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.     

Contractile activities of leukotrienes C4 and D4 on vascular strips from rabbits

Leukotrienes C₄ (LTC₄) and D₄ (LTD₄), major components of slow-reacting substances of anaphylaxis (SRS-A), caused dose-dependent contractions of rabbit coronary arteries in concentrations of 10^?9 to 10^?7 M and 10^?10 to 10^?7 M, respectively. The potency of LTC₄ and LTD₄, when compared with the concentration that elicits half of the contraction induced by 25 mM KCl, was 17 and 76 times, respectively, greater than that of histamine. In contrast, other blood vessels from rabbits were either unresponsive (renal artery and vein, mesenteric artery and thoracic aorta) or only weakly responsive (pulmonary artery and vein and portal vein) to both leukotrienes even at 10^?7 M. The LTD₄-induced coronary contraction was inhibited by FPL 55712 (10^?7 and 10^?6 M), a selective SRS-A inhibitor, in a dose-dependent manner, but not by diphenhydramine (10^?7 M), a histamine H₁-receptor blocker or by indomethacin (10^?5 M), a prostaglandin synthetase inhibitor, suggesting that LTD₄ has a direct effect on the coronary arteries. These results indicate that the leukotrienes may act as potent, selective coronary vasoconstrictors and that SRS-A responsive receptors exist in the rabbit coronary artery.     

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.     

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.     

Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa

The effects of leukotrienes C₄ (LTC₄) and D₄ (LTD₄) on the secretion by human bronchial mucosa of [¹⁴C]glucosamine-labeled, trichloro-acetic acid/phosphotungstic acid-precipitable glycoprotein and lysozyme were evaluated . LTC₄ and LTD₄, in the concentration range of 0.16 to 1600 nM, induced a dose-related increase in the release of radiolabeled glycoprotein, but not of lysozyme. This secretagogue effect was selective for high molecular weight glycoproteins of about 2–5 × 10⁶ daltons, and the median effective concentrations (EC₅₀ of LTC₄ of 9.4 × 10⁻⁹ M and of LTD₄ of 2.44 × 10⁻⁸ M, indicate that these leukotrienes are approximately 100-fold more potent than the cholinergic agonist methacholine. Incubation of [¹⁴C]glucosamine-labeled bronchial mucosal explants with LTC₄ or LTD₄ for six sequential 15-min periods revealed a rapid, progressive decrement in glycoprotein release, compatible with stimulatory action on secretion rather than augmentation of the rate of glycoprotein synthesis. This interpretation is also consistent with the finding that the specific activity (ratio of bound radiolabel: protein content) of the macromolecular glycoprotein secreted by the explants is not changed with stimulation of release by the leukotrienes. Based upon the activity of synthetic leukotriene analogs, the specific C-6 chirality of the sulfidopeptide of LTD₄, the presence of a hydroxyl at C-5 and the presence of eiconsanoid carbons 9–20 were no importance for secretagogue activity. These findings contrast with the stereochemical requirements for the spasmogenic response to sulfidopeptide leukotrienes and suggest that leukotriene-induced secretion is not likely to be mediated via a specific receptor.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C₄ and D₄ in isolated airway smooth muscle was investigated. In rat trachea, neither LTC₄ or D₄ elicited a response. In contrast, LTC₄ was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD₄ in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC₄ was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD₄ in both trachea and parenchyma. As regards LTC₄-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC₄ in parenchyma.     

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.     

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.     

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.     

Pharmacologic characterization of the contractile activity of peptide leukotrienes in guinea-pig pulmonary artery

The actions of the peptide leukotriene (LT) LTC₄, LTD₄ and LTE₄ and phenylephrine (PE) were studied in isolated left branches of the guiena-pig pulmonary artery (GPPA). Indomethacin 5 × 10⁻⁶ M enhanced both the potency and maximal response of all agonists, but the effect on LTD₄ and LTE₄ was larger. The influence of indomethacin suggests the release of an endogenous vasodilating cyclooxygenase product in GPPA. In the pressence of indomethacin the rank-order of potency was LTC₄ > LTD₄ > LTE₄ ≥ PE with respective pD₂ vaues of 7.65, 7.39, 6.35 and 6.26. All further studies were carried out in the presence of 5 × 10⁻⁶ indomethacin. Removal of the endothelium further increased both potency (> 3-fold) and the maximal response of all agonists tested, indicating that a non-clycooxygenase endothelium-dependent relaxing factor may be present in GPPA. In separate studies, GPPA was demonstrated capable of metabolizing ³H-LTC₄ by an L-serine borate inhibitable γ-glutamyl transpeptidase. In contrast, relatively little formation of ³H-LTE₄ was apparent either from ³H-LTC₄ or ³H-LTD₄. The LTD₄-selective antagonists, LY 171,883 and ICI 198,615 had -log molar K_B values of 6.07 ± 0.14 and 9.38 ± 0.32, respectively, against LTD₄ in the absence of endothelium. The ability of LY 171,883 to antagonize LTC₄ was eliminated in the presence of 45 mM serine borate in endothelium denuded tissues. LT receptors in GPPA appear to be heterogeneous and similar to guinea pig ariway receptors.     

