Enhancement of leukotriene D4-induced contraction of guinea-pig isolated trachea by platelet activating factor

The effect of platelet activating factor (PAF), a potent lipid mediator of inflammation, was examined in the induction of airway hyperreactivity to known mediators of anaphylaxis. Concentration-dependent contractions of the isolated guinea-pig trachea to PAF (10(-7)-10(-5) M) were produced and an EC50 value was found to be 7.5 X 10(-7) M. Pretreatment for 30 min with a known PAF inhibitor, CV-3988 (10(-5) or 10(-4) M), produced significant inhibition of PAF contractions; however, at 10(-6) M, CV-3988 had no effect. In the presence of meclofenamic acid (10(-6) M), the concentration-response curve to PAF was shifted significantly upward and to the left. This potentiation could be reversed by pretreating the tissues with the peptidoleukotriene antagonists, FPL 55712 or SK&F 102922 (10(-5) M). Pretreatment with PAF concentrations having essentially no intrinsic activity (10(-8), 10(-7)) significantly enhanced the contraction of guinea-pig trachea to various concentrations of LTD4 and to certain concentrations of a thromboxane mimic (U-46619). Pretreatment with lyso-PAF failed to potentiate the LTD4 response, while pretreatment with CV-3988 reverse the potentiation by PAF of the lower concentrations of LTD4. However, PAF failed to enhance contractions (with or without the presence of meclofenamic acid) to acetylcholine, histamine, PGD2 or LTC4 (in the presence of serine borate). These results indicate a possible role for PAF as a mediator of airway hyperreactivity.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonism of leukotriene (LT) C4-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cyclooxygenase inhibitor indomethacin. NDGA (30 microM) as well as indomethacin (5 microM) inhibited LTC4-induced contractions. But, in the presence of indomethacin NDGA was ineffective to inhibit the LTC4 response, whereas two other lipoxygenase inhibitors, phenidone (3-30 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM), markedly inhibited it. The antagonist action of an LTD4 receptor antagonist FPL55712 against LTC4-induced contractions was significantly reduced by NDGA (10-30 microM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect on the LTC4 antagonism in the presence of indomethacin, but 0.3 microM phenidone and 1 microM ETYA which did not inhibit the LTC4 response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC4, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LTC4 response was not inhibited by indomethacin, 0.3 microM phenidone and 1 microM ETYA. In the presence of a gamma-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC4 antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 microM histamine- and 30 microM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H1-blocker pyrilamine. These results suggest that some putative non-prostanoids are involved in LTC4-induced contractions of guinea-pig trachea and which regulate the effects of LTD4 antagonism and beta-adrenoceptor activation.     

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 acids (HETEs) 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 microgram/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 microM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 microM) 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 for LTD4, since contractions to LTC4 (in the presence of I-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.     

Mechanism of action of platelet-activating factor on guinea-pig lung parenchyma strips

The contribution of thromboxane A2 to platelet-activating factor (PAF)induced contraction of guinea-pig lung parenchyma strips (GPLPS) was investigated using an experimental design that allowed us to record the contractions of the tissues in parallel with the determination of thromboxane B2 (TXB2) levels in the organ baths by enzyme immunoassay. It was found that the first injection of PAF induced the contraction of GPLPS and the release of TXB2. Following subsequent additions of PAF to the same tissue, the contractile response was abolished but TXB2 levels were not significantly reduced. Pretreatment of the tissue with the thromboxane synthetase inhibitor OKY-046 (3.5, 170, and 350 microM) strongly inhibited the release of TXB2 but had no effect on the contraction of the tissues induced by PAF. The mechanism of PAF-induced contraction of GPLPS was further investigated using several drugs that interfere with arachidonic acid metabolism. It was found that pretreatment of the tissues with the cyclooxygenase and thromboxane synthetase inhibitors indomethacin (2.8, 28, and 56 microM) and OKY-046 (170 microM) or with the thromboxane antagonist SKF-88046 (1.25 and 12.5 microM) had no significant effect on the contractile response to PAF. The compound L-655,240 (2.5, 25, and 50 microM), which acts simultaneously as an antagonist of thromboxane and inhibitor of lipoxygenase, significantly reduced GPLPS contractions induced by PAF. Another lipoxygenase inhibitor, nordihydroguaiaretic acid (33 microM), and the inhibitor of both pathways of arachidonic acid metabolism, BW775c (110 microM), both reduced PAF-induced contractions of GPLPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea

Contractions of guinea pig trachea in the absence and presence of indomethacin to LTD4 greater than LTC4 greater than K+ greater than histamine greater than acetylcholine were reduced following a 45 minute exposure of the tissues to calcium-free Krebs' solution (Ca2+-free Krebs' solution), were further reduced by a transient exposure to EGTA (1.25 mM) in Ca2+-free Krebs' solution and were virtually abolished when tested in the presence of EGTA (0.125 mM) in Ca2+-free Krebs' solution. In normal Krebs' solution (2.5 mM Ca2+) the Ca2+ entry blockers nifedipine (N) much greater than D-600 greater than verapamil (V) greater than diltiazem (D) almost completely abolished the contractions to K+ but blocked only a component of the maximum response to the other agonists. After exposure to Ca2+-free Krebs' solution for 45 minutes, any residual contractions to LTC4 & LTD4, were reversed by low concentrations of N (0.3 microM) or D-600 (2.1 microM). Leukotrienes appear to mobilize a superficial and a bound store of Ca2+ which gains entry through at least two types of Ca2+ channels (or mechanisms), one of which is blocked by N and D600. K+-induced contractions appear to be dependent on superficial and tightly bound Ca2+ but entry is solely through channels which are blocked by the Ca2+ entry blockers studied. Contraction to histamine and acetylcholine persisted following exposure of the tissues to Ca2+ free Krebs' solution but contractile activity was virtually abolished in Ca2+ free Krebs' solution containing EGTA. Residual contractions to histamine and part of the residual contractions to acetylcholine in Ca2+-free Krebs' solution were blocked by low dose N (0.3 microM) or D600 (2.1 microM). These findings suggest a major role for extracellular Ca2+ during spasmogen-induced contraction in this tissue.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D4 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 microM), and by arachidonic acid (AA), melittin and LTD4. FPL55712 (0.1 and 1 microM) inhibited contractions induced by AA (100 microM) and the phospholipase A2 activator melittin (3 micrograms/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 micrograms/ml) more than by A23187 (1 microgram/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 microM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Involvement of cyclooxygenase-dependent pathway in contraction of isolated ileum by urotensin II

We previously reported that urotensin II induced biphasic (brief- and long-lasting) contractions and the brief contraction was mediated by acetylcholine release from ganglionic cholinergic neurons in a segment of guinea-pig ileum. In the present work, we studied the mechanism contributing to long-lasting contractions induced by urotensin II. Treatment with 0.1 microM tetrodotoxin, 300 nM omega-conotoxin GVIA (an inhibitor of N-type Ca2+ channels) and 10 microM indomethacin (an inhibitor of cyclooxygenases) markedly inhibited 100 nM urotensin II-induced long-lasting contractions. The addition of 1 microM prostaglandin F2alpha (PGF2alpha) caused a limited brief contraction following long-lasting contraction, while 1 microM PGE2 induced marked biphasic contractions. Treatment with neurotoxins inhibited the long-lasting contractions induced by PGF2alpha and PGE2 without changing the PGE2-induced brief contractions. Treatment with 1 microM atropine markedly inhibited the urotensin II- and PGF2alpha-induced long-lasting contractions, but was less effective on the PGE2 responses. Treatment with a phospholipase A2 inhibitor decreased the urotensin II-induced contractions. These findings suggest that urotensin II induces, at least partially, long-lasting contractions via PG-sensitive cholinergic neurons and muscarinic acetylcholine receptors in the ileum.     

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 characterized 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 contractions 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 microM), LT antagonist, FPL55712 (10 microM), atropine (1 microM), phenoxybenzamine (10 microM), and LTB4 (10 microM) failed to alter LTC4 and D4 concentration-response curves. The results indicate 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.     

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

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)     

Spasmogenic effect of platelet activating factor (PAF) on isolated rat stomach fundus strip

Platelet activating Factor (PAF) produced an increase in resting tension of isolated rat stomach fundus strips. The spasmogenic effect of a 90 nM dose was equivalent to the contraction to 110 nM acetylcholine (ACh). Tissues exposed once to PAF became refractory to re-challenge with a dose of PAF normally producing maximum contraction (desensitization). PAF desensitized tissues remained responsive to the contraction effects of ACh and KCl (80 mM). Lyso-PAF failed to produce any effect. PAF contraction was dose-dependently antagonized by pretreatment of tissues with the PAF receptor antagonist L-652,731. PAF contractions were not blocked by antagonists of cholinergic, adrenergic, histaminergic, and serotonergic receptors, nor by inhibition of cyclooxygenase. PAF is a potent spasmogen on the isolated rat stomach fundus strip, and this effect is PAF and PAF-receptor specific.     

