Heterogeneity of leukotriene receptions in guinea-pig trachea

The selective leukotriene (LT) antagonist FPL 55712 antagonized the contractile activity of synthetic LTD₄ and E₄ on guinea-pig trachea. Schild analysis of the antagonism provided evidence for two distinct receptors for LTD₄: one with significantly higher affinity for FPL 55712 than the other. LTE₄ appears to interact preferentially with the high affinity receptor.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C₄ and D₄ in isolated airway smooth muscle was investigated. In rat trachea, neither LTC₄ or D₄ elicited a response. In contrast, LTC₄ was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD₄ in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC₄ was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD₄ in both trachea and parenchyma. As regards LTC₄-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC₄ in parenchyma.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [³H]-leukotriene D₄ ([³H]-LTD₄), in the presence or absence of unlabeled LTD₄, the diastereoisomer of LTD₄ (5R,6S-LTD₄), leukotriene E₄ (LTE₄) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [³H]-LTD₄ in the crude membrane fraction isolated from guinea pig lung. The time required for [³H]-LTD₄ binding to reach equilibrium was approximately 20 to 25 min at 37°C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [³H]-LTD₄ to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (B_max), derived from Scatchard analysis, was approximately 320±200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5±4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD₄, FPL 55712 and 5R,6S-LTD₄ competed with [³H]-LTD₄. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD₄, did not significantly compete with [³H]-LTD₄ at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

Propranolol potentiates leukotriene D4-induced bronchoconstriction and enhances antagonism by FPL 55712

Aerosol LTD₄-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs was potentiated by either pretreatment with propranolol or bilateral adrenalectomy, whereas bilateral vagotomy did not affect the LTD₄ response. The dose-response curve describing LTD₄-induced changes in dynamic lung compliance (C_{D Y N}) and pulmonary resistance (R_L) [as reflective indices of bronchochonstriction] was shifted to the left by approximately 20-fold by propranolol. Against an equal degree of LTD₄-induced bronchoconstriction, the leukotriene antagonist, FPL 55712, had an apparent 20-fold greater potency in propranolol-pretreated animals vis a vis saline-treated controls. The duration of action of aerosol FPL 55712 was similar in both propranolol-treated and saline-treated animals. These results demonstrate that aerosol LTD₄-induced bronchoconstriction is modulated by an adrenergic compensatory bronchodilator mechanism that is apparently dependent upon the adrenals and independent of vagal influences. Inhibition of the effect of this reflex with propranolol also enhances the apparent potency of an aerosol leukotriene antagonist, FPL 55712, presumably reflecting a constant LTD₄ to antagonist ratio in the saline-treated and propranolol-pretreated guinea pigs.     

Characterization of specific receptors for leukotriene D4 on human alveolar macrophages

Using ³H-leukotriene D₄, a specific receptor assay has been developed for human alveolar macrophages, obtained by broncho-alveolar lavage of patients undergoing fiberoptic bronchoscopy because of suspected bronchial carcinomaa. Lavage was performed in a carcinoma-free lobe of the lung and alveolar macrophages were subsequently isolated and incubated for binding studies. ³H-Leukotriene D₄ was found to bind specifically with high affinity (K_d = 3.8 nM), in a saturable manner (B_max = 90 fmol/10⁶ cells), reversible and selective. Specific binding was linear with protein concentration and equilibrium binding at 4°C was reached at 50 min. Scatchard and Hill analysis revealed a single class of binding sites with no cooperativity among the sites. displacement studies with LTD₄, the selective SRS-A antagonist FPL 55712 and with leukotriene C₄ revealed respective K_i values of 3.4; 16; and 110 nM. The data suggest that human alveolar macrophages may contain a specific receptor type for LTD₄, which has a relatively low affinity for LTC₄, and are discussed in relation to modulatory processes in the lung, apart from direct actions of LTD₄ on smooth muscle receptors. From the data here acquired, it may be apparent that the study of characteristics of receptors specific for a broncho-active substance like LTD₄ on huma alveolar macrophages, which play an important role in immuno-inflammatory processes seen in many chronic lung diseases, may yield major insights into the pathogenesis and therapy decisions involved in these diseases.     

Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A₄ and D₄ displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD₄ was much higher than that of LTA₄. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD₄ was very active whereas LTA₄ was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA₄ by the smooth muscle preparations was a prerequisite to its biological activity. LTA₄ was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA₄ to LTB₄, LTC₄, LTD₄ and LTE₄. Incubation of LTA₄ with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB₄ and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA₄ undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activites of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA₄ is related to the tissue levels of enzymes which catalyse its biotransformation.     

