Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa

The effects of leukotrienes C4 (LTC4) and D4 (LTD4) on the secretion by human bronchial mucosa of [14C]glucosamine-labeled, trichloro-acetic acid/phosphotungstic acid-precipitable glycoprotein and lysozyme were evaluated in vitro. LTC4 and LTD4, in the concentration range of 0.16 to 1600 nM, induced a dose-related increase in the release of radiolabeled glycoprotein, but not of lysozyme. This secretagogue effect was selective for high molecular weight glycoproteins of about 2-5 x 10(6) daltons, and the median effective concentrations (EC50) of LTC4 of 9.4 x 10(-9) M and of LTD4 of 2.44 x 10(-8) M, indicate that these leukotrienes are approximately 100-fold more potent than the cholinergic agonist methacholine. Incubation of [14C]glucosamine-labeled bronchial mucosal explants with LTC4 or LTD4 for six sequential 15-min periods revealed a rapid, progressive decrement in glycoprotein release, compatible with stimulatory action on secretion rather than augmentation of the rate of glycoprotein synthesis. This interpretation is also consistent with the finding that the specific activity (ratio of bound radiolabel: protein content) of the macromolecular glycoprotein secreted by the explants is not changed with stimulation of release by the leukotrienes. Based upon the activity of synthetic leukotriene analogs, the specific C-6 chirality of the sulfidopeptide of LTD4, the presence of a hydroxyl at C-5 and the presence of eicosanoid carbons 9-20 were of no importance for secretagogue activity. These findings contrast with the stereochemical requirements for the spasmogenic response to sulfidopeptide leukotrienes and suggest that leukotriene-induced secretion is not likely to be mediated via a specific receptor.     

Prostaglandin F2 stimulates the generation of lipoxygenase products in guinea pig isolated trachea

The generation of lipoxygenase products on the contraction elicited by prostaglandin (PG) F2 alpha was investigated in the guinea-pig isolated trachea. Indomethacin (5 x 10(-6) M) inhibited the response at low concentrations of PGF2 alpha while enhanced the response at higher concentrations of PGF2 alpha. Phenidone (10(-4) M) and nordihydroguaiaretic acid (NDGA, 3 x 10(-5) M) appeared to inhibit the PGF2 alpha response. The PGF2 alpha response augmented by indomethacin was dose-dependently inhibited by NDGA and a leukotriene (LT) antagonist, FPL55712. NDGA had no effect on the contraction elicited by histamine but markedly inhibited the contraction elicited by LTD4. The inhibition by NDGA of the LTD4-induced contraction was abolished in the presence of indomethacin (5 x 10(-6) M). FPL55712 inhibited the LTD4-induced contraction but the extent of the antagonism was not changed by indomethacin. In the presence of indomethacin PGF2 alpha (10(-8) M) did not affect the LTD4 (3 x 10(-9) M) response but significantly enhanced the arachidonic acid (AA, 6.6 x 10(-5) M)-induced contraction. FPL55712 (3 x 10(-6) M) completely inhibited the AA response augmented by PGF2 alpha. These results suggest that lipoxygenase-mediated LT-like substances are released in the response at higher concentrations of PGF2 alpha on the guinea-pig isolated trachea, and the mode of action of PGF2 alpha is different from those of histamine and LTD4.     

Effects of a newly synthesized leukotriene antagonist, NZ-107, on immediate-type hypersensitivity reaction in rats and guinea-pigs.

The effects of 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone (NZ-107) on immediate type hypersensitivity reactions in rats and guinea-pigs were studied. 1. When NZ-107, at a dose of 50 mg/kg (i.p.) or 100 mg/kg (orally), was administered to rats, 48-h homologous passive cutaneous anaphylaxis (PCA) reaction and histamine-, leukotriene C4 (LTC4)- and leukotriene D4 (LTD4)-induced skin reactions were suppressed by the agent. 2. NZ-107 (10(-6) g/ml) inhibited both LTC4- and LTD4-induced contractions of isolated rat stomach smooth muscle. 3. NZ-107 inhibited antigen-induced histamine release from rat peritoneal mast cells by 26% at a concentration of 10(-4) g/ml. 4. NZ-107, at doses of 25 and 50 mg/kg (orally), significantly inhibited guinea-pig 3-h heterologous PCA reaction. 5. NZ-107 inhibited antigen-induced histamine release from guinea-pig lung tissue by 17% and 48% at concentrations of 5 x 10(-5) and 10(-4) g/ml, respectively. 6. NZ-107, at doses of 25 and 50 mg/kg (i.p.), inhibited antigen-induced bronchoconstriction and eosinophil accumulation in the bronchoalveolar lavage fluid (BALF) of guinea-pigs. These results suggest that NZ-107 has anti-allergic action including inhibition of eosinophil accumulation in an antigen-challenged airway lesion in rats and guinea-pigs. The anti-allergic action of this agent is thought to be due to its action as a histamine and LT antagonist and its consequent inhibition of antigen-induced histamine release.     

