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.     

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

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

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.     

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.     

Melanocyte-Stimulating Properties of Arachidonic Acid Metabolites: Possible Role in Postinflammatory Pigmentation

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b-locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D₂, leukotriene (LT) B₄, LTC₄, LTD₄, LTE₄, thromboxane (TX) B₂ and 12-hydroxy eicosatetraenoic acid (12-HETE). The effect of LTC₄ was particularly strong compared to that of PGE₂, about which we have previously reported. On the other hand, PGE₁, PGF_2α and 6-ketoPGF_1α did not show any significant stimulatory effect. These data suggest that arachidonate-derived chemical mediators, especially LTC₄, may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.     

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 .     

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.     

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites

Leukotriene D₄ (LTD₄)-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D₂-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD₄ and PGD₂ were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD₄-induced bronchoconstriction and thromboxane (TX) A₂ release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT₁ receptor activation by LTD₄ was established by measurements of immunoreactive 6-keto PGF_1α (a stable metabolite of PGI₂) and PGE₂. In contrast, TP receptor-mediated bronchoconstriction by PGD₂ was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE₂ was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD₂ nor U-46619 actively released PGE₂, but continuous production of PGE₂ and PGI₂ was established, and the response to PGD₂ was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD₄ and PGD₂ in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD₄ was amplified predominantly by secondarily released TXA₂, that induced by PGD₂ was attenuated by bronchoprotective PGE₂ and PGI₂, presumably tonically produced in the airways.     

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.     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S)

The effects of prostacyclin (PGI₂) and its stable metabolite 6-oxo-PGF_1α on various bioassay tissues are compared with those of PGE₂ and PGF_2α, using the cascade superfusion method. On vascular smooth muscle, PGI₂ caused relaxation of all tissues tested except the rabbit aorta. PGE₂ relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF_1α was ineffective at the concentrations tested.On gastro-intestinal smooth muscle, PGI₂ contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE₂ or PGF_2α. None of these substances contracted that cat terminal ileum. 6-oxo-PGF_1α was inactive on these tissues at the doses tested.PGI₂ was less active than PGE₂ or PGF_2α in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF_1α was active only on the rat uterus. Thus, PGI₂ can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

Intraperitoneal injection of zymosan in mice induces pain,inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma proteins in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE₂ (measured by RIA) which reached maximum levels of 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC₄, while later, LTE₄ was the major component. LTD₄ levels remained low throughout and no LTB₄ was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, recued PGE₂ levels and inhibited writhing. Phenidone reduced peptido-LT levels. $\text{In}$$\text{vitro}$ studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE₄, LTD₄, LTB₄ or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the $\text{in}$$\text{vivo}$ metabolism of LTC₄ to LTD₄ and LTE₄ could not be identified.     

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.     

Sensitivity of ovine myometrial tissue to various eicosanoids in vitro

The sensitivity of sheep myometrial tissue to prostaglandin F_2α (PGF_2α), PGE₂, the thromboxane analog U-44069, and leukotrienes C₄ (LTC₄) and LTD₄ was investigated in a superfusion system. Tissues were obtained from eight oophorectomized ewes, with or without pretreatment with estradiol-17β. After equilibration, spontaneous activity was abolished by adding indomethacin to the superfusion fluid. The dose needed to induce a contraction with a peak level of 50% of the median peak level of spontaneous contractions increased from PGE₂ to PGF_2α, U-44069, LTC₄, and LTD₄. The differences between the doses required were significant for all compounds, except between LTC₄ and LTD₄. Estradiol-17β pretreatment caused an increase in the required dose of PGF_2α. The results of this study do not support the hypothesis that leukotrienes are involved in the regulation of myometrial activity.     

Leukotriene D4 and E4 formation by plasma membrane bound enzymes

The homogenate of rat basophilic leukemia cells produces both the dihydroxy-leukotrienes and the peptido-leukotrienes (LT) C₄, D₄ and E₄. The enzymes responsible for the formation of LTA₄ and LTB₄ are in the soluble fraction while the enzymes for LTC₄, LTD₄ and LTE₄ are particulate (10, 000 × g pellet). Centrifugation of the 10, 000 × g pellet over a sucrose gradient resulted in two subfractions, a membrane fraction and a pellet (sucrose pellet.) The fractions were incubated with LTC₄, and the products were identified by bioassay, HPLC and UV spectra. The membrane fraction contained the enzymes γ-glutamyl transpeptidase and amino peptidase which convert LTC₄ to LTD₄ and LTD₄ to LTE₄, respectively. When incubated with LTC₄, the membrane fraction showed a dose dependent formation of LTD₄ and a time course which reached a plateau at 30 to 45 minutes. Addition of serine borate blocked the formation of LTD₄, and cysteine blocked LTE₄. We conclude that the γ-glutamyl transpeptidase and the amino peptidase which produce LTD₄ and LTE₄ respectively are plasma membrane bound.     