Leukotriene F4: Comparison of its pharmacological profile with that of the other cysteinyl-containing leukotrienes in guinea-pig ileum and lung parenchyma in vitro

The biological effects of leukotriene (LT)F₄ were compared, on a molar basis, with those of LTC₄, LTD₄ and LTE₄ on isolated superfused strips of guinea-pig ileum smooth muscle (GPISM) and lung parenchyma (GPP). LTF₄ was 1–2 orders of magnitude less active than the other leukotrienes on GPISM (LTD₄ > LTC₄ > LTE₄ > LTF₄) whereas, in the GPP, the activity of LTF₄ was comparable with that of LTE₄, both leukotrienes being about one order of magnitude less active than LTC₄ or LTD₄ (LTC₄=LTD₄ > LTE₄=LTF₄). Further, LTF₄ caused protracted contractions of the GPP which were indistinguishable from those due to LTE₄ and of a much longer duration than responses elicited by either LTC₄ or LTD₄.FPL 55712 (1.9μM) antagonised actions of LTF₄ in both tissue preparations. Indomethacin (2.8μM) inhibited contractions induced by LTF₄ in GPP indicating that part of the bronchoconstriction due to LTF₄, like that elicited by the other leukotrienes, is mediated via release of cyclo-oxygenase products.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contraction elicited by leukotriene (LT) C₄ and D₄ on isolated guinea pig trachea were characterized under conditions in which LTC₄ to LTD ₄ metabolism was blocked by presence of 45 mM ?-serine-borate complex (SB). The presence of Sb caused a shift of the LTC₄-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC₄ to LTD₄ by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC₄-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD₄-induced contractions. In contrast, another LT antagonist, Sk&F 101132, equally antagonized the contractions elicited by LTC₄ and LTD₄ in either the presence or absence of SB. The differential antagonism of LTC₄ and LTD₄ implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 μM, suppressed the maximal LTC₄-induced contraction by no more than 20%. whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC₄ concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD₄. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

Differential activity of leukotrienes upon human pulmonary vein and artery

Responses to leukotrienes B₄, C₄, D₄ and E₄ were examined in human pulmonary artery and pulmonary vein preparations from surgical specimens. Leukotrienes C₄ (LTC) and D₄ (LTD) were potent contractants of pulmonary vein over the dose range of 10⁻¹⁰M to 10⁻⁶M, whereas they produced minimal contractions of human pulmonary artery only at concentrations of 10⁻⁸M or greater. Leukotriene E₄ was less potent than LTC or LTD, and leukotriene B₄ (LTB) at concentrations up to 10⁻⁶M had no effect upon either pulmonary veins or pulmonary arteries. Contractions of pulmonary vein by LTD were inhibited in a competitive manner by FPL 55712. Dose response characteristics of LTD and inhibition by FPL 55712 were similar for pulmonary venous and bronchial smooth muscle. We conclude that pulmonary vein smooth muscle has leukotriene receptors comparable to those of bronchial smooth muscle whereas pulmonary artery does not.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B₄ were compared, on a molar basis, with those LTC₄, LTD₄, LTE₄, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD₂, PGE₁, PGF_2α, PGI₂, 6-oxo-PGF_1α, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series.LTB₄ similar to LTC₄ and LTD₄ on GPP, in relation to potency and contractions induced, but differed from LTE₄ in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB₄ produced contractions which were resistant to FPL 55712 (1.9μM) and, when given repeatedly, caused tachyphylaxis in GPP,LTB₄ was considerable more active on GPP than the other substances investigated. Further, PGD₂, PGF_2α and PGI₂ contracted GPP, the order of potency being PGD₂ > PGF_2α ? PGI₂ whereas PGE₁ and PGE₂ relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD₄ displaying the highest activity, LTB₄ was inactive on this tissue. 5-HETE and 6-oxo-PGF_1α were inactive on both GPP and GPISM.On the basis of differential effects of LTB₄ on GPP and GPISM, this assay repressents a simple and selective means to distinguish LTB₄-like materials from other naturally-occuring substances likely to be generated in inflammatory fluids.     

