 10 -3 M) was investigated on the sustained contractions of preparations to carbachol (10 -6 M) and histamine (10 -5 M). In the first part of the study pD 2 values obtained with carbachol were 6.48 ± 0.09, 5.42 ± 0.05, and 6.48 ± 0.02 for guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial tissues, respectively. pD 2 values obtained with histamine were 5.34 ± 0.10, 5.74 ± 0.06, and 6.32 ± 0.03 for guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial tissues, respectively. No significant difference was observed between the pD 2 values before and after incubation with carnitine (P >. 05). In the second part of the study, only 10 -4 M L-carnitine exerted an insignificant relaxant effect (6.16% ± 1.22% on carbachol induced contractions and 4.48% ± 0.85% on histamine induced contractions) in guinea pig trachea. Our results show that L-carnitine exerts no in vitro relaxant effect in guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial preparations.

An improved procedure for the preparation of the endothelin-B-receptor ligand sarafotoxin 6c.

Sarafotoxin 6c (S6c) is a specific agonist for the endothelin-B- (ETB) receptor, being largely used as a standard compound in pharmacological studies related to ETB-receptors. We have developed an improved peptide synthesis prncedure. Using the Fmoc chemistry methodology and an appropriate orthogonal protection scheme, we obtained a crude peptide with a major component identified as [Cys(Acm)(1,15)]S6c. A silver salt (AgBF4) treatment of this crude material completely removed the Acm moieties without the appearance of any major side-products. The free peptide was cyclized using a Fe3+/Fe2+ oxidizing method. Besides the formation of the bridges, no other modification was observed. Thus, the peptide purification was facilitated and an excellent yield of pure sarafotoxin was obtained. The material was characterized by analytical reverse phase-high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Pharmacological properties of the peptide were evaluated in an ETB preparation (guinea pig lung parenchyma strips) and were identical to those of an S6c standard.

Pharmacological antagonism of Anchietia salutaris extracts on the contraction induced by prostaglandin D2 and U46619 in guinea-pig lung parenchymal strips.

Anchietia salutaris tea is traditionally used in Brazil to treat allergies, suggesting it contains compounds with antagonistic activity on the allergic mediators. We have evaluated extracts and semi-purified fractions of Anchietia salutaris as a source of compounds having this type of antagonism on the contraction induced in guinea-pig lung parenchymal strips and on platelet aggregation and shape change. After 10 min pre-incubation dichloromethane extracts containing 30 or 100 microg mL(-1) inhibited the contraction induced by prostaglandin D2 (PGD2) in guinea-pig lung parenchymal strips with dose ratios (DR) of 0.76+/-0.14 and 0.93+/-0.19, respectively; the amount of inhibition depended both on the concentration and on the time of pre-incubation (DR after 30 min pre-incubation was 1.21+/-0.51). The dichloromethane extract and its semi-purified fractions also inhibited the contractions induced by U46619, a more potent, stable, synthetic agonist of thromboxane A2 (TxA2) prostanoid (TP) receptors, the receptors acted upon by PGD2 to produce lung contractions. The dichloromethane extract did not inhibit the lung parenchymal contractions induced by histamine, leukotriene D4 (LTD4) or platelet-activating factor (PAF). Platelet aggregation induced by U46619, adenosine 5'-diphosphate (ADP) or PAF was not inhibited by the dichloromethane extract. Indeed, the extract potentiated platelet aggregation induced by low concentrations of these agonists and also potentiated the shape change induced by U46619. These results imply that the dichloromethane extract of Anchietia salutaris and its semi-purified fractions contain an active principle that competitively inhibits TxA2 TP receptors, the stimulation of which causes lung parenchymal contraction. The inhibition seems to be selective for this receptor subtype, because the extract fails to inhibit platelet aggregation or shape change. This provides additional support of earlier reports suggesting the occurrence of TP receptor subtypes.

Responses of guinea-pig lung parenchymal strips to tracheobronchial lavage fluid from horses affected with summer pasture-associated obstructive pulmonary disease.

