Bronchoconstrictor effects of leukotriene B4 in the guinea pig

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous $\text{in vitro}$ studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

The lung parenchymal strip as a sensitive assay for leukotriene B4

Leukotriene B₄ (LTB₄) (3 × 10⁻¹¹ to 1.5 × 10⁻⁹ mole; 10 ng to 500 ng) contracted the guinea-pig lung parenchymal strips in a dose-dependent manner. The contractile effect of LTB₄ was not affected by methysergide (0.2 μg/ml), propranolol (3.0 μg/ml), phenoxybenzamine (0.1 μg/ml), atropine (0.1 μig/ml), diphenhydramine (0.1 μg/ml) and FPL-55712 (1.0 μg/ml), but was nearly completely abolished by indomethacin (20 μg/ml). It is concluded that the contraction of the parenchymal strip to LTB₄ may constitute a simple, sensitive and selective bioassay which could be either used for the determination of LTB₄ in biological material or for studies on structure-activity relationship.     

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.     

Structural requirement for the action of leukotriene B4 on the guinea-pig lung:importance of double bond geometry in the 6, 8, 10-triene unit

The action of four synthetic 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acids has been compared to the action of natural leukotriene B₄ (LTB₄) in perfused guinea-pig lung and in the parenchymal strip preparations. Synthetic LTB₄ (Fig. 1) having the 6-$\text{cis}$, 8, 10-$\text{trans}$ triene unit was found to be as powerful as natural LTB₄ both for contracting the parenchymal strip and for releasing prostaglandins and thromboxanes from the perfused lung while three other isomers were inactive. The results indicate that the action of LTB₄ on the lung is highly dependent on the geometry of the conjugated triene.     

Spasmogenic activity of C5adesArg anaphylatoxin on guinea pig lung parenchymal strips: Sensitivity of the leukotriene-mediated component to cyclooxygenase inhibitors

The leukotriene-dependent component of C5a_desArg-induced contractile activity on guinea pig lung parenchymal strips is inhibited by cyclooxygenase inhibitors. Indomethacin simultaneously increased leukotriene release while inhibiting both cyclooxygenase-dependent mediator release and the contractile force generated. Tissue responses to LTC₄ and LTD₄ are also inhibited by cyclooxygenase blockade, while contractions induced by the thromboxane A₂ analog, U-46619, histamine or acetylcholine are not affected. These data indicate a functional role for cyclooxygenase metabolites in leukotriene-induced contractile responses in lung.     

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.     

Leukotriene F4 and the release of arachidonic acid metabolites from perfused guinea pig lungs in vitro

Radioimmunoassay and bioassay techniques have been used to investigate the ability of leukortriene (LT)F₄ to release products of arachidonic acid metabolism from guinea pig isolated lungs perfused via the pulmonary artery. Also, the abilities of LTC₄, LTD₄ LTE₄ and LTE₄ to contract guinea pig ileal smooth muscle (GPISM) was studied. Each of the LT's contracted GPISM. The rank order of potency was LTD₄ > LTC₄ > LTE₄ > > LTF₄ in a ratio 1:7:170:280 respectively. Bioassay of pulmonary effluents indicated the passage of LTF₄ through the lungs caused a contraction of rabbit aorta as well as an FPL-55712 sensitive contraction of GPISM. The contractions of rabbit aorta were inhibited by pretreatment of the lungs with Indomethacin but not with the thromboxane synthetase inhibitor Dazoxiben. Radioimmunoassay of the lung effluents indicated LTF₄ to cause a 70-fold increase in thromboxane B₂ (TXB₂), 4-fold increase in prostaglandin (PG)E₂ and a 16-fold increase in 6-keto PGF_1α levels. The LTF₄-induced increments of these immunoreactive metabolites was inhibited by pretreatment of the lungs with Indomethacin. Pretreatment of lungs with Dazoxiben inhibited the LTF₄-induced increment in TXB₂ and enhanced the effluet levels of PGE₂ 24-fold (compared with untreated lungs). There were no detectable differences in either immunoreactive LTC₄ or immunoreactive LTB₄ levels. It is concluded LTF₄ is a relatively weak agonist on GPISM and can induce the release of cyclooxygenase products of arachidonic acid metabolism from guinea pig perfused lung.     