Pharmacology of peptide leukotrienes on ferret isolated airway smooth muscle

The contractile activities of peptide leukotrienes (LT) on isolated spiral strips of ferret trachea were chracterized pharmacologically. LTC₄ and LTD₄ contracted ferret tracheal strips in a concentration-related manner and were 3- to 8-fold more potent than carbachol. In contrast, high concentrations of LTE₄ evoked either weak contraction or none at all, whereas LTC₄ and D₄ were partial agonists compared to carbachol. In tissues which were unresponsive to LTE₄, this compound antagonized contractile responses to LTC₄ and D₄ in an apparently competitive manner: Carbachol-induced contractions were not altered by LTE₄. The cyclooxygenase inhibitor, indomethacin (5 μM), LT antagonists, FPL55712 (10 μM), atropine (1 μM), phenoxybenzamine (10 μM), and LTB₄ (10 μM) failed to alter LTC₄ and D₄ concentration-response curves. The results in dicate that ferret trachea is sensitive to the contractile activity of LTC₄ and LTD₄ but not LTE₄. The LT-induced contractions appear to be mediated by a direct action of the LT rather than indirectly through release of secondary mediators such as thromboxane, prostaglandin, or acetylcholine. LT receptors in ferret trachea are insensitive to FPL55712 but are antagonized by LTE₄.     

Differing mechanisms for leukotriene D4-induced bronchoconstriction in guinea pigs following intravenous and aerosol administration

Leukotriene D₄ (LTD₄) when administered intravenously or by aerosol to guinea pigs produced changes in pulmonary mechanics including a decrease in dynamic compliance and an increase in pulmonary resistance. The effects of intravenous LTD₄ (0.5 μg kg⁻¹) were short lived and abolished by pretreatment of the animal with either cyclooxygenase inhibitors, a thromboxane synthetase inhibitor (OKY 1555) or an SRS-A antagonist (FPL 55712). These findings suggest that bronchoconstriction produced by the intravenous infusion of LTD₄ at 0.5 μg kg⁻¹ is due to the release of thromboxane A₂. However, in animals treated with indomethacin, LTD₄ at higher doses (>0.8 μg kg⁻¹) still elicited a bronchoconstriction which could be blocked by FPL 557112. Nebulization of 0.1 – 1.0 μg of LTD₄ into the lung produced prolonged changes in pulmonary mechanics which were inhibited by FPL 55712 and were potentiated indomethacin. LTD₄, therefore, when administered by aerosol produced effects on the lung which were not mediated by cyclooxygenase products. Responses to nebulized rather than intravenous LTD₄ in the guinea pig may more closely resemble those seen in human tissues.     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substances (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C₄ (LTC₄) and D₄ (LTD₄) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD₄ were indistinguishable. LTC₄ and LTD₄ had similar actions although LTD₄ was more potent than LTC₄. Indo-methacin (1 μg/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC₄ and LTD₄. The residual contraction on the GPP was abolished by FPL 55712 (0.5 – 1.0 μg/ml). It appears, therefore, that a major part of the constrictor actions of LTC₄ and LTD₄ in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A₂ (TxA₂) and prostaglandins (PGs).In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 μg/ml failed to antagonise leukotriene-induced contractions.     

Heterogeneity of leukotriene receptors in guinea-pig trachea

The selective leukotriene (LT) antagonist FPL 55712 antagonized the contractile activity of synthetic LTD4 and E4 on guinea-pig trachea. Schild analysis of the antagonism provided evidence for two distinct receptors for LTD4: one with significantly higher affinity for FPL 55712 than the other. LTE4 appears to interact preferentially with the high affinity receptor.     

Pharmacological comparison of L-serine borate and glutathione as inhibitors of metabolism of LTC4 to LTD4 by the isolated guinea pig trachea

