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 15 microM) 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, trifluoperazine (1-200 microM), 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 (2-3 microM) than the IC50 for inhibition of leukotrienes (75 microM). 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 compounds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

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.     

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.     

Inhibitory effect of a peptide leukotriene antagonist ONO-1078 on LTD4- and antigen-induced thromboxane B2 production in guinea pig lungs

The effect of a peptide leukotriene receptor antagonist ONO-1078 on the production of thromboxane (Tx) B₂ induced by leukotriene (LT) D₄ and antigen challenge was examined in guinea pig lungs. LTD₄ (1–1,000 nM) induced a concentration-dependent production of TxB₂ in nonsensitized guinea pig lungs and ovalbumin challenge (0.01–100 μg/ml) produced TxB₂ and peptide leukotrienes in a concentration-dependent manner in ovalbumin-sensitized guinea pig lungs. ONO-1078 inhibited LTD₄ (100 nM)-induced TxB₂ production with the IC₅₀ value of 0.24 μM. Furthermore, ONO-1078 inhibited antigen (10 μg/ml)-induced TxB₂ production with the IC₅₀ value of 0.14 μM without effect on the production of peptide leukotrienes. These results suggest that ONO-1078 may prevent the antigen-induced production of TxB₂ through the blockade of the activation of receptors by endogenously generated peptide leukotrienes.     

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.     

The effect of calcium antagonists on histamine and leukotriene-induced tracheal microvascular permeability in the guine pig

The effects of several calcium antagonists, i.e., nifedipine, verapamil adn 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), were evaluated on agonist-induced increases in permeability of the airway microvasculature in anesthetized guinea pigs. Vascular permeability was measured as tracheal extravascular albumin content by using ¹²⁵I-bovine serum albumin and the utilization of ⁵¹Cr labelled-erythrocytes to correct for blood volume. Intratracheal injections of histamine (1, 10 and 100 μg) or leukotriene (LT) D₄ (1, 10 and 100 ng) produced dose-dependent increases in extravasated radiolabelled albumin in the trachea. Although histamine produced a greater maximal response than LTD₄, the latter provocation was tent times more potent than the former. Nifedipine, a dihydropyridine calcium slow channel blocker, exhibited dose-dependent (30, 100 and 300 μg/kg) inhibitory activity against histamine-induced increases in extravascular albumin, while another calcium slow channel blocker, verapamil (100, 300 and 1000 μg/kg), exhibited much less activity. TMB-8, a purported intracellular calcium antagonist (1 and 10 mg/kg), was observed to have some inhibitory activity versus histamine. Similar doses of all three calcium antagonists failed to significantly inhibit increases in tracheal microvascular permeability evoked by LTD₄. These results suggest that differences in mediator-induced microvascular permeability in the guinea pig trachea are evident depending upon the agonist selected and the pool of calcium utilized.     

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.     

Contraction of guinea pig gail bladder strips by leukotrienes and other agonists

In the presence of indomethacin, Leukotriene C₄ (LTC₄), LTD₄ and LTE₄ were shown to be contractile agents on guinea pig gall bladder strips. The respective pD₂ values for LTC₄, LTD₄ ad LTE₄ were 9.1, 9.1 and 7.7. The contractile effects of LTD₄ were not mediated through the generation of cyclooxygenase products and were antagonized by the SRS-A antagonist FPL-55712. The effects of PGE₁, PGF_2α, the endoperoxide analogue U44069 and histamine on gall bladder strips were also examined. All these agents caused dose-related contractions but were considerably less potent than the leukotrienes. Leukotrienes are therefore potent contractile agents on the guinea pig gall bladder and may contribute to gall bladder contractions or spasms .     