The response of parenchymal strips from guinea-pig lungs to tracheobronchial lavage fluid (TBLF) collected from 8 normal horses and from 8 affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) was determined. TBLF was collected during the summer (July) and winter (February) seasons. The serum/TBLF urea nitrogen ratio was used to standardize the mediator concentration in the TBLF. Four strips were used from each guinea-pig. The first strip did not receive any antagonist and served as the control. The second, third and fourth strips received antagonists of PGE2, LTD4 and PAF, respectively at 10(-6) mol/L for 30 min. The tissues were then precontracted with a dose of histamine (10(-5) mol/L) and their responses to 1 ml of TBLF were determined. The study showed that TBLF obtained in the summer from unaffected horses produced a significantly greater relaxation than that from the affected horses, whereas TBLF obtained in the winter from unaffected or affected horses did not cause a significantly different degree of relaxation. Among the antagonist-treated strips, only those exposed to the PGE2 blocker showed a significant reduction in the relaxation caused by TBLF obtained in the summer from SPAOPD horses. This suggests that PGE2 is an important mediator present in the summer in the TBLF from horses affected with SPAOPD.

Glucocorticoid modulation of muscarinic and beta-adrenergic receptors in guinea pig lung.

We investigated the effect of the in vivo treatment of guinea pigs with methylprednisolone, 10 mg/kg daily, on lung muscarinic and beta-adrenergic receptors. Receptor densities were assessed by saturation experiments of tritiated N-methylscopolamine and dihydroalprenolol binding to lung membranes. After 3 h of treatment, methylprednisolone induced a decrease of 19.2% (P < 0.05) of muscarinic receptors but was without effect on beta-adrenergic receptor density. After 24 h, an increase of 39.7% (P < 0.01) and 16.9% (P < 0.05) was observed for muscarinic and beta-adrenergic receptors, respectively. For muscarinic receptors, this increase reached 53.4% (P < 0.01) within 48 h and stayed at this level until 96 h. The increase of beta-adrenergic receptors was maximal (24.9%) after 72 h and returned to the control value after 96 h. The dissociation constant (Kd) values of both ligands were not affected by the glucocorticoid treatment. Functional studies showed that the 96 h treatment did not affect the contractile response of guinea pig lung parenchymal strips to carbachol since the 50% concentration value (EC50) and the maximal contraction value (Emax) were not significatively different from control values. These data show that glucocorticoids control the expression of both muscarinic and beta-adrenergic receptors in guinea pig lung but with different time courses and to a larger extent for muscarinic receptors. The glucocorticoid treatment did not modify the contractile response of lung strips to carbachol, confirming the absence of effect on the affinity of muscarinic receptors and suggesting that the receptor reserve exceed the increase of their density by the steroid.

Bombesin-induced contractions of guinea pig lung strips are modulated by endogenous nitric oxide.

We have investigated the effects of a nitric oxide (NO) biosynthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) on the bombesin-evoked contraction of guinea pig parenchymal lung strips. The bombesin-induced contractions of lung strips were significantly increased after L-NAME (300 micro)) pre-treatment. The maximal response was increased (P < 0.01) by 37% after L-NAME treatment when compared with the control group. The pD2 value was not influenced by L-NAME pre-treatment. The enhancement of the bombesin-induced contraction caused by L-NAME was reversed by addition of an excess of the NO precursor L-arginine (600 microM) but not by the addition of its inactive enantiomer D-arginine (600 microM). Like L-NAME, methylene blue (1 microM), an agent that inhibits the soluble guanylyl cyclase activated by NO, significantly increased (P < 0.01) the maximal contraction induced by bombesin (183 +/- 16 mg) when compared with the control group (141 +/- 15 mg). When tested against other agonist-induced contractions, L-NAME did not change the responsiveness of parenchymal lung strips to bradykinin or carbachol but significantly increased lung contraction induced by histamine. NO synthesis inhibition resulted in a pronounced increase in the bombesin-induced contraction of guinea-pig lung strips. Our results suggest that bombesin contributes to NO synthesis and release which then acts to reduce the contraction of the lungstrip in response to bombesin.