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.     

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.     

The modulatory effects of leukotrienes C4 and D4 on methacholine- and substance P-induced salivary secretion in rats

Although certain prostaglandins have been found to be inhibitory to nerve-evoked salivary flow, little is known of the effects the leukotrienes on salivary secretion. It was the purpose of this investigation to examine the effects of leukotrienes C₄ (LTC₄) and D₄ (LtD₄) on salivary secretion in the rat, using methacholine or substance P to induce basal secretion, and to test whether or not the observed effects of these eicosanoids were receptor-mediated by using the leukotriene receptor blocker FPL-55712.Methacholine (3 × 10⁻⁴ M), or substance P (1 × 10⁻⁶ M) was infused intra-arterially to stimulate secretion and saliva was collected separately from the parotid gland and the submandibular gland of anesthetized rats. LTC₄ and LTD₄ (each at 1 × 10⁻⁹ to 1 × 10⁻⁶ M) were found to reduce methacholine- and substance P-induced salivary flow in a dose-related manner. Salivary protein concentration and amylase activity were not significantly altered by the leukotrienes; however, arginine-esterase activity, stimulated by substance P, was increased by both leukotrienes. FPL-55712 (1 × 10⁻⁸ M) was shown to reduced the inhibitory effects of LTC₄ and LTD₄, suggesting the involvement of leukotriene receptors for these agents in their action.     

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.     

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.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: Release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D₄ (LTD₄) or leukotriene C₄ (LTC₄) resulted in concentration dependent increases in prostacyclin release measured as 6-ketoprostaglandin F_1α. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B₄ and leukotriene E₄ (LTE₄) were inactive at con- centrations up to 10 μM. The induction of prostacyclin synthesis by LTC₄ and LTD₄ was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23107 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

Formation of cysteinyl-containing leukotrienes by human arterial tissues

Human arterial rings incubated in modified Tyrode solution released small amounts of leukotriene (LT) C₄-like material spontaneously and larger amounts upon stimulation with the ionophore A23187 as determined by radioimmunoassay. By reversed phase high pressure liquid chromatography (HPLC) LTC₄-like material was found to comigrate with authentic LTC₄, LTD₄ and LTE₄. Nordihydroguaiaretic acid (NDGA) significantly inhibited the ionophore A23187-induced release of LTC₄-like material, while indomethacin was without effect. Simultaneously the arterial rings released much larger amounts of 6-keto-prostaglandin (PG) F_1α, which were significantly decreased by indomethacin. The results demonstrate that human arterial tissue has the capacity to synthesize cysteinyl-containing LT from endogenous arachidonic acid.     

Species difference in increased vascular permeability by synthetic leukotriene C4 and D4

The activity of synthetic LTC₄ was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 × 10^?11 M- 3 × 10^?9 M). The activities of LTC₄ and LTD₄ in increased vascular permeability in guinea pigs, rats and rabbits were compared with those histamine, bradykinin and prostaglandin (PG) E₂. LTC₄ was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5–100 times more potent than histamin. LTD₄ was about 10 times more potent than LTC₄ in guinea pigs and as equipotent to LTC₄ in rats. On the contrary, in rabbits, neither LTC₄ (upto 30 nmole/site) nor LTD₄ (1 nmole/site) induced the dye exduation. These results show that species difference is present in activity of LTC₄ and LTD₄ in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC₄ was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC₄ and LTD₄ did not potenciate the activity of bradykinin in vascular permeability.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contraction elicited by leukotriene (LT) C₄ and D₄ on isolated guinea pig trachea were characterized under conditions in which LTC₄ to LTD ₄ metabolism was blocked by presence of 45 mM ?-serine-borate complex (SB). The presence of Sb caused a shift of the LTC₄-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC₄ to LTD₄ by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC₄-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD₄-induced contractions. In contrast, another LT antagonist, Sk&F 101132, equally antagonized the contractions elicited by LTC₄ and LTD₄ in either the presence or absence of SB. The differential antagonism of LTC₄ and LTD₄ implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 μM, suppressed the maximal LTC₄-induced contraction by no more than 20%. whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC₄ concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD₄. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

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.