Interaction between leukotriene C4 and histamine in the guinea-pig

Lipoxygenase metabolites have proposed as potential chemical mediators of the bronchial hyperractivity which characterizes asthma (2,6). In addition to the possibility that leukotrienes (LTs) sensitize airways smooth muscle to the contractile actions of other mediators such as histamine (1–3), a number of studies have provided evidence for LT-induced enhancement of bronchoconstriction by a vagal dependent mechanism (4–6). In the present study the effects of exposure of the airway to LTC₄ on subsequent responsiveness to histamine have been investigated in both and experiments. LTC₄, in a concentration eliciting threshold contractile responses of the isolated trachea (1.7 nM), had no effect on either the EC₅₀ or maximal contractile response to histamine. At a concentration eliciting an approximately EC₅₀ contractile response, LTC₄ (10 nM) shifted the histamine concentration-response curve rightwards altering the maximum response. In anaesthetized, mechanically ventilated guinea pigs LTC₄ (0.1–0.4 nMole/kg, i.v.) injected 20 s beforehand, failed to alter histamine (9–36 nMole/kg, i.v.)-induced bronchoconstriction whereas, under the same conditions, LTD₄ (0.05–0.2 nMole/kg, i.v.) dose-dependently enhanced histamine-induced bronchoconstriction. On the other hand, LTC₄ or LTD₄ (16 uM, 30 s) aerosols potentiated histamine (9.36 nMole/kg, i.v.) in a concentration-dependent manner (Table). Both LTC₄ and LTD₄ aerosols enahance airway reactivity to histamine whereas only LTD₄ has this action when administered intravenously. Neither LTC₄ nor LTD₄ (6) enhances the contractile effects of histamine on isolated airways smooth muscle. It is concluded that the broncho-constriction enhancing action of these leukotrienes may be indirectly mediated.     

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-induced contraction is mediated by cysteinyl leukotriene receptor CysLT1 in guinea pig fundus but by CysLT1 and CysLT2 in antrum

AimsLeukotriene D₄ (LTD₄) causes contraction of the stomach through unclear receptors. The aim of the present study is to characterize the cysteinyl leukotriene receptor (CysLT) mediating leukotriene-induced muscle contraction in the stomach.Main methodsWe measured contraction of gastric muscle strips isolated from the guinea pig fundus and antrum caused by cysteinyl leukotrienes, including LTC₄, LTD₄ and LTE₄, as well as the dihydroxy leukotriene LTB₄ in vitro.Key findingsIn both fundic and antral muscle strips, LTC₄ and LTD₄ caused marked whereas LTE₄ caused moderate, concentration-dependent contractions. In contrast, LTB₄ caused only small contraction. The relative potencies for cysteinyl leukotrienes to cause contraction in both fundus and antrum were LTC₄ = LTD₄ > LTE₄. The LTD₄-induced contraction was not affected by tetrodotoxin or atropine, suggesting that the action is not neurally mediated. The LTD₄-induced contraction in the fundus was almost abolished by the CysLT₁ selective antagonist montelukast. In contrast, the LTD₄-induced contraction in the antrum was only partially inhibited by montelukast or the dual CysLT₁ and CysLT₂ antagonist BAY u9773. This antral contraction was almost abolished by the combination of montelukast and BAY u9773, indicating enhancement of inhibition.SignificanceThe results of the present study demonstrate that cysteinyl leukotrienes LTC₄, LTD₄ and LTE₄ cause moderate to marked whereas the dihydroxy leukotriene LTB₄ causes small muscle contraction in the stomach in vitro. The leukotriene-induced contraction is mediated by CysLT₁ in fundus but by CysLT₁ and CysLT₂ in antrum.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [³H]leukotriene D₄ (LTD₄) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD₄. The values for fifty percent inhibition of bound [³H]LTD₄ was 1.5 nM for LTD₄, 45 nM for LTC₄ and 24 nm for LTE₄. LTB₄ at 3.0 × 10⁻⁵ M had no effect on [³H]LTD₄ binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC₄ and LTD₄. However, in the presence of 20 nM serine-borate this method was highly specific for LTD₄. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD₄. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient ‘r’ of 0.992. The assay was also validated by quantitation of LTs released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD₄ than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD₄ specifically or LTC₄ plus LTD₄ simultaneously in one cell system.     

And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Evidence for multiple leukotriene D4 receptors in smooth muscle

Using pure leukotriene D₄ (LTD₄) as the agonist, we determined the dissociation constants, K_B and pA₂ values, for the selective leukotriene antagonist FPL 55712 on guinea pig ileum, trachea, and parenchyma. Responses of the 3 tissues to LTD₄ were competitively antagonized by FPL 55712. K_B and pA₂ values were similar for trachea and parenchyma. However, these values differed from those obtained in ileum. We propose the existence of multiple LTD₄ receptors, with those in lung differing from LTD₄ receptors in ileum.     