Cumulative dose-response curyes to leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD)₄ were obtained on indomethacin (5 μM) treated isolated guinea pig tracheal spiral strips. LTC₄ curves, in the presence of either glutathione (GSH; 10 mM) or L-serine borate (SB; 45 mM), were not antagonized by FPL-55712 (3 μM), a selective LTD₄ receptor antagonist. LTC₄ curves on trachea treated with a lower concentration of GSH (1 mM), and LTD₄ curves were competitively antagonized by FPL-55712. LTC, curves on GSH (10 mM) treated trachea were 2 fold to the left of those on SB treated tissues. This effect of GSH was blocked by pretreatment with nordihydro-guiaretic acid (30 μM), an inhibitor of 5-lipoxygenase.GSH (10 μM) and SB (45 mM) are effective inhibitors of conversion of LTC₄ into functionally important levels of LTD₄ by the guinea pig trachea. In addition, GSH appeares to enhance LTC₄ responsiveness by increasing synthesis of a contractile 5-lipoxygenase product(s), possibly LTC₄. From the data it is suggested that for inhibition of LTC₄ metabolism, SB may be more usefull when examining responses to exogenously applied LTC₄, while GSH (10 mM) may be useful when examining responses to endogenously generated LTC₄.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A₄, B₄, C₄, D₄ and E₄ have potent myotropic activity on guinea-pig lung parenchymal strip . The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB₄, the tissues became desensitized to LTB₄ but were still responsive to histamine, LTA₄, LTC₄, LTD₄ and LTE₄. When LTD₄ was infused continuously, the lung strips contracted to LTB₄ and histamine but were no longer responsive to LTA₄, LTC₄, LTD₄ and LTE₄. Furthermore, FPL-55712 (10 ng ml⁻¹− 10 ug ml⁻¹) produced dose-dependent inhibitions of LTA₄, LTC₄, LTD₄ and LTE₄ without inhibiting the contraction to LTB₄ and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB₄ and another for LTD₄; LTA₄, LTC₄ and LTE₄ probably act on the LTD₄ receptor.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C4 and D4 in isolated airway smooth muscle was investigated. In rat trachea, neither LTC4 or D4 elicited a response. In contrast, LTC4 was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD4 in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC4 was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD4 in both trachea and parenchyma. As regards LTC4-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC4 in parenchyma.     

Detection of leukotriene C4 and D4 in the exudate of rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used as an acute exudative inflammatory model. The crude ethanol extract of the pleural fluid at 5 hr after carrageenin injection caused the very slow contraction of guinea-pig ileum, which was antagonized by FPL 55712 (1 μg/ml). The ethanol extract was cleaned by LH-20 and was rendered for separation of LTC₄ and LTD₄ by reversed-phase high-performance liquid chromatography (HPLC). Two peaks which showed the same retention time on HPLC as those of LTC₄ and LTD₄ had the contractile activity of guinea-pig ileum and the ratios of the contractile activity to the height on HPLC agreed with those of synthetic LTC₄ and LTD₄. Two peaks of Δ⁶-trans-LTB₄, 5S,12R-(E,E,E,Z)-diHETE and 5S, 12S-(E,E,E,Z)-diHETE, were detected, but the appreciable amount of LTB₄ was smaller than that of each Δ⁶-trans-LTB₄ in the pleural fluid at 5 hr.     

Comparative effects of platelet activating factor, leukotriene D4 and histamine on guinea pig trachea, bronchus and lung parenchyma

The myotropic effect of platelet activating factor (PAF), leukotriene D₄ (LTD₄) and histamine were compared on guinea pig pulmonary tissues. The initial administration of PAF induced a contraction of strips of trachea, bronchus and lung parenchyma. However subsequent injections were characterized by relaxation of trachea and bronchus and a highly reduced (if any) contraction of the parenchyma. The three tissues of the guinea pig respiratory system contracted strongly to leukotriene D₄ and histamine. Indomethacin blocked PAF-induced relaxation of the trachea and bronchus and reduced the contraction of the lung parenchyma. The injection of PAF in the pulmonary circulation stimulated the release of substance(s) causing the contraction of the trachea, bronchus and parenchyma. This study suggests that PAF is not a direct agonist of bronchoconstriction.     

The activity of synthetic leukotriene C-1 on guinea pig trachea and ileum

The effects of synthetic leukotriene C-1 (LTC-1) on isolated guinea pig trachea and ileum have been determined and compared to histamine. LTC-1 produced a slow contraction of the trachea and the ileum with pD2 values of 8.7 +/- 0.1 (n = 14) and 8.5 +/- 0.1 (n = 13), respectively. In comparison, the pD2 values for histamine were 5.6 +/- 0.1 (n = 6) and 6.2 +/- 0.1 (n = 6), indicating LTC-1 was 2-3 orders of magnitude more potent. LTC-1 was antagonised by FPL 55712 with pA2 values of 6.9 +/- 0.1 (n = 5) and 6.4 +/- 0.1 (n = 7) on the trachea and ileum, respectively. Incubation with lipoxidase produced a time and enzyme dependent loss of biological activity and a concurrent shift in U.V. absorption spectrum.