Tachykinin receptors mediating contraction of guinea pig lung strips

The aim of this study was to determine which tachykinin receptors mediate contraction in guinea pig lung parenchymal strips in vitro. Contraction caused by selective neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 (NK-3) receptor agonists and the natural agonists substance P (SP) and neurokinin A (NKA) was measured in the absence or presence of the NK-1 antagonist CP-96,345 and/or the NK-2 antagonist SR 48968. The NK-1 agonist [Sar 9, Met(O 2) 11]-substance P and the NK-2 agonist [Nle 10]-neurokinin A 4–10 caused similar concentration-dependent contractions that were inhibited by CP-96,345 and SR 48968, respectively. The NK-3 agonist [MePhe 7]-neurokinin B also caused contraction, albeit at 10-fold higher concentrations, and this contraction was unaffected by either the NK-1 or NK-2 antagonist or the combination of both antagonists. Either CP-96,345 or SR 48968 alone had little effect on NKA-mediated contraction but administration of both antagonists virtually eliminated force generation. SP-induced tension was partially inhibited by SR 48968 and unaffected by CP-96,345. A second NK-1 receptor antagonist CP-99,994 also had no effect on SP-mediated tension. Therefore, NK-1, NK-2, and NK-3 agonists can all cause contraction in guinea pig lung strips, NKA-induced tension is mediated by both NK-1 and NK-2 receptors, and SP-induced contraction is mediated in part by NK-2 receptors. Both the SP and the [MePhe 7]-neurokinin B data suggest that activation of a third neurokinin receptor subtype that is unaffected by an NK-1 receptor antagonist or an NK-2 receptor antagonist can cause contraction in guinea pig lung strips.

Structure-Activity Studies of the C-Terminal Segment of Structurally Reduced Analogues of ET-1

Structurally reduced analogues of endothelin-1 (ET-1) were synthesized by linking via aminocaproic acid (Aca) the segment 3-11 of ET-1 to C-terminal fragments of various lengths [16-21, 17-21,...,21]. Analogues were studied in their linear or cyclic form in the absence or presence of a formyl group on the Trp21 side-chain, and their biologic activities were tested using guinea pig lung parenchymal strips. The absence of the first disulfide bridge and the presence of the Aca spacer caused a slight decrease in activity. The presence of His16 is essential for the contractile activity of the monocyclic peptides. Formylation of these monocyclic analogues did not modify this behavior. Similarly, linear analogues carrying S-acetamidomethyl (Acm) functions and an Aca linker were still active. However, most formylated linear derivatives were partial agonists, whereas the addition of His16 caused a decrease in contractile activity. In these latter analogues, molecular modeling studies suggested that formylation produces different conformations.

Reversibility of tachyphylaxis to C5a in guinea pig tissues, perfused human placental lobule, and umbilical artery

The spasmogenic effect of C5a is mediated by histamine and/or eicosanoids. Tachyphylaxis to this effect of C5a occurs rapidly, but the spasmogenic effects of C5a on a guinea pig lung parenchymal strips, field-stimulated ventricular papillary muscle, and human umbilical artery were completely restored by a 1-h period of drug-free rest, whereas that of guinea pig ileum was not. Perfusion of the isolated human placental lobule with C5a caused a transient pressor response that was largely abolished by indomethacin (5 microM), indicating mediation by cyclooxygenase metabolites. This pressor response to C5a was also completely restored following a 1-h rest period. The results show that tissue rest reverses tachyphylaxis to the spasmogenic effects of C5a in tissues where the response is mediated by cyclooxygenase metabolites. Where the response is mediated by histamine released by mast cells, restoration does not occur, presumably because of the catastrophic nature of mast cell degranulation. Histamine released in guinea pig papillary muscle by C5a may be from non-mast-cell sources.

Lipoxygenase stimulating effects of hydroxylated docosahexaenoates produced by human platelets

Human platelet suspensions are capable of lipoxygenating docosahexaenoic acid (22:6n3) to an 11(S)-OH-, 14(S)-OH- or 17(S)-OH-22:6n3. The structure and stereochemical purity of these derivatives were confirmed by GC/MS and chiral phase LC analysis. The purified OH-22:6n3 positional isomers which are formed by human platelets were capable of inducing a concentration-dependent contractile response in the guinea-pig lung parenchymal strip at sub-micromolar concentrations. OH-22:6n3 may act in part through stimulation of leukotriene (LT) production as an increase in peptidyl-LT levels (LTC 4, LTD 4 and LTE 4) occurred during the OH-22:6n3-induced contraction in this preparation. Both specific lipoxygenase inhibitors (caffeic acid, 20 uM and NDGA, 50 uM) and a LT receptor antagonist (FPL55712, 20 uM) significantly inhibited the contractile response. Moreover, the OH-22:6n3 positional isomers induced a concentration-dependent increase in LTB 4 and LTC 4 production in the guinea-pig chopped lung preparation. Other hydroxylated fatty acids and parent fatty acids which were tested (12-OH-20:4n6, 5-OH-20:4n6, 12-OH-20:5n3, 20:5n3 and 22:6n3) did not significantly contract this airway smooth muscle preparation or alter LT production. The hydroxylated 22:6n3 metabolites may modulate airway smooth muscle function in part through the release of peptidyl-LTs from the guinea-pig lung.