Modulation of autonomic neurotransmission by PGD2: Comparison with effects of other prostaglandins in anesthetized cats

Experiments with anesthetized cats were done to study possible roles of different prostaglandins (PGs) in modulating sympathetic neuroeffector transmission. We recorded contractions of the nictitating membrane (n.m.), blood flow in the carotid artery, heart rate and blood pressure, both under control conditions and while stimulating the cut cervical sympathetic nerve. Intra-carotid arterial injection (i.a.) of PGD₂ depressed sympathetic transmission to the n.m. without depressing the effects of exogenous norepinephrine (NE). In contrast, PGE₂ enhanced the effects of nerve transmission or exogenous NE on the stimulated n.m. PGI₂ had similar but shorter effects to PGE₂. PGF_2α or a stable PGH₂ analog, contracted the n.m. smooth muscle with no detected effect on nerve transmission. Carotid blood flow was increased by PGD₂, PGE₂ and PGI₂. PGD₂ and PGI₂ caused bradycardia that could be blocked by atropine. This ability of PGD₂ to modulate autonomic nerve activity is of particular interest because of recent reports that nerve tissue synthesizes PGD₂.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-¹⁴C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF_1α, TxB₂ and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF_2α, PGE₂, PGD₂, LTB₄ and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB₄, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB₄ is considerably increased after adrenalectomy. In the spleen, PGD₂ and 12-HETE are decreased after adrenalectomy.The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA₂ inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

Leukotrienes cause eosinophil emigration into conjunction tissue

The ability of LTB₄, LTC₄, the 5S,6R and 5R,6S LTD₄ stereoisomers, and LTE₄ to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological andl ight microscopy techniques. LTD₄ and LTE₄ demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10ng to 1000ng), while there was only a minimal response to LTC₄. LTB₄ produced marked eosinophil inflitrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB₄. The 5R,6S LTD₄ stereoisomer did not evoke any leukocyte infiltrantion. The SRS-A antagonist, FPL 55712, abolished pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10μg to 1000μg. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Quantitation of luekotrienes C4, D4 amd E4 released by pathophysiological stimuli

In order to examine the modulation of leukotriene (LT) release, the PAF-acether-mediated stimulation of these compounds in rat lung was studied. Release of LTC₄, LTD₄ and LTE₄ in both perfused and chopped lung preparations was measured using HPLC and radioimmunoassay. Pre-incubation or pre-infusion of the tissue with indomethacin and PGE₂ was conducted to investigate the effect of cyclooxygenase inhibitors and products on the lipoxygenase pathway. In addition, the effects of LT levels of pre-incubation with vasoactive intenstinal polypeptide (VIP) in chopped lung were observed.In perfused rat lung, indomethacin reduced the levels of LTC₄ relative to LTD₄ as measured in the first 2 min after stimulation of the lung by PAF-acether. Chopped lung preparations, incubated for 15 min. exhibited higher levels of LTC₄ and LTD₄ in indomethacin-treated samples, this increases being effectively reversed by PGE₂.In the VIP pre-incubation experiments clear inhibition of peptido -leukotriene synthesis was observed, with no LTC₄ and only low levels of LTD₄ and LTE₄ observed in VIP-incubated samples. In preliminary experiments using rabbit C5a des arg and PAF-acether on rabbit lung parenchyma strips to stimulaet LT release, disodium cromoglycate pre-incubation was observed to inhibit this release.Inhibition of the 5-lipoxygenase pathway of PGE₂ is supported by these experiments. VIP appears to act as an inhibitor of LTC₄ and LTD₄ biosynthesis or release in this model. Too little is known that peptidergic actions to postulate a mechanism by which a neuroendocrine peptide exerts control of release of arachidonate metabolites; however, VIP is associated with muscarinic stimulation (1) and has been found in mast cells (2).     

Specific enhancement of LTD4-induced contractions of isolated guinea pig trachea by 5-hydroxyeicosatetraenoic acid (5-HETE)

In view of the likely production of monohydroxyeicosatetraenoic acid (HETE's) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 μg/ml), a 17-fold increase (0.97 ± 0.34 ng/ml to 16.73 ± 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 μM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 μM) enhanced subsequent LTD₄-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD₄ concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD₄ responses. Also, the 5-HETE-induced enhancement seemed specific fot LTD₄, since contractions to LTC₄ (in the presence of l-serine borate), acetylcholine, histamine, PGD₂ or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD₄ to exacerbate airway smooth muscle contraction in asthma.