A formyl peptide contracts guinea pig lung: role of arachidonic acid metabolites

We studied the role of cyclooxygenase and lipoxygenase products of arachidonic acid metabolism in mediating N-formyl-methionyl-leucyl-phenylalanine- (FMLP) induced contractions of guinea pig lung parenchymal strips. The cyclooxygenase inhibitors indomethacin (10(-5) M) and aspirin (3 X 10(-5) to 10(-4) M), the lipoxygenase inhibitor nordihydroguaiaretic acid (10(-5) to 3 X 10(-5) M), and the combined cyclooxygenase/lipoxygenase inhibitors 1-phenyl-3-pyrazolidinone (Phenidone) (3 X 10(-5) to 3 X 10(-4) M) and BW 755C (10(-5) to 10(-4) M) each caused a decrease in the maximum force induced by FMLP (Fmax) and an increase in the concentration of FMLP required to produce 50% of Fmax (EC50). The thromboxane synthesis inhibitor imidazole (3 X 10(-3) M) also decreased Fmax. The leukotriene D4 receptor antagonist FPL 55712 (5.7 X 10(-6) to 1.9 X 10(-5) M) increased the EC50 for FMLP, whereas desensitization of lung parenchymal strips to leukotriene B4 by pretreatment with this leukotriene (10(-7) M) had no effect on FMLP-induced contraction. After exposure to FMLP (10(-6) M), guinea pig lung produced (as determined by high-performance liquid chromatography and radioimmunoassay) leukotrienes C4 and B4, thromboxane A2 (as measured by its stable degradation product thromboxane B2), and prostaglandin F2 alpha. Lung strips not exposed to FMLP showed no evidence of leukotriene production. We conclude that thromboxane A2 and leukotriene C4 generated in response to FMLP mediate a substantial fraction of the force induced by this peptide in guinea pig lung parenchymal strips.     

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.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: Myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, Δ⁶-trans-leukotriene B₄, 12-epi-Δ⁶-leukotriene B₄, ω-hydroxy-leukotriene B₄, ω-carboxy-leukotriene B₄, 15S-hydroxy-eicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to TLB₄ on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The ω-hydroxy-LTB₄ appeared more potent than LTB₄ both for inducing a contraction and for releasing prostanoids whereas the ω-carboxy-LTB₄ was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by PFL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB₄ (and other hydroxy acids) actions on the guinea-pig lungs.     

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 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 substance (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. Indomethacin (1 microgram/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 of the GPP was abolished by FPL 55712 (0.5 - 1.0 microgram/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 microgram/ml failed to antagonise leukotriene-induced contractions.     

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.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B4 (LTB4) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB4 showed a pronounced neutrophil infiltration. These results confirm previous in vitro studies in which LTB4 was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A2.     

Leukotriene B4 Enhances NOD2-Dependent Innate Response against Influenza Virus Infection

Leukotriene B₄ (LTB₄), a central mediator of inflammation, is well known for its chemoattractant properties on effectors cells of the immune system. LTB₄ also has the ability to control microbial infection by improving host innate defenses through the release of antimicrobial peptides and modulation of intracellular Toll-like receptors (TLRs) expression in response to agonist challenge. In this report, we provide evidences that LTB₄ acts on nucleotide-binging oligomerization domain 2 (NOD2) pathway to enhance immune response against influenza A infection. Infected mice receiving LTB₄ show improved survival, lung architecture and reduced lung viral loads as compared to placebo-treated animals. NOD2 and its downstream adaptor protein IPS–1 have been found to be essential for LTB₄-mediated effects against IAV infection, as absence of NOD2 or IPS–1 diminished its capacity to control viral infection. Treatment of IAV-infected mice with LTB₄ induces an increased activation of IPS-1-IRF3 axis leading to an enhanced production of IFNβ in lungs of infected mice. LTB₄ also has the ability to act on the RICK-NF-κB axis since administration of LTB₄ to mice challenged with MDP markedly increases the secretion of IL–6 and TNFα in lungs of mice. TAK1 appears to be essential to the action of LTB₄ on NOD2 pathway since pretreatment of MEFs with TAK1 inhibitor prior stimulation with IAV or MDP strongly abrogated the potentiating effects of LTB₄ on both IFNβ and cytokine secretion. Together, our results demonstrate that LTB₄, through its ability to activate TAK1, potentiates both IPS–1 and RICK axis of the NOD2 pathway to improve host innate responses.     

Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment

Eicosanoids are bioactive lipid mediators derived from arachidonic acid¹ (AA), which is released by cytosolic phospholipase A₂ (cPLA₂). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA₂ knockout vs wild-type mice, we demonstrated that prostaglandins (PGE₂, PGD₂ and PGF_2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB₄, LTC₄, LTD₄, LTE₄) production required cPLA₂ expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.     

Thromboxanes: Synthase and receptors

Thromboxane A₂ is a biologically potent arachidonate metabolite through the cyclooxygenase pathway. It induces platelet aggregation and smooth muscle contraction and may promote mitogenesis and apoptosis of other cells. Its roles in physiological and pathological conditions have been widely documented. The enzyme that catalyzes its synthesis, thromboxane A₂ synthase, and the receptors that mediate its actions, thromboxane A₂ receptors, are the two key components critical for the functioning of this potent autacoid. Recent molecular biological studies have revealed the structure-function relationship and gene organizations of these proteins as well as genetic and epigenetic factors modulating their gene expression. Future investigation should shed light on detailed molecular signaling events specifying thromboxane A₂ actions, and the genetic underpinning of the enzyme and the receptors in health and disease.     

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.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B₄ (LTB₄), leukotriene D₄ (LTD₄) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15uM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB₄, LTD₄ and histamine. An antagonist of calmodulin, triflouperazine (1–200 uM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC₅₀ of this compound for inhibition of histamine was much lower (2–3uM) than the IC₅₀ for inhibition of leukotrienes (75 uM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compunds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.