Leukotriene D4 receptors are not negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma.

1. The possibility that receptors for the peptide-containing leukotrienes may be negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma was investigated by comparing the effect of leukotriene D4 (LTD4) on the intracellular cyclic nucleotide (cyclic AMP and cyclic GMP) content and on the activity of cyclic AMP-dependent protein kinase (PKA). In addition, the potential association between changes in the cyclic nucleotide content and the ability of LTD4 to increase lung parenchymal tone was also evaluated. 2. Non-cumulative challenge of parenchymal lung strips with LTD4 elicited concentration-dependent contractions (pD2 = 8.23) that were paralleled by concentration-related increases in the intracellular level of cyclic AMP and cyclic GMP, and in the activation state of PKA (Kact = 33 nM). Temporally, these biochemical effects of LTD4 were transient, peaking after approximately 5 min drug contact thereafter decaying, despite the continued generation of tone. Both the biochemical and mechanical effects of LTD4 were antagonized by the LTD4-receptor blocking drug, ICI 198,615 (1 microM for 60 min), indicating that they were receptor-mediated events. 3. Challenge of guinea-pig lung with LTD4 (200 nM; EC100 for tension generation) stimulated a 150 and 70 fold increase in the elaboration of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) respectively, relative to that generated spontaneously. 4. Pretreatment of lung strips for 60 min with an irreversible inhibitor of cyclo-oxygenase, flurbiprofen,at a concentration (8 microM) that abolished both basal and LTD4 (200 nM)-induced TXB2 and 6-keto-PGF1alpha release, relaxed rapidly the spontaneous tone of the tissues, reduced the cyclic AMP content by ~50%and lowered the PKA activity ratio from 29% to 17%. In addition, flurbiprofen abolished the ability of LTD4 (200 nM) to increase the cyclic AMP content and to activate PKA. Functionally, the magnitude of LTD4 (200 nM)-induced tone and the increase in cyclic GMP content were attenuated by approximately 20% and 50% respectively in flurbiprofen-treated tissues.5. In flurbiprofen-treated tissues, isoprenaline (10 microM for 10 min) increased the cyclic AMP content(from 4 to 27 pmol mg-1 protein) and activated PKA (from 15% to 26%). Preincubation (30 s or 5 min)of lung with LTD4 (200 nM) did not inhibit (or enhance) these isoprenaline-induced effects.6. Pretreatment of lung strips for 60 min with the thromboxane synthetase inhibitor, dazmegrel (10 microM),relaxed the spontaneous tone of the tissues, abolished the LTD4 (200 nM)-stimulated release of TXB2 and significantly enhanced (~two fold) the elaboration of 6-keto-PGF1alpha. In addition, dazmegrel attenuated (by ~50%) LTD4 (200 nM)-induced cyclic GMP accumulation but approximately doubled both the cyclic AMP content and PKA activity ratio. LTD4-induced contractions, in contrast, were not affected by dazmegrel.7. EP 092 (1 microM for 60 min), a selective TP-receptor blocking drug, had no effect on spontaneous tone,eicosanoid formation or on the cyclic GMP content of guinea-pig lung parenchymal strips. Likewise,EP 092 exerted no significant mechancial effect in lung challenged with LTD4 (200 nM) although it did potentiate, to a small extent, the ability of LTD4 (200 nM) to increase the cyclic AMP content.8. It is concluded that LTD4 can increase the intracellular level of cyclic AMP in guinea-pig parenchyma and activate PKA by a leukotriene-receptor-mediated mechanism sensitive to ICI 198,615. However,these biochemical actions of LTD4 are induced indirectly by an arachidonic acid-derived cyclo-oxygenase product(s) other than TXA2. Thus, contrary to reports of other investigators, no evidence was found to corroborate the finding that stimulation of leukotriene receptors on guinea-pig lung parenchyma results in a rapid lowering of the cyclic AMP content even in cyclo-oxygenase-blocked tissues. These data,therefore, do not support the hypothesis that leukotriene-induced tension generation is dependent upon a prior reduction in the cyclic AMP content.

Pharmacological and histological examinations of regional differences of guinea-pig lung: a role of pleural surface smooth muscle in lung strip contraction.