Pharmacological characterization using selected antagonists of the leukotriene receptors mediating contraction of guinea-pig trachea

The effects of six leukotriene (LT) antagonists on LTC4-, D4- and E4-induced contraction of isolated guinea-pig tracheal spirals were examined. Concentration-response effects of the leukotrienes were determined by cumulative addition in the presence of indomethacin (5 microM) alone for LTE4, or with 10 mM of either glutathione or L-cysteine to inhibit metabolism of LTC4 or LTD4, respectively. Concentration-response curves to the LTs were obtained in the absence and presence of Wy-45,911, Wy-44,329, FPL-55,712, Ly-171,883, Wy-48,252 and ICI-198,615 representing three structurally different chemical groups of LT antagonists. At 30 microM, the antagonists produced little or no antagonism of LTC4-induced contractions. Analysis of the Schild plots for antagonism of LTD4 and E4 suggested two receptors for the agonist effects of LTD4 and a single receptor for the agonist effects of LTE4. Comparison of pA2 values for Wy-45,911, FPL-55,712, LY-171,883 and Wy-48,252 provided evidence that LTE4 is acting at the antagonist high affinity LTD4 receptor to produce contractile effects. From the data, we conclude that there are three LT receptors (one for LTC4 and two LTD4 subtypes) through which exogenously applied LTs evoke contraction of the isolated guinea-pig trachea.     

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 were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to inhibit metabolic conversion of the leukotrienes. NDGA (30 microM) and ETYA (100 microM) 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 microM) 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.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1-100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC50 was 1 nM. The response to oxytocin (0.01-10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desensitization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1–100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC₅₀ was 1 nM. The response to oxytocin (0.01–10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desentization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

The effect of synthetic leukotrienes on tracheal microvascular permeability

The effect of synthetic leukotrienes (LT) C4, D4 and E4 on the permeability of the airway microvasculature to plasma albumin was quantitatively evaluated using an in situ guinea pig tracheal model. Vascular permeability was measured as extravascular albumin content by employing 125I-bovine serum albumin and, in order to correct for blood volume, 51Cr-erythrocytes were used. Intratracheal injection of synthetic LTC4, LTD4 and LTE4 (0.1-1000 ng) produced dose-dependent increases in tracheal extravascular albumin content. The leukotrienes were approximately 100-1000 fold more potent than histamine, although histamine did produce a greater maximal increase in extravascular albumin than the leukotrienes. Methacholine did not increase extravascular albumin content. The microvascular permeability effect of LTD4 was antagonized by FPL 55712 but not by mepyramine; conversely, the effect of histamine was antagonized by mepyramine and not by FPL 55712. Additionally, indomethacin did not alter the LTD4-induced increases in tracheal vascular permeability. These results suggest that the effect of LTD4 on tracheal microvascular permeability is directly mediated and is not the indirect result of cholinergic stimulation, histamine release or de novo synthesis of cyclooxygenase products.     

U19052 (ICIAm): a novel leukotriene analog which antagonizes LTC4, LTD4, and LTE4

Chemically stable analogs of peptide leukotrienes (LT) have been developed in our laboratories by replacement of the natural triene backbone with a C7H15 substituted aromatic moiety (1). These analogs are potent agonists of airway smooth muscle. Substitution in the peptide region resulted in U19052, an LT receptor antagonist. U19052 antagonized LT-induced contractions of guinea-pig tracheal spirals in a concentration-related manner. The pA2 values versus LTD4 and LTE4 were 6.0 and 5.7, respectively, with slopes which were not significantly different from unity. LTC4-induced contractions were antagonized by U19052 with a pKB of 5.6 obtained either in the absence or presence of L-serine borate. In contrast, carbachol and histamine concentration-response curves were not altered by U19052. LTD4 or LTE4 contractions of isolated guinea-pig ileum were antagonized by U19052 with pKB values of 7.2. The results indicate that potent selective LT antagonists can be developed from stable analogs of leukotrienes. U19052, an example of this series, appears to be as effective in antagonizing LTC4- as well as LD4- and LTE4-induced contractions in guinea-pig tracheal spirals.     

Peptidoleukotrienes induce an endothelium-dependent relaxation of guinea pig main pulmonary artery and thoracic aorta

The purpose of our investigation was to assess the role of the endothelium in the vasomotor effects of leukotrienes. Norepinephrine-preconstricted rings isolated from guinea pig main pulmonary artery and thoracic aorta responded to LTC4 and LTD4 with a concentration-dependent relaxation. In endothelium-denuded rings, both LTC4 and LTD4 caused a concentration-dependent contraction. The LTD4 receptor antagonist ICI 198,615 inhibited both LTC4- and LTD4-induced relaxation and contraction. Inhibition of gamma-glutamyl transpeptidase with AT-125 prevented the effects of LTC4, but not those of LTD4. The relaxant effect of LTD4 was not modified by indomethacin, but was abolished by methylene blue. We conclude that: 1) LTD4 induces a receptor-mediated endothelium-dependent relaxation of cavian pulmonary artery and aorta; 2) the vasorelaxant effect of LTC4 requires its conversion to LTD4; 3) the vasorelaxant effect of LTD4 is unrelated to PGI2 release, and is probably due to the release of an "EDRF"; 4) the removal of the endothelium reveals a direct receptor-mediated vasoconstricting effect of leukotrienes.