Synthesis and vascular actions of an arachidonic acid ethylene-diamino-triethyl-ester (AA-EDTA) derivative

An ethylene-diamino-triethyl-ester derivative of arachidonic acid (AA-EDTA) was newly synthesized and tested for its coronary vasoactivity in isolated perfused cat coronary arteries. This arachidonic acid analog exerted a coronary vasodilator effect and significantly antagonized the coronary vasoconstrictor effect of LTD4. The constrictor response to the thromboxane analog carbocyclic thromboxane A2 was unaffected by AA-EDTA. These properties of AA-EDTA may be useful in counteracting the vasoconstrictor influence of leukotrienes in situations such as coronary artery vasospasm.     

Comparative effects of leukotrienes on porcine pulmonary circulation in vitro and in vivo

The present study examined the effect of leukotrienes on porcine pulmonary vasculature both in vivo and in vitro. In vitro studies using isolated vascular strips demonstrated that pulmonary arterial smooth muscle contracted to leukotriene C4 (LTC4), whereas pulmonary vein smooth muscle did not. Pulmonary arterial contraction was due to both the direct action of LTC4 and secondarily generated thromboxane A2 (TxA2). In vivo, LTC4 injection caused a pronounced but transient increase in pulmonary arterial pressure and pulmonary arterial wedge pressure (Ppw), with a smaller effect on left ventricular end-diastolic pressure. Effects of LTD4 were smaller with comparable pressure changes at all three sites, suggesting a primary cardiac effect. Like LTC4, histamine caused a disproportionate increase in Ppw vs. left ventricular end-diastolic pressure. These observations suggest that LTC4 causes pulmonary venoconstriction in vivo despite its lack of effect on pulmonary vein smooth muscle in vitro. This discrepancy may be due to venoconstrictor effects of TxA2 generated from upstream pulmonary arterial vessels.     

Differential activity of leukotrienes upon human pulmonary vein and artery

Responses to leukotrienes B4, C4, D4 and E4 were examined in human pulmonary artery and pulmonary vein preparations from surgical specimens. Leukotrienes C4 (LTC) and D4 (LTD) were potent contractants of pulmonary vein over the dose range of 10(-10) M to 10(-6) M, whereas they produced minimal contractions of human pulmonary artery only at concentrations of 10(-8) M or greater. Leukotriene E4 was less potent than LTC or LTD, and leukotriene B4 (LTB) at concentrations up to 10(-6) M had no effect upon either pulmonary veins or pulmonary arteries. Contractions of pulmonary vein by LTD were inhibited in a competitive manner by FPL 55712. Dose response characteristics of LTD and inhibition by FPL 55712 were similar for pulmonary venous and bronchial smooth muscle. We conclude that pulmonary vein smooth muscle has leukotriene receptors comparable to those of bronchial smooth muscle whereas pulmonary artery does not.     

Actions of lipoxygenase metabolites in isolated rat lungs

Leukotrienes constrict smooth muscle and could be important for the regulation of the pulmonary circulation. We examined the production and action of lipoxygenase metabolites in isolated lungs, where we controlled the perfusing fluid used. Arachidonate injected into isolated rat lungs perfused with cell- and protein-free physiological salt solution caused a transient pressor response. Following indomethacin, arachidonate caused a delayed slow pressure rise followed by edema. The lung effluent contracted the guinea pig ileum. High-pressure liquid chromatography (HPLC) analysis of the perfusate demonstrated the presence of leukotrienes (LTC4 and LTD4). Diethylcarbamazine, a leukotriene synthesis inhibitor, prevented the slow pressure rise and edema seen after indomethacin plus arachidonate. In lungs perfused with cell- and protein-free physiological salt solution, LTC4, but not LTD4, caused a transient pressure rise followed by a sustained pressure rise. The sustained rise was abolished by a leukotriene-receptor blocker (FPL 55712) but not by indomethacin. In blood-perfused lungs, LTC4 caused only the transient pressure rise that was not blocked by FPL 55712. In lungs perfused with physiological salt solution containing albumin, LTC4 had no effect. We concluded that 1) perfused nonsensitized rat lungs produced LTC4 and LTD4; 2) LTC4 may be a major pulmonary vasoconstrictor; and 3) albumin binding limits the pressor effect of LTC4.     