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.     

Inhibition of leukotriene-induced contraction of guinea pig trachea by 5-lipoxygenase inhibitors

The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC₄ or LTD₄ were examined on the isolated guinea pig trachea. Responses to either LTC₄ or LTD₄ or LTD₄ were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to exhibit metabolic conversion of the leukotrienes. NDGA (30 μM) and ETYA (100 μM) produced a selective competitive antagonism of LTD₄ - induced contractions, while phenidone antagonized both LTC₄- and LTD₄ - induced responses in a non-competitive manner. In contrast, BW-755c (30 μM) did not significantly antagonize LTC₄ or LTD₄ concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors.     

The effect of calcium antagonists on contractions of the sensitized and normal guinea pig ileum

The purpose of this study was to learn wether a number of Ca²⁺ antagonists were effective in reducing contractile response of the isolated ileum of the sensitized and normal guinea pig. Contractions of the normal ileum in response to LTD₄, acetylcholine, histamine, and potassium chloride were obtained before and after verapamil, diltiazen and papaverine. Ovalbumin-induced contractions of the ovalbumin-sensitized ileum were obtained in the presence of the three Ca²⁺ antagonists. In the normal ileum, all the Ca²⁺ antagonists were highly effective in diminishing the contractile responses to LTD₄, acetylcholine, histamine and potassium chloride. In the sensitized ileum, ovalbumin-evoked contractions, with subsequent release of a potent contractile mediator (presumably SRS-A), were Ca²⁺-dependent since verapamil, diltiazem and papaverine caused a concentration-related reduction of contractions. Thus, the influx of extracellular Ca²⁺ plays a key role in the contractile responses of the normal and sensitized guinea pig ileum when stimulated by various potent agonists acting on specific receptors or on the cell membrane.     

Peptidoleukotriene binding in guinea pig uterine membrane preparations

Peptidoleukotrienes are known to be potent smooth muscle contractile agents in many tissues, including guinea pig uterus. In order to characterize the receptors at which the leukotrienes interact, guinea pig uteri were homogenized in 50nM Tris-HCl, pH 7.4 at 40°C and centrifuged at 1000xg fpr 10 min. The supernatant was centrifuged at 40,000 xg and the washed pellet was used to measure the binding of ³H-LTC₄ and ³H-LTD₄. Specific binding of ³H-LTD₄ was not detected, but specific, saturable binding of ³H-LTC₄ was measured at 40°C, was complete in 10 min. and was rapidly reversible on addition of unlabeled LTC₄. Binding was linear with protein concentration and stimulated by CaCl₂ and L-serine borate. Scatchard and kinetic analysis of binding in the presence of calcium suggested a K_d of 10–12 nM. LTC₄ was a more potent competitor of binding than LTD₄ (IC₅₀ − 40nM and 30 μM, respectively). FPL 55712 inhibited binding from 10–100 μM but stimulated binding at lower concentrations. Thus, the guinea pig uterus has specific receptors for LTC₄, but not LTD₄, that can be demonstrated by radioligand binding.     

Contraction of guinea pig uterus by synthetic leukotrienes

Synthetic leukotrienes (LT) C₄ and D₄ elicited concentration-dependent contractions of the guinea pig uterus between 10^?8-10^?6M, whereas LTE₄ appeared 1000-fold weaker. The potencies of LTC₄ and LTD₄ were similar to that of acetylcholine and PGF_2α but weaker than that of PGE₂. The maximal contractions elicited by LTC₄ and LTD₄ were 66.0 ± 2.1% and 63.8 ± 4.6% that elicited by acetylcholine. FPL 55712 (10^?5M) antagonized the uterine contractile activity of LTD₄, while meclofenamic acid at 10^?5M but not at 10^?6M also antagonized the LTD₄-induced contration. Radioimmunoassay of the uterine tissue bathing fluid following LTD₄ indicated the variable presence of low concentrations of PGE₂, PGF_2α and TXB₂. These results demonstrate the LTC₄ and LTD₄ possess significant uterine contractile activity, which may only partially be mediated indirectly via prostaglandin products.     

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 activities 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.