Thromboxane A2 antagonist inhibits leukotriene D4-induced smooth muscle contraction in guinea-pig lung parenchyma, but not in trachea

Although the bronchoconstriction induced by leukotriene D₄ (LTD₄) has been reported to be partly mediated by thromboxane A₂ (TXA₂) in the guinea-pig airway, it is not known which part of the airway is susceptible to TXA₂. In order to determine the role of TXA₂ in the central and peripheral airways, we compared the effect of a TXA₂ antagonist on tracheal strips to its effect on parenchymal strips of guinea-pigs. Tracheal and parenchymal strips were mounted in a 3.5 ml organ bath filled with Krebs-Henseleit solution aerated with 95% O₂, 5% CO₂ and kept at 37°C. After equilibration for 60 min in Krebs solution, the strip was contracted by exposure to 10⁻⁵ M of acetylcholine (ACh). Sixty minutes after ACh was eliminated, the concentration-response curve to LTD₄ (10⁻⁹ M–10⁻⁷ M) was obtained, and the LTD₄-induced contractions were expressed as the percent of the contraction evoked by 10⁻⁵ M of ACh. We measured the contractile response to LTD₄ in the presence or absence of the TXA₂ antagonist, BAY u3405 (10⁻⁸ M–10⁻⁶ M). In the tracheal strips, BAY u3405 had no effect on the LTD₄-induced contraction. However, in parenchymal strips, BAY u3405 significantly suppressed the contractile response to LTD₄. These results suggest that in the central airway LTD₄ contracts smooth muscle directly, but that in the peripheral airway LTD₄ induces smooth muscle contraction both directly and indirectly, via TXA₂.     

Autoradiographic localization of leukotriene C4 and D4 binding sites in guinea-pig lung

Binding of [3H] leukotriene C4 and D4 to guinea-pig lung sections was charaterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar charateristics to binding in a membrane preparation. Autoradiography revealed that the distribution of LTC4 and D4 binding sites was markedly different. Smooth muscle and epithelium of central and peripheral airways were densely labelled with [3H]LTC4; vascular smooth muscle and alveolar walls were also labelled. With [3H]LTD4, however, there was no detectable labelling of airways or vessels but subtantial labelling of alveolar walls. This lends futher support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.     

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 − 10⁻⁵M) were produced and an EC₅₀ value was found to be 7.5 × 10⁻⁷M. Pretreatment for 30 min with a known PAF inhibitor, CV-3988 (10⁻⁵ or 10⁻⁴M), produced significant inhibition of PAF contractions; however, at 10⁻⁶M, CV-3988 had no effect. In the presence of meclofenamic acid (10⁻⁶M), 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 (10⁻⁵M). 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.     

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD₄. This material however had a different retention time from LTD₄ when subjected to HPLC and co-chromatographed with synthetic LTE₄. In addition to LTE₄ a substance which had properties indistinguisable from those of LTB₄ when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB₄. The adventitia and intima were the richest source of LTE₄, the adventitia releasing slightly more than the intima. The output of LTB₄ and LTE₄ was inhibited by 6,9-deepoxy-6,9-(phenylimino)-Δ^6,8 prostaglandin I₁ (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE₄.     

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.     

-alkoxyphenol leukotriene B4 receptor antagonists

A series of -alkoxyphenols containing a tetrazole acid sidechain have been prepared as antagonists of leukotriene B₄ receptors. These compounds were tested as receptor antagonists of human neutrophil and guinea pig lung membrane leukotriene B₄ receptors. Compounds in this series were found to be up to 18-fold more potent than LY255283. These results indicate that the acyl group of the 1,2,4,5 substituted hydroxyacetophenone class of LTB₄ antagonists is not critical to antagonist potency.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotreine B₄ has been developed. The assay is sensitive; 5 pg LTB₄ caused significant inibition of binding of [³H]-LTB₄ and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B₂ and arachidonic acid do not exhibit detectable cross-reactions (< 0.03%). Flowever, some non-cyclic dihydroxy- and monohydroxy-eicosatetraeonic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-$\text{trans}$-14-$\text{cis}$-elcosatetraenoic and 12-hydroxy-5,8,10,14-elcosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB₄ from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB₄ by reverse-phase high liquid chromatography follwing solvent extraction and silinic acid chromatography.     

Characterization of leukotriene A4 and B4 bioysnthesis

We have studied LTA₄ and LTB₄ synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB₄ from arachidonic acid are localized in the soluble fraction (100, 000 x g supernatant) of these cells. The formation of LTA₄ and LTB₄ is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 μM, the synthesis of LTB₄ leveled off at 30 μM and of LTA₄ at 100 μM while 5-HETE had not reached a plateau at 300 μM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA₄ and only a relatively small amount of LTB₄. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA₄ to LTB₄. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA₄ synthesis did not produce any additional LTA₄ or LTB₄. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

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.     

Synthesis of thromboxane B2 metabolites

This paper reports the synthesis of 11-dehydrothromboxane B₂ methyl ester (II), 15-dehydrothromboxane B₂ methyl ester (III), 15-dehydro-13,14-dihydrothromboxane B₂ (XII) and 2,3-dinorthromboxane B₂ methyl ester (XV). These compounds, as their free acids, have been reported to be thromboxane metabolites.     

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.     

Studies on the conjugation of leukotriene B4 with proteins for development of a radioimmunoassay for leukotriene B4

Leukotriene B₄ (LTB₄) (I) has been converted to its N-(3-aminopropyl)amide derivative (III) and to its hydrazide derivative (VII) via LTB₄ δ-lactone. The amide (III) was coupled with Bovine Serum Albumin using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. The hydrazide (VII), was coupled with Hemocyanin (Keyhole Limpet) (KLH) using 6-N-maleimidohexanoic acid chloride as coupling agent.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

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.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .     

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production

The metabolism of thromboxane B₂ was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5, 6, 7, 8, 9, 11, 12, 14, 15-³H₈]-Thromboxane B₂ was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods.In urine the major metabolites were identified as 11-dehydro-TXB₂ and 2, 3, 4, 5-tetranor-TXB₁, and other prominent products were 11-dehydro-2, 3, 4, 5-tetranor-TXB₁, 2, 3-dinor-TXB₁ and 2, 3-dinor-TXB₂. In the circulation, TXB₂ was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB₂, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB₂ was not converted into 11-dehydro-TXB₂ by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver.The results of the present study suggest that 11-dehydro-TXB₂ maybe a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB₂ and 11-dehydro-TXB₂.     

Isomers of leukotriene B4 possess different biological potencies

Rat elicited polymorphonuclear leucocytes (PMNs), when exposed to the ionophore A23187, release three isomers of leukotriene B₄. The three isomers have been purified and tested for their ability to induce the chemokinesis of human PMNs in vitro, the aggregation of rat PMNs in vitro and changes in vascular permeability in rabbit skin in vivo in the presence of PGE₂. The results demonstrate that all three isomers are biologically active and that the enzymatically produced isomer, in which the conjugated triene contains one and two double bonds, is more potent than the two diastereoisomers of LTB₄ which contain all double bonds in the conjugated triene and which are produced by non-enzymatic hydrolysis.     

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B₄ (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [³H]-leukotriene B₄ resulted in the rapid conversion of leukotriene B₄ to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B₄) and 5(S),12(R),19-trihydroxy-6,8,10,14-eicosatetraenoic acid (19-hydroxy-leukotriene B₄). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B₄) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoic acid (Δ16,17–18-carboxy-19,20-dinor-leukotriene B₄) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B₄ by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B₄ from inflammatory sites.