Vasoconstrictor effects of leukotrienes C4 and D4 in the feline mesenteric vascular bed

The effects of leukotriene C4 (LTC4) and leukotriene D4 (LTD4) in the feline mesenteric vascular bed were investigated under conditions of controlled blood flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Intra-arterial injections of LTC4 and LTD4 (0.3-3.0 micrograms) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC 4 and LTD4 were similar to norepinephrine (NE) whereas mesenteric vasoconstrictor response to the thromboxane analog, U46619, was markedly greater than were responses to LTC4 and LTD4. Meclofenamate in a dose that greatly attenuated the systemic depressor response to arachidonic acid was without effect on vasoconstrictor responses to LTC4 and LTD4, NE and U46619 in the mesenteric vascular bed. The present data show that LTC4 and LTD4 possess significant vasoconstrictor activity in the feline mesenteric vascular bed. In addition, the present data suggest that products of the cyclooxygenase pathway do not mediate vasoconstrictor responses to LTC4 and LTD4 in the intestinal circulation of the cat.     

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.     

Differential activity of leukotrienes upon human pulmonary vein and artery

Responses to leukotrienes B₄, C₄, D₄ and E₄ were examined in human pulmonary artery and pulmonary vein preparations from surgical specimens. Leukotrienes C₄ (LTC) and D₄ (LTD) were potent contractants of pulmonary vein over the dose range of 10⁻¹⁰M to 10⁻⁶M, whereas they produced minimal contractions of human pulmonary artery only at concentrations of 10⁻⁸M or greater. Leukotriene E₄ was less potent than LTC or LTD, and leukotriene B₄ (LTB) at concentrations up to 10⁻⁶M had no effect upon either pulmonary veins or pulmonary arteries. Contractions of pulmonary vein by LTD were inhibited in a competitive manner by FPL 55712. Dose response characteristics of LTD and inhibition by FPL 55712 were similar for pulmonary venous and bronchial smooth muscle. We conclude that pulmonary vein smooth muscle has leukotriene receptors comparable to those of bronchial smooth muscle whereas pulmonary artery does not.     

Actions of synthetic leukotrienes on platelets and blood vessels in the anesthetised pig: the release of a platelet derived vasodilator

The actions of leukotrienes (LT's) C4, D4, E4 and F4 have been investigated in the perfused hind-limb of the anesthetized pig. In the blood perfused hind limb LTC4, D4 and E4 increased the perfusion pressure in a dose-dependent fashion whereas LTF4 decreased perfusion pressure. In the Tyrode perfused hind limb all LT's increased perfusion pressure (rank order potency LTC4 = LTD4 much greater than LTF4). The actions of LTF4 were not affected by a wide variety of pharmacological treatments, including indomethacin, methysergide and FPL-55712. The LT's aggregated porcine platelets (rank order potency LTC4 greater than LTF4 greater than LTD4) and induced the release of a platelet-derived vasodilatory mediator. The results provide pharmacological evidence of specific leukotriene receptors in vivo and that leukotrienes can independently modulate blood flow. These data suggest that important interactions may occur between platelets, the arachidonate lipoxygenase products and platelet-derived substances in response to inflammatory stimuli in the cardiovascular system.     

Studies on the mechanism of leukotriene induced coronary artery constriction

Leukotriene (LT) C4, D4, and E4 at concentrations of 10 to 100 ng/ml were found to be potent coronary artery constrictors in the perfused cat coronary artery and perfused rat heart. In contrast, LTB4, was essentially inactive. The coronary constrictor effect of leukotrienes was not related to thromboxane release, but rather appeared to be due to a calcium mediated activation of specific leukotriene receptors.     

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 characterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar characteristics 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 substantial labelling of alveolar walls. This lends further support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.     

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.     

Ocular responses to leukotriene C4 and D4 in the cat

The effects of leukotriene C4 (LTC4) and D4 (LTD4) on the iridial smooth muscles, intraocular pressure, blood-aqueous barrier and regional blood flow in the eye have been studied in cats. The test compounds were injected into the anterior chamber. Both LTC4 and LTD4 caused a dose-dependent constriction of the pupil, the agents being about equipotent. The effect on the iridial sphincter muscle was not dependent on nerve conduction, cyclo-oxygenase products or muscarinic receptors. Maximal constriction was achieved with 0.1-1 microgram of the test compounds. The smallest dose to induce a decrease in pupil diameter was 0.01 microgram. After intracameral injection of 4 micrograms the miotic response was markedly delayed. This indicates that in high concentrations LTC4 and LTD4 probably also stimulate the iridial dilator muscle. The blood flow in the anterior uvea decreased after intracameral injection of 4 micrograms LTC4/LTD4. Smaller doses had no clear effect. There was no effect on the blood-aqueous barrier as judged from the aqueous humor protein concentration. The intraocular pressure decreased slightly after injection of the test compounds.     

