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.     

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.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A₄, B₄, C₄, D₄ and E₄ have potent myotropic activity on guinea-pig lung parenchymal strip . The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB₄, the tissues became desensitized to LTB₄ but were still responsive to histamine, LTA₄, LTC₄, LTD₄ and LTE₄. When LTD₄ was infused continuously, the lung strips contracted to LTB₄ and histamine but were no longer responsive to LTA₄, LTC₄, LTD₄ and LTE₄. Furthermore, FPL-55712 (10 ng ml⁻¹− 10 ug ml⁻¹) produced dose-dependent inhibitions of LTA₄, LTC₄, LTD₄ and LTE₄ without inhibiting the contraction to LTB₄ and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB₄ and another for LTD₄; LTA₄, LTC₄ and LTE₄ probably act on the LTD₄ receptor.     

The effect of leukotrienes C4 and D4 on the microvasculature of guinea-pig skin

The effects of chemically-synthesised leukotrienes C₄ and D₄ (5(S) hydroxy-6(R)-δ-glutamylcysteinylglycinyl-7,9,11,14-eicosa-⁴tetraenoic acid, LTC₄; 5(S) hydroxy-6(R)-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, LTD₄) on the microvasculature have been measured in guinea-pig skin using [¹²⁵I]-albumin accumulation to measure plasma exudation and ¹³³Xe clearance to measure blood flow changes. As previously shown using biosynthetic material, LTD₄ caused vasoconstriction resulting in reduced blood flow. Similarly, LTC₄ was found to have vasoconstrictor activity but was more potent and had a steeper dose-response curve than LTD₄. There was no evidence of conversion of exogenous arachidonic acid to vaso-constrictor activity in the skin $\text{in vivo}$ (in the absence of another stimulus): intradermally injected arachidonic acid produced vasodilatation, but induced little change in blood flow in animals pretreated with indomethacin. The vasodilator effect of arachidonic acid is presumed to be due to conversion to either PGE₂ or PGI₂. These results suggest that cyclo-oxygenase is normally active in the skin, whilst lipoxygenase requires activation in some way. As reported in a previous study, LTD₄ induced plasma exudation when injected into the skin, but pronounced responses could only be induced by LTD₄ mixed with a vasodilator prostaglandin such as PGE₂. In contrast, LTC₄ induced no exudation when tested alone and little when PGE₂ was added. However, evidence was obtained that LTC₄ has some permeability-increasing activity which is marked by its potent vasoconstrictor activity.     

Pharmacological study of the effects of leukotrienes C4, D4, E4 & F4 on guinea pig trachelis: Interaction with FPL-55712

Leukotrienes D₄ ? C₄ > E₄ ? F₄ produced qualitatively similar contractions of guinea-pig trachealis, which were antagonized by the SRS-antagonist FPL-55712. Schild analyses indicated that FPL-55712 when tested in a low concentration range (0.57–5.7 × 10^?6M) competitive antagonist of LTC₄, LTE₄ and LTF₄ (slope not significantly different from one). The interaction of FPL-55712 with LTD₄ may be noncompetitive (slope < 1). Comparison of the calculated dissociation constants (?log K_B) indicated that FPL-55712 was more effective at blocking LTE₄ and LTF₄ compared to LTC₄ and LTD₄. In the presence of higher concentrations of FPL-55712 (1.9 × 10^?5M) the antagonism of LTC₄ became noncompetitive. These findings indicate that important differences exist in the interaction of FPL-55712 with the various peptido leukotrienes in guinea pig trachealis. Discovery of more selective antagonists will be needed to determine if multiple receptor subtypes are present in this tissue.     

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).     

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.     

Positive interaction between leukotriene C4 and histamine and other mediators on vascular tissues

The interaction of leukotriene C₄ (LTC₄) with the contractile activity of histamine (H), serotonin (5HT) and norepinephrine (NE) has been investigated in isolated vascular preparations. Threshold concentration of LTC₄ (5 × 10⁻⁹ M) significantly potentiated the vasoconstricting effect of these compounds on guinea-pig pulmonary artery (GPPA). This phenomenon was long-lasting for H since it was still present 40 min after LTC₄ had been washed. FPL-55712 (10⁻⁵M) counteracted the increased H response on GPPA induced by LTC₄. Potentiation of H activity due to LTC₄ was also observed on guinea-pig thoracic aorta (GPTA) indicating that LTC₄-induced hyperreactivity is not a phenomenon restricted to the pulmonary vascular bed. In the experiments carried out in presence of indomethacin (3 × 10⁻⁶M), LTC₄ still potentiated H-induced vasoconstriction on GPPA, however the time course of the phenomenon was significantly shorter than that observed in absence of the cyclooxygenase inhibitor. The contractile activity of H and NE on guinea-pig portal vein (GPPV) was not potentiated by LTC₄ These results demonstrate that LTC₄ induces hyperreactivity of the arterial vascular tissue to vasoactive compounds and suggest that cysteinyl-leukotrienes may have pathological significance in the hemodynamic changes occurring during anaphylactic reactions. Preliminary experiments carried out on human intralobar pulmonary artery strongly support this hypothesis.     