Leukotriene and anti-IgE induced contractions of human isolated trachea: studies with leukotriene receptor antagonists and a novel 5-lipoxygenase inhibitor

The pharmacological actions of three leukotriene D4 (LTD4) receptor antagonists, FPL-55712, L-648,051, and L-649,923, and a novel inhibitor of leukotriene biosynthesis, L-651,896, have been investigated on isolated human tracheal smooth muscle. In the order of potency L-648,051 greater than FPL-55712 greater than L-649,923, these agents antagonized contractions to LTD4 and produced parallel rightward shifts in the dose-response curves. Mean -log KB values against LTD4 were 6.9 +/- 0.1, 6.5 +/- 0.3, and 6.0 +/- 0.1 for L-648,051, FPL-55712, and L-649,923, respectively. FPL-55712 also antagonized contractions to LTC4 (-log KB value, 6.4 +/- 0.3) and this activity was not decreased by the gamma-glutamyl transpeptidase inhibitor, L-serine borate. In the presence of 1 x 10(-7) M atropine, 7 x 10(-6) M mepyramine, and 1.4 x 10(-6) M indomethacin, L-648,051 at 2 x 10(-5) and 2 x 10(-6) M produced complete and partial blockade, respectively, of the contraction to goat anti-IgE. L-649,923 and FPL-55712 produced partial but significant inhibition at 2 x 10(-5) M, whereas the 5-lipoxygenase inhibitor, L-651,896, produced almost complete inhibition at 3.5 and 35 x 10(-6) M. L-Serine borate (15 mM) did not alter the the activity of FPL-55712 versus anti-IgE. These findings indicate that LTD4 receptors mediate contraction of human trachea to exogenously applied and endogenously (anti-IgE) released leukotrienes. LTD4 antagonists, such as L-648,051, may be useful in assessing the role of leukotrienes in respiratory disease.     

Guinea-pig liver leukotriene A4 hydrolase. Purification, characterization and structural properties

Leukotriene A4 hydrolase from perfused guinea-pig liver was purified 1200-fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37 degrees C (27 microM and 68 mumol x mg-1 x min-1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at -10 degrees C, 0 degree C and +10 degrees C (Arrhenius plots). Physical properties including Mr (67,000-71,000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N-terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S,6R)-5,6-dihydroxy-7,9-trans-11,14-cis-icosatetraenoic acid.     

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 in vitro. 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.     

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

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 degrees 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 (Bmax), 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.     

Pharmacological characterization using selected antagonists of the leukotriene receptors mediating contraction of guinea-pig trachea

The effects of six leukotriene (LT) antagonists on LTC4-, D4- and E4-induced contraction of isolated guinea-pig tracheal spirals were examined. Concentration-response effects of the leukotrienes were determined by cumulative addition in the presence of indomethacin (5 microM) alone for LTE4, or with 10 mM of either glutathione or L-cysteine to inhibit metabolism of LTC4 or LTD4, respectively. Concentration-response curves to the LTs were obtained in the absence and presence of Wy-45,911, Wy-44,329, FPL-55,712, Ly-171,883, Wy-48,252 and ICI-198,615 representing three structurally different chemical groups of LT antagonists. At 30 microM, the antagonists produced little or no antagonism of LTC4-induced contractions. Analysis of the Schild plots for antagonism of LTD4 and E4 suggested two receptors for the agonist effects of LTD4 and a single receptor for the agonist effects of LTE4. Comparison of pA2 values for Wy-45,911, FPL-55,712, LY-171,883 and Wy-48,252 provided evidence that LTE4 is acting at the antagonist high affinity LTD4 receptor to produce contractile effects. From the data, we conclude that there are three LT receptors (one for LTC4 and two LTD4 subtypes) through which exogenously applied LTs evoke contraction of the isolated guinea-pig trachea.     

Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine

The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion.     

Enhancement of leukotriene D4-induced contraction of guinea-pig isolated trachea by platelet activating factor

The effect of platelet activating factor (PAF), a potent lipid mediator of inflammation, was examined in the induction of airway hyperreactivity to known mediators of anaphylaxis. Concentration-dependent contractions of the isolated guinea-pig trachea to PAF (10(-7)-10(-5) M) were produced and an EC50 value was found to be 7.5 X 10(-7) M. Pretreatment for 30 min with a known PAF inhibitor, CV-3988 (10(-5) or 10(-4) M), produced significant inhibition of PAF contractions; however, at 10(-6) M, CV-3988 had no effect. In the presence of meclofenamic acid (10(-6) M), the concentration-response curve to PAF was shifted significantly upward and to the left. This potentiation could be reversed by pretreating the tissues with the peptidoleukotriene antagonists, FPL 55712 or SK&F 102922 (10(-5) M). Pretreatment with PAF concentrations having essentially no intrinsic activity (10(-8), 10(-7)) significantly enhanced the contraction of guinea-pig trachea to various concentrations of LTD4 and to certain concentrations of a thromboxane mimic (U-46619). Pretreatment with lyso-PAF failed to potentiate the LTD4 response, while pretreatment with CV-3988 reverse the potentiation by PAF of the lower concentrations of LTD4. However, PAF failed to enhance contractions (with or without the presence of meclofenamic acid) to acetylcholine, histamine, PGD2 or LTC4 (in the presence of serine borate). These results indicate a possible role for PAF as a mediator of airway hyperreactivity.     

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methane sulfonamide) an orally active leukotriene D4 antagonist: pharmacological characterization in vitro and in vivo in the guinea pig

The following communicates the pharmacology of Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide) a chemically novel and orally potent leukotriene (LT) D4 receptor antagonist. In the isolated guinea-pig trachea pretreated with indomethacin (5 microM) and L-cysteine (10 mM), Wy-48,252 antagonized TD4-induced contraction with a pKB = 7.6. Against LTC4 on tissues pretreated with IND and glutathione (10 mM), Wy-48,252 had a pKB greater than 5. Wy-48,252 (10 microM) did not antagonize pilocarpine-, histamine- or PGF2 alpha-induced tracheal contraction. Further, in the presence of indomethacin and chlorpheniramine (1 microM), Wy-48,252 dose-dependently inhibited the antigen-induced contraction of guinea-pig trachea in a manner consistent with antagonism at the LTD4 receptor and inhibition of LT synthesis. In the Konzett-Rossler model of i.v. LTD4-induced bronchoconstriction in indomethacin treated guinea pigs, intragastric Wy-48,252 (2 hr) had an ID50 of 100 micrograms/kg and a functional half-life of 5 hr. Against i.v. antigen-induced bronchoconstriction in guinea pigs treated with indomethacin and chlorpheniramine, intragastric Wy-48,252 (2 hr) had an ID50 of 0.6 mg/kg and a 5 hr half life. Intragastric Wy-48,252 also selectively blocked the cutaneous wheal reaction to intradermal LTD4 but not histamine. We conclude that Wy-48,252 is distinguished from other selective LTD4 receptor antagonists by its oral potency and should be useful in ascertaining the role of LTD4 mediated processes in asthma, allergy and animal models.     

Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.     

Novel phthalimide derivatives,designed as leukotriene D(4) receptor antagonists

A series of phthalimide acid derivatives was synthesized and evaluated as leukotriene D(4) receptor antagonists. The tetrazolephthalimide LASSBio 552 (7) was shown to be able to inhibit the contractile activity induced by 100 nM of LTD(4) in guinea-pig tracheal strips with an IC(50) = 31.2 microM. In addition, LASSBio 552 (7) has been showed to present a better efficacy than zafirlukast (1) used as standard.     