1. Parenchymal lung strip preparations have been widely used as an in vitro model of peripheral airway smooth muscle. The present study examined functional responses of 4 consecutive guinea-pig lung parenchymal strips isolated from the central region (segment 1) to the distal edge (segment 4) of the lower lung lobe. The middle two segments were designated as segments 2 and 3. 2. Lung segments 1 and 4 exhibited significantly greater contraction than the other 2 segments to KCl when responses were expressed as mg force per mg tissue weight. Contractile responses to bronchospastic agents including histamine, carbachol, endothelin-1, leukotrienes (LT) B4 and D4, and the thromboxane A2-mimetic U46619 demonstrated no significant difference in EC50 values among the 4 lung segments. 3. Contractile responses of segments 1 and 4 to antigen-challenge (ovalbumin), ionophore A23187 and substance P were significantly greater than the other 2 segments with respect to either sensitivity or maximum responsiveness. 4. U46619-induced contractions of the 4 lung segments were relaxed in similar manner by papaverine and theophylline up to 100%, salbutamol up to 80%, and sodium nitroprusside by only 20%. In contrast, sodium nitroprusside markedly reversed U46619-induced contraction of pulmonary arterial rings and bronchial rings. 5. Histological studies identified 2-4 layers of smooth muscle cells underlying the lung pleural surface. Mast cells were prominent in this area. Moreover, morphometric studies showed that segment 4 possessed the least amount of smooth muscle structures from bronchial/bronchiolar wall and vasculatures as compared to the other 3 segments, and a significant difference in this respect was evident between segment 1 and segment 4.6. Since lung segments 1 and 4 are covered with larger surface area of lung pleura, the present results suggest that the significantly greater intrinsic contractile responses of segments 1 and 4 are associated with the presence of increased lung pleural surface possibly together with more mast cells. Thus, a primary contribution to the net contraction of the lung parenchymal strips may be smooth muscle from the lung pleura, alveolar ducts and interstitial contractile cells rather than from bronchi/bronchioles and microvasculatures.

Antiasthmatic Activity of a Novel Thromboxane A&lt;sub&gt;2&lt;/sub&gt; Antagonist, S-1452, in Guinea Pigs

We examined the effect of a potent thromboxane (Tx) A2 receptor antagonist, calcium (1R, 2S, 3S, 4S)-(5Z)-7-(((phenylsulfonyl)amino)bicyclo[2.2.1] hept-2-yl)-5-heptenoate dihydrate (S-1452), on antigen- and various allergic-spasmogen-induced contractions of guinea pig lung parenchymal strips and on the increase in insufflation pressure, an index of bronchoconstriction, in anesthetized guinea pigs. In isolated guinea pig lung parenchymal strips, S-1452 showed competitive antagonism of the contractile activity of U-46619, a TxA2 mimetic, with a pA2 value of 8.9. The compound also inhibited the contraction induced by prostaglandin (PG) D2 and PGF2 alpha, but a TxA2 synthetase inhibitor, OKY-046, did not. In contrast, both drugs inhibited not only leukotriene (LT) D4-induced contraction but also antigen-induced contraction in the presence of a histamine antagonist. In anesthetized guinea pigs, oral administration of S-1452 markedly inhibited the bronchoconstrictions induced by intravenous injection of U-46619, PGD2, PGF2 alpha, LTD4 and platelet-activating factor (PAF) with ED50 values of 0.006, 0.031, 0.112, 0.033 and 0.115 mg/kg, respectively, but OKY-046 inhibited only that by LTD4 and PAF. Additionally, bronchoconstriction following intravenous injection of antigen was almost completely suppressed by S-1452 (0.1 mg/kg) and partially by OKY-046 (300 mg/kg) in passively sensitized guinea pigs which were treated with diphenydramine and propranolol. The inhibitory effect of S-1452 against U-46619-induced broncho-constriction persisted up to 7 h after oral administration.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vitro Antagonism of ONO-1078, a Newly Developed Anti-Asthma Agent, against Peptide Leukotrienes in Isolated Guinea Pig Tissues