Studies on the mechanism of leukotriene induced coronary artery constriction

Leukotriene (LT) C₄, D₄,and E₄ at concentrations of 10 to 100 ng/ml were found to be potent coronary artery constrictors in the perfused cat coronary artery and perfused rat heart. In contrast, LTB₄, was essentially inactive. The coronary constrictor effect of leukotrienes was not related to thromboxane release, but rather appeared to be due to a calcium mediated activation of specific leukotriene receptors.     

Effect of ICI 198,615, SK+F 104,353, MK-571 and CGP45715A on cysteinyl leukotriene-induced responses in guinea-pig heart

Peptidoleukotrienes (LTs), LTC4 and LTD4, cause potent vasoconstriction and myocardial depression in a range of species including man. The recent availability of specific LTD4 antagonists has allowed the evaluation of LT involvement in disease states and the characterisation of LT receptors in the airways. We decided to study the actions of four LT antagonists; ICI 198,615, SK + F 104,353, MK-571 and CGP45715A on LTD4-, LTC4- and U46619-induced effects in the coronary vasculature and on cardiac contractility in the guinea-pig isolated heart. We found a difference in the actions of the antagonists in the GP heart compared with the lung. ICI 198,615 retained its selectivity towards LTD4 whereas SK + F 104,353 antagonised both LTD4 and LTC4. MK-571 and CGP45715A had a non specific action against the LTs. Our results also indicated a direct action of the LTs on cardiac contractility which was not associated with the constriction of the coronary vasculature. These studies indicate that if the leukotrienes are involved in cardiac disease antagonists specific for the peptidoleukotrienes may be of therapeutic benefit in many of the disease states of the heart.     

Synthesis and vascular actions of an arachidonic acid ethylene-diamino-triethyl-ester (AA-EDTA) derivative

An ethylene-diamino-triethyl-ester derivative of arachidonic acid (AA-EDTA) was newly synthetized and tested for its coronary vasoactivity in isolated perfused cat coronary arteries. This arachidonic acid analog exerted a coronary vasodilator effect and significantly antagonized the coronary vasoconstrictor effect of LTD₄. The constrictor response to the thromboxane analog carbocyclic thromboxane A₂ was unaffected by AA-EDTA. These properties of AA-EDTA may be useful in counteracting the vasoconstrictor influence of leukotrienes in situations such as coronary artery vasospasm.     

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 leukotriene (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 LTF4 to contract guinea pig ileal smooth muscle (GPISM) was studied. Each of the LT's contracted GPISM. The rank order of potency was LTD4 greater than LTC4 greater than LTE4 much greater than LTF4 in a ratio of 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 PGF1 alpha 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 effluent 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.     

Effect of hypoxia on responses of respiratory smooth muscle to histamine and LTD4

The aim of the present study was to compare the effect of reduced oxygenation on the contractions of pulmonary vascular and airway smooth muscle induced by leukotriene D4 (LTD4) with those induced by histamine (an agonist with similar mechanisms of smooth muscle contraction) and KCl (a voltage-dependent stimulus). During hypoxia (PO2: 40 +/- 4 Torr) the responses of isolated porcine pulmonary artery and vein spiral strips to LTD4 increased approximately three- and two-fold, respectively, and the vein also exhibited an augmented response to histamine. The augmentation was blunted (LTD4) or reversed (histamine) during anoxia (PO2: 0 +/- 2 Torr). Responses to KCl were not systematically altered by reduced oxygenation. In contrast, the contractions of the guinea pig parenchymal lung strip by all three agonists were generally suppressed by reduced oxygenation. After reoxygenation, the contractile responses of each of the three smooth muscle preparations were generally increased compared with previous and concurrent base-line observations, particularly the LTD4-induced pulmonary vein contraction that increased approximately sevenfold after reoxygenation after anoxia. The contribution (if any) of leukotrienes to hypoxic pulmonary vasoconstriction may reflect increased vascular responsiveness to leukotrienes during hypoxia as well as (or instead of) increased leukotriene release.     

Metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney.

The metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney was investigated. For this purpose LTC4, LTD4 or LTE4 were studied in separate experiments. The isolated perfused rat kidney metabolized all cysteinyl leukotrienes to the final metabolite N-acetyl-LTE4. In the presence of 5% albumin 50% of LTC4 was metabolized to LTD4 (22%), LTE4 (15%) and N-acetyl-LTE4 (13%) within 60 min. Excretion of radioactivity into urine was less than 1%. In contrast, in the absence of albumin, LTC4 was completely metabolized within 45 min to N-acetyl-LTE4, the sole and final metabolite of LTC4 found in the perfusion medium as well as in urine. After 60 min 19% and 42% of total radioactivity were found in the perfusion medium and in urine, respectively. Isolated glomeruli metabolized LTC4 to LTD4 and to LTE4 but not to N-acetyl-LTE4 at a rate comparable to the rate observed by the isolated perfused kidney in the absence of albumin. In contrast to isolated glomeruli isolated tubuli metabolized LTE4 to N-acetyl-LTE4 at a rate comparable to that observed by the isolated perfused kidney in the absence of albumin. The present study shows that the isolated perfused rat kidney metabolizes cysteinyl leukotrienes to the sole and final metabolite N-acetyl-LTE4. In the presence of albumin metabolism is slowed down and excretion of N-acetyl-LTE4 into urine is prevented.     

Characterization of sulfidopeptide leukotriene responses in sheep tracheal smooth muscle in vitro.

We have investigated the effects of leukotrienes (LTs) on isolated tracheal smooth muscle from sheep sensitive to Ascaris suum antigen. LTC4 and LTD4 produced dose-dependent contractions of sheep trachea, but LTE4 was virtually inactive. YM-17690, a non-analogous LT agonist, produced no contractile response up to 100 microM. Indomethacin (5 microM) had no effect on LTC4- and LTD4-induced contractions. L-Serine borate (45 mM), an inhibitor of gamma-glutamyl transpeptidase, shifted the dose-response curve of LTC4 to the left by 161-fold, and L-cysteine (6 mM), an inhibitor of aminopeptidase, shifted the dose-response curves of LTC4 and LTD4 to the left by 67- and 23-fold, respectively. YM-16638 (1 microM), an LT antagonist, shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.57 and 7.13, respectively. YM-16638 did not affect LTC4-induced contractions of L-serine borate-treated tissues, indicating that the compound acts only on LTD4 receptors in sheep trachea, LTE4 (1 microM) shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.87 and 7.31, respectively. YM-17690 (10 microM) showed effects similar to LTE4, suggesting that the compound acts as an LTE4 agonist in sheep trachea. These results suggest that in sheep tracheal smooth muscle (a) LTC4 and LTD4 produce contractions, (b) these LT-induced contractions are not mediated by cyclooxygenase products, (c) LTC4 is converted to LTD4 and then to LTE4, and (d) the potency of the LTC4- and LTD4-induced contractions is increased when their conversion to LTE4 is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotriene C4 action and metabolism in the isolated perfused bullfrog heart

The effects of leukotrienes (LTs) have been widely studied in the isolated perfused mammalian heart; however, little is known about the effect or metabolism of LTs in the isolated bullfrog heart. Isolated perfused bullfrog hearts were administered randomized doses of LTC4, LTD4, or LTE4. The cardiac parameters of heart rate, developed tension, and its first derivative (dT/dt) were recorded. LTC4 was the most potent of the leukotrienes tested in eliciting positive inotropic effects. LTD4 and LTE4 were equally effective but about one order of magnitude less potent than LTC4. None of the LTs showed any chronotropic effects in this preparation. A series of [3H]LTC4 metabolism experiments were carried out using whole perfused hearts and minced bullfrog heart tissue. Isolated perfused bullfrog hearts administered [3H]LTC4 converted significant amounts to [3H]LTD4, and to a lesser degree, [3H]LTE4, during the 6-min course of collection. Both minced atrial and ventricular tissue converted [3H]LTC4 to radioactive metabolites that co-migrated with authentic LTD4 and LTE4 standards. In both tissues, the major product was [3H]LTD4, with smaller amounts of [3H]LTE4 produced. The atrium converted significantly more [3H]LTC4 to its metabolites than did the ventricle. The metabolism of [3H]LTC4 to [3H]LTD4 by both tissues was virtually abolished in the presence of serine borate. Cysteine had no effect on [3H]LTE4 production. The data in this study demonstrate that leukotrienes have the opposite inotropic effect on the heart when compared with mammals. Also in contrast to mammals, frogs metabolize LTC4 to a less potent compound and may use the LTC4 to LTD4 conversion as a mechanism of LTC4 inactivation.