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(-10) M to 10(-6) M, whereas they produced minimal contractions of human pulmonary artery only at concentrations of 10(-8) M or greater. Leukotriene E4 was less potent than LTC or LTD, and leukotriene B4 (LTB) at concentrations up to 10(-6) M 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.     

Mechanisms of leukotriene E4 partial agonist activity at leukotriene D4 receptors in differentiated U-937 cells

Leukotriene E4 (LTE4) is shown to be a partial agonist of leukotriene D4 (LTD4) in differentiated U-937 cells. The data that support this conclusion are: 1) LTE4 completely displaced [3H]LTD4 from its receptors in U-937 cell membranes. 2) LTE4 induced only 30 +/- 4% of the maximal Ca2+ transient induced by LTD4 in the presence of 1 mM extracellular Ca2+ and 60 +/- 4% of the maximal LTD4 response in the absence of extracellular Ca2+. 3) LTE4 induced only a fraction of the inositol phosphates metabolized by LTD4. Moreover, LTE4 resulted in essentially no production of the inositol 1,4,5-trisphosphate isomer, while LTD4 induced a rapid and substantial transient increase in this isomer. The generation of inositol phosphates by both agonists was unaffected by extracellular Ca2+. 4) The EC50 values for Ca2+ mobilization for LTD4 and LTE4 corresponded with their affinity (Kd values) for the LTD4 receptor. 5) A series of structurally diverse LTD4 receptor antagonists blocked the Ca2+ mobilization responses to LTD4 and LTE4 with identical rank orders of potency. 6) LTE4 acted as an antagonist of LTD4 of potency. 6) LTE4 acted as an antagonist of LTD4 effects when they were coadministered. 7) LTE4 and LTD4 acutely desensitized Ca2+ mobilization to each other. All of the effects of LTE4 are explained by its partial agonist activity at the LTD4 receptor as shown by the following data. 1) Neither LTD4 nor LTE4 had any effect on the agonist activity of fMet-Leu-Phe, LTB4, or platelet-activating factor. 2) None of the above agonists or antagonists to the above receptors affected any of the activities of LTD4 or LTE4. 3) Neither LTD4 nor LTE4 induced desensitization of Ca2+ mobilization to any of the non-LTD4 receptor agonists tested. 4) Under the conditions studied, we have not observed any evidence of multiple subclasses of LTD4 receptors in U-937 cells. LTE4 is a partial agonist of the LTD4 receptor, because it can only couple the LTD4 receptor to a portion of the signaling system available to the receptor when occupied by LTD4. Specifically, LTD4 caused the activation of receptor-operated calcium channels, mobilization of intracellular Ca2+, the activation of phosphatidylinositol-phospholipase C, and the liberation of an additional, as yet undefined, intracellular mediator. To do this, LTD4 receptors couple to at least two and perhaps more guanine nucleotide binding proteins. LTE4 is unable to activate the phosphatidylinositol-phospholipase C but can mimic the other effects of LTD4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and LTD4-antagonist activity of desamino-2-nor-leukotriene analogs

A series of desamino-2-nor-leukotriene analogs has been prepared by the reaction of various thiols with several methyl trans-4,5-epoxy-6Z-alkenoates, followed by deprotection. The products were assessed for their ability to antagonize the LTD4-induced contraction of the isolated guinea pig trachea. Several compounds displayed potent leukotriene antagonist activity, i.e., KB values in the sub-micromolar range, while only minimally affecting basal airway tone. The most potent analog, 4-hydroxy-5-(2-carboxyethylthio)-6Z-nonadecenoic acid, antagonized both LTD4- and LTE4-induced contractions of the trachea in an apparently competitive fashion. These agents possess increased potency relative to SK&F 101132, the first leukotriene analog identified as having LT-antagonist activity. Thus, these results demonstrate that deletion of the peptide amino group can produce leukotriene analogs which have minimal intrinsic contractile activity on the isolated guinea pig trachea, yet possess potent leukotriene-antagonistic effects.     