We evaluated the antagonist activity of ON01078 against peptide leukotrienes (LTs) by a radioligand binding assay and functional experiments in guinea pigs. In the radioligand binding assay, ONO-1078 inhibited [3H]LTD4 and [3H]LTE4 bindings to lung membranes (Ki = 0.99 and 0.63 nM, respectively) and was 2,000- to 3,000-fold more potent than FPL55712. Antagonism of ONO-1078 against [3H]LTC4 binding (Ki = 5640 nM) was approximately twofold more potent than that of FPL55712. The antagonism of ONO-1078 against [3H]LTD4 binding was competitive. In functional experiments, ONO-1078 showed competitive antagonism against the LTC4- and LTD4-induced contractions of guinea pig trachea and lung parenchymal strips with a pA2 range of 7.70 to 10.71 and was approximately 400- to 3,300-fold more potent than FPL55712. Interestingly, in the presence of an inhibitor of the bioconversion of LTC4 to LTD4, ONO-1078 also antagonized the LTC4-induced contraction of guinea pig trachea (pA2 = 7.78). ONO-1078 significantly reversed the LTD4-induced prolonged contraction without effect on the KCl- and BaCl2-induced contractions of guinea pig trachea. Furthermore, ONO-1078 antagonized the antigen-induced SRS-A mediated contraction of guinea pig trachea. On the other hand, ONO-1078 showed no antagonism against histamine, acetylcholine, 5-hydroxytryptamine, prostaglandin D2 and U-46619. In addition, ONO-1078 showed little or no effect on the activities of cyclooxygenase, 5-lipoxygenase and thromboxane synthetase. These in vitro studies indicate that ONO-1078 is a highly potent, selective and competitive antagonist of peptide leukotrienes that acts with higher affinity at LTD4 and LTE4 receptors than LTC4 receptors.

Effects of vasoactive intestinal peptide, helodermin and galanin on responses of guinea‐pig lung parenchyma to histamine, acetylcholine and leukotriene D4

1. The effect of vasoactive intestinal peptide (VIP) was studied on the contractile response of guinea-pig lung parenchymal strips (GPP) induced by bronchoconstrictor agonists, such as leukotriene D4 (LTD4), histamine and acetylcholine (ACh). This effect of VIP was compared with helodermin, a peptide that is structurally related to VIP, and galanin, another neuropeptide that is thought to co-exist with VIP. 2. VIP (10 nM) induced a potent and reversible inhibition of the contractions of GPP induced by LTD4 (1-30 pmol) but did not affect those due to ACh (1-100 nmol) or histamine (1-30 nmol). A ten fold higher concentration of VIP (100 nM) did not further inhibit LTD4-induced responses or reduce those induced by histamine or ACh. 3. Helodermin (10 nM) had a similar inhibitory effect on contractions of GPP induced by LTD4 (3-30 pmol) but did not affect contractions induced by histamine (1-10 nmol). 4. Indomethacin (2.8 microM) and salbutamol (10 nM) significantly reduced responses elicited by LTD4 and histamine but not those due to ACh. A ten fold higher concentration of salbutamol (100 nM) further inhibited the contractions due to LTD4 and histamine and at this concentration responses induced by ACh were inhibited. 5. VIP (10 nM) and helodermin (10 nM) significantly reduced the LTD4-induced release of thromboxane A2 (TXA2), measured as TxB2 by radioimmunoassay, from GPP. The smaller release of TxA2 induced by histamine was not significantly reduced in the presence of VIP. 6. In comparative studies, galanin (10-100 nM) did not affect contractions of GPP induced by either LTD4, histamine or ACh. In contrast to VIP and helodermin, both at 0.1-3 nmol, which induced doserelated relaxations of guinea-pig trachea, galanin was inactive on this preparation in doses of up to 3 nmol.7. In conclusion, our results show that contractions of GPP induced by LTD4 are more sensitive to inhibition by VIP and helodermin than are contractions due to histamine or ACh. This inhibition appears to be associated with the different contribution of released TxA2 to contractions evoked by the agonists. VIP and helodermin inhibit the cyclo-oxygenase-dependent component of the LTD4-induced response, as in the case of indomethacin.

Inhibitory effect of econazole on the release of thromboxanes.

The effect of econazole on the release of thromboxanes was investigated. It was found that econazole inhibited concentration-dependently the aggregation of guinea pig platelets stimulated with arachidonic acid. The compound also reduced significantly the LTB4-induced contraction of guinea pig lung parenchyma strips and the contraction of rabbit aorta to the effluent of LTD4-stimulated guinea pig lungs, both effects mediated mostly by thromboxane generation. The concentration of TXB2 in the effluents from LTD4 stimulated lungs, assayed by EIA, was significantly reduced following pretreatment of the lungs with 10(-4) M and 10(-5) M of econazole, whereas the levels of PGE2 were increased. These results demonstrate that econazole is a selective inhibitor of thromboxane synthesis.