Some newly synthesized leukotriene B4 analogs inhibit LTB4-induced lysozyme release from rat polymorphonuclear leukocytes

Antagonistic activities of newly synthesized LTB4 analogs, which possess the same olefinic geometry and the same hydroxyl group stereochemistry as natural LTB4, were investigated by studying lysozyme release from rat polymorphonuclear leukocytes (PMNLs). 14,15-Dihydro LTB4(LTB3) induced lysozyme release from PMNLs as well as LTB4, while 14,15-dehydro LTB4 did not cause lysozyme release but instead clearly inhibit LTB4-induced lysozyme release. Compounds containing aminocarbonyl groups partially retained lysozyme releasing activity. A displacement of the hydrocarbon chain at C13-20 by cyclohexenyl and beta-cyclohexylethyl groups had little effect on lysozyme release, but did strongly inhibit release induced by LTB4. These results may be useful in developing LTB4 antagonists.     

Inhibition of leukotriene B4-induced neutrophil degranulation by leukotriene B4-dimethylamide

Leukotriene B₄ (LTB₄) is a potent mediator of pro-inflammatory responses including neutrophil degranulation. Leukotriene B₄ dimethylamide has been synthesized and shown to inhibit neutrophil degranulation induced by LTB₄. The inhibition required time to develop (～60 secs), and had a K_D of circa 2 × 10^?7M, and occurred at concentrations where LTB₄ dimethylamide had negligible agonist activity.     

Bronchodilator drugs and their combinations antagonize the dose-related leukotriene D4-induced airway obstruction in guinea pigs

The effects of theophylline (THEO), terbutaline (TER), and ipratropium bromide (IPRA), given i.v. alone or in combination, were studied on leukotriene D4 (LTD4)-induced airway obstruction in anaesthetized guinea pigs. LTD4 (0.1-1.6 microgram/kg i.v.) obstructed small airways more than large ones as assessed in terms of relative changes of lung resistance (RL) and dynamic lung compliance (CDyn). A slight tachyphylaxis to LTD4 was observed after repeated administration, especially in the responses of large bronchi. The airway effects of LTD4 were almost totally abolished by prior administration of indomethacin (5 mg/kg i.v.) suggesting a central role of secondarily released cyclo-oxygenase products in this model. THEO (1 to 20 mg/kg) and TER (10 to 400 micrograms/kg) antagonized dose-dependently the LTD4 (0.4 microgram/kg i.v.) induced rise in RL and decrease in CDyn, whereas IPRA (10 to 400 micrograms/kg) failed to show comparably activity. THEO 5 and 20 mg/kg proved highly efficient also on the dose-related airway challenge by LTD4 (0.6 and 1.5 microgram/kg i.v.). Combined treatment with THEO 5 mg/kg + Ter 80 micrograms/kg resulted in an additive effect on RL and CDyn. The combination to THEO 20 mg/kg + TER 80 micrograms/kg was about as effective as THEO 20 mg/kg alone suggesting a nearly maximal effect by the latter treatment. It is concluded that THEO is considerably more efficient on the LTD4-induced airway obstruction than previously observed on the cholinergic model in guinea pigs. Combined treatment with THEO and beta 2-adrenoceptor agonist may antagonize in an additive manner the LTD4 effects on large and small airways.     

Electromechanical coupling of ferret airway smooth muscle in response to leukotriene C4

We investigated the possible electrophysiological basis for the slow, prolonged force generation by airway smooth muscle (ASM) produced by leukotriene C4 (LTC4). Preparations of ASM were made from ferret trachea and placed in tissue microchambers for study. Some of these preparations were arranged so that force transducers and intracellular microelectrodes (with tip resistances of 30-80 M omega) could be used to measure isometric force and cell membrane potential (Em) simultaneously from ASM cells stimulated by LTC4. We found that ferret tracheal muscle was relatively sensitive to LTC4 and that this sensitivity was not significantly affected by atropine (1 microM), phentolamine (1 microM), propranolol (3 microM), and pyrilamine (1 microM). In a 1 nM solution of LTC4, Em was -54.0 +/- 1.2 mV from 18 impalements (n) from 6 animals (N) compared with a base-line value of -61.6 +/- 0.8 mV (n/N = 29/8, P less than 0.0005). This change did not lead to force generation, however. Higher concentrations of LTC4 led to progressive decreases in Em to which force generation was closely coupled. Concentrations greater than or equal to 70 nM led to phasic oscillations in Em of 0.6-0.8 Hz and 1.7 mV in amplitude, which were abolished by 10 microM verapamil, although the base-line Em was unaffected by this concentration. Although 300 nM LTE4 by itself caused only a small depolarization of ferret trachealis, it substantially antagonized the electromechanical responsiveness of this smooth muscle to LTC4. We conclude that ferret ASM is relatively sensitive to LTC4 and that there is an electrical basis for the slow, prolonged force generation caused by this mediator.     