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.     

Leukotrienes C4 and D4 psoriatic skin lesions

Chemoattractant arachidonate lipoxygenase products have been recovered from the skin lesions of psoriasis, and may play a role in eliciting the intra-epidermal neutrophil infiltrate that characterises this disease. In view of evidence for lipoxygenase activity in psoriasis, the characteristic vasolidation in psoriatic lesions, and the vasodilator properties of leukotriene (LT) C₄ and D₄ in human skin, the presence of these LTs in psoriatic lesions has been investigated. Skin chamber fluid from abraded psoriatic lesions contained significantly greater amounts of immunoreactive material than that from clinically normal skin, as determined by a double antibody radioimmunoassay (RIA) that uses antiserum cross-reacting with both LTC₄ and LTD₄. Purification of lesional chamber fluid and scale extracts by high performance liquid chromatography (HPLC) and RIA of fractions showed immunoreactivity which co-eluted with standard LTC₄ and LTD₄. These findings suggest that LTC₄ and LTD₄ may play a role in mediating the vasodilation and increased blood flow that characterise psoriatic skin lesions.     

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.     

Pharmacological comparison of L-serine borate and glutathione as inhibitors of metabolism of LTC4 to LTD4 by the isolated guinea pig trachea

Cumulative dose-response curyes to leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD)₄ were obtained on indomethacin (5 μM) treated isolated guinea pig tracheal spiral strips. LTC₄ curves, in the presence of either glutathione (GSH; 10 mM) or L-serine borate (SB; 45 mM), were not antagonized by FPL-55712 (3 μM), a selective LTD₄ receptor antagonist. LTC₄ curves on trachea treated with a lower concentration of GSH (1 mM), and LTD₄ curves were competitively antagonized by FPL-55712. LTC, curves on GSH (10 mM) treated trachea were 2 fold to the left of those on SB treated tissues. This effect of GSH was blocked by pretreatment with nordihydro-guiaretic acid (30 μM), an inhibitor of 5-lipoxygenase.GSH (10 μM) and SB (45 mM) are effective inhibitors of conversion of LTC₄ into functionally important levels of LTD₄ by the guinea pig trachea. In addition, GSH appeares to enhance LTC₄ responsiveness by increasing synthesis of a contractile 5-lipoxygenase product(s), possibly LTC₄. From the data it is suggested that for inhibition of LTC₄ metabolism, SB may be more usefull when examining responses to exogenously applied LTC₄, while GSH (10 mM) may be useful when examining responses to endogenously generated 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 μg) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC₄ 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.     

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.     

Bioconversion of leukotriene C4 by rat glomeruli and papilla

Since leukotriene C₄ (LTC₄) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added |³H| LTC₄ by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted |³H| LTC₄ mainly into |³h| LTE₄ (83%) and, at a smaller extent, into |³H| LTD₄ (4%). Intact |³H| LTC₄ represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD₄. In contrast, there was nearly no conversion of |³H| LTC₄ (87% ntact) in the presence of homogenized papilla. The metabolism of |³H| LTC₄ by the glomeruli was time- and temperature- dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize |³H| LTC₄. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform |³H| LTC₄ into its metabolites, LTD₄ and LTE₄. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected |³H| LTC₄ from its bioconversion. These results demonstrate that both glomeruli and papilla possess the γ-glutamyl transpeptidase and dipeptidase necessary to process LTC₄. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.     

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.     

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.     

Possible involvement of thromboxane in bronchoconstrictive and hypertensive effects of LTC4 and LTD4 in guinea pigs

The actions of leukotriene (LT) C₄ and D₄ on the systemic arterial pressure and the insufflation pressure in guinea pigs and rabbits were examined. In guinea pigs, 0.3 – 3 nmole/kg of LTC₄ and 0.1 – 1.0 nmole/kg of LTD₄ administrated from left jugular vein caused dose-dependent increase of the airway resistance measured by the Konzett-Rössler method and a triphasic blood pressure response; an initial hypotension, a secondary hypertension and a third long-lasting hypotension. All of the hypertensive phase and 100 – 150% of the increase of the airway resistance by LTC₄ and LTD₄ were inhibited by a selective thromboxane synthetase inhibitor, OKY-1581 (10 mg/kg, i.v.) and only the hypertension was observed. Indomethacin (10 mg/kg, i.p.) also inhibited not only the airway resistance increase, but also the prolonged hypotension by LTC₄ and shortened the duration of the hypotension by LTD₄. It is suggested that thromboxane might be involved in bronchoconstriction and hypertensive effects by LTC₄ and LTD₄ and that hypotensive prostaglandin might be involved in the hypotensive phase after LTC₄ and LTD₄. In rabbits, the increse of the airway resistance by LTC₄ and LTD₄ (upto 100 nmole/kg, i.v.) was negligible and only the hypotension was observed.     

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

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

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.