Abstract
Analysis of leukotriene B4 production by purified rat and human neutrophil leukotriene (LT) A4 hydrolases in the presence of 5(S)-trans-5,6-oxido-7,9-trans-11-cis-eicosatrienoic acid (leukotriene A3) demonstrated that this epoxide is a potent inhibitor of LTA4 hydrolase. Insignificant amounts of 5(S), 12(R)-dihydroxy-6-cis-8,10-trans-eicosatrienoic acid (leukotriene B3) were formed by incubation of rat neutrophils with leukotriene A3 or by the purified rat and human LTA4 hydrolases incubated with leukotriene A3. Leukotriene A3 was shown to be a potent inhibitor of leukotriene B4 production by rat neutrophils and also by purified rat and human LTA4 hydrolases. Covalent coupling of [3H]leukotriene A4 to both rat and human neutrophil LTA4 hydrolases was shown, and this coupling was inhibited by preincubation of the enzymes with leukotriene A4. Preincubation of rat neutrophils with leukotriene A3 also prevented labeling of LTA4 hydrolase by [3H]leukotriene A4. This result indicates that leukotriene A3 prevents covalent coupling of the substrate leukotriene A4 and inhibits the production of leukotriene B4 by blocking the binding of leukotriene A4 to the enzyme.
Highlights
Analysis of leukotriene B4production bypurified rat lase to date is thaot f Stenson et al [10] in which an unidenand human neutrophil leukotriene (LT) A4 hydrolases tified product of 5-lipoxygenase metabolism of 5,8,11-eicosain the presence of 5(S)-trans-5,6-oxido-7,9-trans-11tr-ienoate inhibited leukotrieneB4production in a 10,000 X g cis-eicosatrienoic acid demonstrated supernatant fractionof a homogenate of RBL-1 cells
Leukotriene AS was shown to be a potent inhibitor of leukotriene B4 production by rat neutrophils and by purified rat and human LTA, hydrolases
In this paper,we have showed that theepoxide leukotriene A3 is a verypoor substrate for LTA, hydrolase but is an excellent inhibitor of leukotriene B4 productioinn rat neutrophils and of leukotriene B4 production by purified rat and human LTA, hydrolases
Summary
Neutrophil cell suspensions (>85% polymorphonuclear leukocytes) were prepared in Eagle's minimal medium (GIBCO) buffered a t p H 7.4 with 30 mM Hepes from peritoneal exudates obtained 18-20 h. Suspensions were centrifuged a t 150 x g for 15 min, resuspended in Selective inhibition of leukocyte LTA, hydrolase' is of major theHepes-buffered medium, filteredthroughcoarselenspaper (Fisher), and recentrifuged a t 150 X g for 15 min. The final pellet importance in determining the contributionf leukotriene B4 was resuspended in the Hepes-buffered medium a t a concentration to the maintenanceof the inflammatory state in suchcondi- of 1X io' cells/ml. Drolase; HPLC, high performance liquid chromatography; SDS, so- leukotriene A, were analyzed by collecting 0.5-ml fractions and after dium dodecyl sulfateH; epes, 4-(2-hydroxyethyl)-l-piperazineeth- addition of 5 ml Aquasol (New England Nuclear) scintillation deteranesulfonic acid.
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