Pharmacology of aerosol leukotriene C4- and D4-induced alteration of pulmonary mechanics in anesthetized cynomolgus monkeys

Aerosol administration of solutions of 900 micrograms/ml of leukotriene C4 (LT) or D4 to cynomolgus monkeys produced dose-dependent, equipotent increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn). Time to peak response was, in part, related to dose and ranged from 4 to 20 min. Both LTC4 and LTD4 were less potent than histamine. Aerosol pretreatment with the cyclooxygenase inhibitor indomethacin had no significant effect on either LTC4 or LTD4 dose-response curves; however, at the highest doses of these agonists a notable, nonsignificant inhibition of effects on both Rp and Cdyn was seen. Intravenous dl-propranolol had no effect on responses to LTD4. Aerosol pretreatment with FPL 55712 significantly (P less than 0.05) inhibited airway responses to both LTC4 and D4. In contrast, an intravenous infusion of FPL 55712 failed to block the bronchospastic activity of LTD4. In conclusion, cynomolgus monkeys are responsive to aerosol administration of LTC4 and LTD4, and the pharmacology of their responses appears to resemble that of man.     

Pranlukast protects leukotriene C4- and D4-induced epithelial cell impairment of the nasal mucosa in vitro

To investigate the effect of pranlukast on leukotriene- induced airway mucosal epithelial dysfunction, samples of human nasal mucosa obtained during surgery for facial trauma were exposed to leukotriene C4 and/or D4 and observed on a TV screen magnified x 2,500. Leukotriene C4- and D4-induced ciliary inhibition and delayed mucosal surface alterations appeared several hours later. Pranlukast prevented both the mucosal epithelial cell dysfunction and the delayed epithelial cell alteration.     

Visualization and comparison of molecular dynamics simulations of leukotriene C4, leukotriene D4, and leukotriene E4

Molecular dynamics simulations of leukotriene C₄ (LTC₄), leukotriene D₄ (LTD₄), and leukotriene E₄ (LTE₄) were carried out, and the data were visualized in an animated video format. Three-dimensional ghost images show the positions of the heavy atoms of all three molecules throughout the simulations. The ghost images can be superimposed to give a single three-dimensional image in which the shapes of the most populated conformers of each molecule are apparent and can be compared. Leukotriene D₄ was found to occupy mostly T-shaped conformations, while LTC₄ occupied mostly cup-shaped conformations, and LTE₄ occupied a wide range of conformations spanning the LTD₄ and LTC₄ types. Digital filtering and graphing of the internal geometries of the molecules as a function of time revealed differences in dynamic behavior. The results are discussed in light of current knowledge about leukotriene receptors.     

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.     

Secondary release of thromboxane A2 in aerosol leukotriene C4-induced bronchoconstriction in guinea pigs

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01–1.0μg/ml of leukotriene C4 and 12.5–400μg/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0μg/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6, 7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole     

Secondary release of thromboxane A2 in aerosol leukotriene C4-induced bronchoconstriction in guinea pigs

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01-1.0 micrograms/ml of leukotriene C4 and 12.5-400 micrograms/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0 micrograms/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6,7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole.     

Behavioral and physiological effects of leukotriene C4

Leukotriene C4 (LTC4), a lipoxygenase metabolite of arachidonic acid, is a biological mediator of vasoregulation, pulmonary activity, shock, and inflammation, that has been demonstrated to have radioprotective efficacy. The effects of LTC4 on locomotor activity, rectal temperature and hematocrit were examined. Subcutaneous administration of doses of 1.0 micrograms LTC4/mouse or less did not affect locomotor activity. Doses of 5 or 10 micrograms LTC4/mouse, however, resulted in almost complete cessation of locomotion within 12-14 min following treatment. At these doses, activity was suppressed for 2 h with complete recovery by 3 h postinjection. While a dose as high as 10 micrograms LTC4 did not affect rectal temperature, 5 and 10 micrograms LTC4 resulted in hematocrit increases of 10% and 40% respectively. Hematocrit returned to baseline within 1 h after a 5 micrograms pretreatment of LTC4, and by 3 h following a 10 micrograms pretreatment. The duration of LTC4-induced locomotor suppression did not correlate with previously determined durations of LTC4-induced radioprotection.     

2-nor-leukotriene analogs: antagonists of the airway and vascular smooth muscle effects of leukotriene C4, D4 and E4

A structural analog of LTD4, 4R-hydroxy-5S-cysteinylglycyl-6Z-nonadecenoic acid (4R, 5S, 6Z-2-nor-LTD1) has been synthesized and pharmacologically characterized. It significantly antagonized the contractile action of LTD4, LTC4 and LTE4 in guinea pig airways. In addition, this compound antagonized the in vitro vasoconstrictive effects of LTD4 in the guinea pig pulmonary artery. The study of a series of structural analogs of 4R, 5S, 6Z-2-nor-LTD1 suggests that the spatial separation of the C-1 (eicosanoid) carboxyl relative to the hydroxyl is a critical determinant in LTD4 agonist/antagonist activity.     