Leukotriene biosynthesis and metabolism detected by the combined use of HPLC and radioimmunoassay

A radioimmunoassay for leukotriene D4 (LTD4) has been developed which exhibits sufficiently high sensitivity to be useful in conjunction with RP-HPLC in the detection of LTC4, LTD4 and LTE4 in physiological samples. The detection limit of the assay was approximately 240 amoles, using antiserum TG1 at a dilution of 6 X 10(3), with 50% displacement at 70 fmoles. Antiserum NW1, also at a dilution of 6 X 10(3), displayed a detection limit of 9 fmoles with 50% displacement at 100 fmoles. The two antisera have similiar crossreactivities, both manifesting useful affinities for LTE4 and LTC4, and low or negligible affinities for other arachidonic acid metabolites, or their derivatives. The radioimmunoassay was used to detect 1) LTC4, LTD4 and LTE4 released from perfused rat lung in response to platelet-activating factor (PAF) stimulation, 2) conversion of exogenous LTD4 to LTE4 in human blood, and 3) endogenous leukotrienes in human blood samples.     

Pharmacology of the leukotriene antagonist verlukast: the (R)-enantiomer of MK-571.

Verlukast (MK-679) (3-[(3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl)[3-(dimethylamino)- 3- oxopropyl)thio)methyl)-thio)propionic acid) is a potent and selective inhibitor of [3H]leukotriene D4 binding in guinea-pig (IC50 = 3.1 +/- 0.5 nM) and human (IC50 = 8.0 +/- 3.0 nM) lung homogenates and dimethyl sulfoxide differentiated U937 cell membrane preparations (IC50 = 10.7 +/- 1.6 nM) but is essentially inactive versus [3H]leukotriene C4 binding in guinea-pig lung homogenates (IC50 values of 19 and 33 microM). Functionally, when tested at 60 nM, it antagonized contractions of guinea-pig trachea (GPT) induced by leukotriene C4, leukotriene D4, and leukotriene E4 (respective-log KB values of 8.6, 8.8, and 8.9) and contractions of human trachea (HT) induced by leukotriene D4 (-log KB value 8.3 +/- 0.2). In contrast, verlukast (20-200 nM) failed to antagonize contractions of GPT induced by leukotriene C4 in the presence of 45 mM L-serine borate. Intravenous (i.v.) and aerosol verlukast antagonized bronchoconstriction (BC) induced in anaesthetized guinea pigs by i.v. leukotriene D4 but did not block BC to arachidonic acid or histamine. Intraduodenal verlukast (0.25 mg/kg) antagonized leukotriene D4 (0.2 micrograms/kg) induced BC in guinea pigs. Oral and aerosol administration blocked leukotriene D4-induced BC in conscious squirrel monkeys. Orally administered compound also blocked ovalbumin-induced BC in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile for verlukast is similar to that of the racemic compound, MK-571. Verlukast is currently in clinical development for the treatment of asthma and related diseases.     

Positive interaction between leukotriene C4 and histamine and other mediators on vascular tissues

The interaction of leukotriene C4 (LTC4) with the contractile activity of histamine (H), serotonin (5HT) and norepinephrine (NE) has been investigated in isolated vascular preparations. Threshold concentration of LTC4 (5 X 10(-9) M) significantly potentiated the vasoconstricting effect of these compounds on guinea-pig pulmonary artery (GPPA). This phenomenon was long-lasting for H since it was still present 40 min after LTC4 had been washed. FPL-55712 (10(-5) M) counteracted the increased H response on GPPA induced by LTC4. Potentiation of H activity due to LTC4 was also observed on guinea-pig thoracic aorta (GPTA) indicating that LTC4-induced hyperreactivity is not a phenomenon restricted to the pulmonary vascular bed. In the experiments carried out in presence of indomethacin (3 X 10(-6) M), LTC4 still potentiated H-induced vasoconstriction on GPPA, however the time course of the phenomenon was significantly shorter than that observed in absence of the cyclooxygenase inhibitor. The contractile activity of H and NE on guinea-pig portal vein (GPPV) was not potentiated by LTC4. These results demonstrate that LTC4 induces hyperreactivity of the arterial vascular tissue to vasoactive compounds and suggest that cysteinyl-leukotrienes may have pathological significance in the hemodynamic changes occurring during anaphylactic reactions. Preliminary experiments carried out on human intralobar pulmonary artery strongly support this hypothesis.