Inhibitors of protein kinase C selectively enhanced leukotriene D4-induced calcium mobilization in differentiated U-937 cells

U-937 cells differentiated with dimethylsulphoxide for 3-4 days express receptors for leukotriene D4 (LTD4), which are coupled to Ca2+ mobilization and phosphatidylinositol (PI) metabolism. Treatment of U-937 cells with an inhibitor of protein kinase C (PKC) [staurosporine (100 nM)] augmented the Ca2+ mobilized by LTD4. The peak concentration of the LTD4-induced increase in [Ca2+]i was 1500 nM in untreated cells and 3000 nM in cells treated with staurosporine for 30 s. Maximal mobilization responses were observed at 1-10 microM LTD4 in both control and staurosporine-treated cells. The increased Ca2+ response to LTD4 after staurosporine treatment occurred within 30 s and was attributable to both intracellular and extracellular stores. Additionally, a second phase of Ca2+ mobilization occurred after stimulation with LTD4, which was elevated by pretreatment with staurosporine--this effect was maximal after 5-10 min of treatment. Staurosporine either had no effect or decreased the Ca2+ mobilization response of differentiated U-937 cells to other agonists, such as LTB4, platelet activating factor, ATP or the chemotactic peptide f-Met-Leu-Phe. Although staurosporine alone had no effect on basal PI metabolism it increased LTD4-induced PI metabolism. Staurosporine did not prevent the tachyphylaxis observed upon second challenge with LTD4, nor did it prevent LTD4-induced homologous densensitization. Other compounds which inhibit PKC (sphingosine and 1-O-hexadecyl-2-O-methylglycerol), also enhanced the Ca2+ response of U-937 cells to LTD4, but not to other agonists. These data show that inhibition of PKC enhanced responses of LTD4, suggesting that PKC plays a role in determining the responsiveness of LTD4 receptors.     

Propranolol potentiates leukotriene D4-induced bronchoconstriction and enhances antagonism by FPL 55712

Aerosol LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs was potentiated by either pretreatment with propranolol or bilateral adrenalectomy, whereas bilateral vagotomy did not affect the LTD4 response. The dose-response curve describing LTD4-induced changes in dynamic lung compliance (CDYN) and pulmonary resistance (RL) [as reflective indices of bronchoconstriction] was shifted to the left by approximately 20-fold by propranolol. Against an equal degree of LTD4-induced bronchoconstriction, the leukotriene antagonist, FPL 55712, had an apparent 20-fold greater potency in propranolol-pretreated animals vis a vis saline-treated controls. The duration of action of aerosol FPL 55712 was similar in both propranolol-treated and saline-treated animals. These results demonstrate that aerosol LTD4-induced bronchoconstriction is modulated by an adrenergic compensatory bronchodilator mechanism that is apparently dependent upon the adrenals and independent of vagal influences. Inhibition of the effect of this reflex with propranolol also enhances the apparent potency of an aerosol leukotriene antagonist, FPL 55712, presumably reflecting a constant LTD4 to antagonist ratio in the saline-treated and propranolol-pretreated guinea pigs.     

Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.     

Membrane localization and topology of leukotriene C4 synthase

Leukotriene C(4) (LTC(4)) synthase conjugates LTA(4) with GSH to form LTC(4). Determining the site of LTC(4) synthesis and the topology of LTC(4) synthase may uncover unappreciated intracellular roles for LTC(4), as well as how LTC(4) is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC(4) synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC(4) synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC(4) synthase. The active site of LTC(4) synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB(4) and LTC(4) occurs in different subcellular locations and suggests that LTC(4) must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane.     

Cyclooxygenase mediated airway response to leukotriene D4 in the cat

The effects of leukotriene D4 (LTD4) on pulmonary mechanics were investigated in anesthetized, paralyzed cats under conditions of controlled ventilation. Intravenous injections of LTD4 in doses of 3, 10, and 30 micrograms caused significant increases in transpulmonary pressure (PTP) and lung resistance (RL) while decreasing dynamic compliance (Cdyn). LTD4 also increased systemic arterial pressure (PAo). The changes in PTP, RL, and Cdyn in response to LTD4 were blocked by sodium meclofenamate, a cyclooxygenase inhibitor. However, there was no significant change in the increase in PAo following cyclooxygenase blockade. U 46619, a thromboxane mimic, was 30 to 100 times more potent than LTD4 in increasing PTP, RL and decreasing Cdyn in the cat. These data show that LTD4 has significant smooth muscle constrictor activity in central airways as well as peripheral portions of the feline lung. In addition, these data suggest that in the cat the actions of intravenously administered LTD4 on lung mechanics are mediated by release of cyclooxygenase products while the systemic pressor effects are not dependent upon the integrity of the cyclooxygenase pathway.