Abstract
We recently identified a novel pathway for the metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by human neutrophils, resulting in oxidation of the 5-hydroxyl group to give 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) (Powell, W. S., Gravelle, F., and Gravel, S. (1992) J. Biol. Chem. 267, 19233-19241). This pathway is quite specific for 5-HETE and other eicosanoids containing a 5(S)-hydroxyl group followed by a 6-trans double bond. In the present study we have shown that 5-oxo-ETE is very potent in raising cytosolic calcium levels in human neutrophils. This effect was reproducibly observed at concentrations as low as 0.3 nM, and the EC50 was found to be 2 nM. The mechanism of action of 5-oxo-ETE on neutrophils appeared to be distinct from that of leukotriene B4 (LTB4), since it was not blocked by the LTB4 antagonist LY255283 at a concentration which completely prevented the response to LTB4. As would be expected for a receptor-mediated mechanism, the response to 5-oxo-ETE was subject to homologous desensitization and was completely abolished by prior treatment of neutrophils with 5-oxo-ETE (100 nM) but was not affected by pretreatment of these cells with the same concentration of LTB4. 5-Oxo-15(S)-hydroxy-6,8,11,13- eicosatetraenoic acid (5-oxo-15-hydroxy-ETE), formed from 5(S),15(S)-dihydroxy-6,8,11,13- eicosatetraenoic acid (5,15-di-HETE) by the pathway responsible for the formation of 5-oxo-ETE, also raised cytosolic calcium levels in human neutrophils, with an EC50 of about 15 nM. 5-HETE, the precursor of 5-oxo-ETE, also had this effect but was about 100 times less potent than the latter substance. Desensitization experiments indicated that both 5-oxo-15-hydroxy-ETE and 5-HETE act by a mechanism similar to that of 5-oxo-ETE, but different from that of LTB4. In addition to their effects on calcium levels, both 5-oxo-ETE and 5-oxo-15-hydroxy-ETE had chemotactic effects on human neutrophils. Related eicosanoids, including 15-oxo-5,8,11,13-eicosatetraenoic acid, 5,15-diHETE, and 5(S)-hydroxy-15-oxo-6,8,11,13-eicosatetraenoic acid were much less potent, as both chemotactic and calcium-mobilizing agents. These results suggest that neutrophils possess a specific recognition mechanism for 5-oxo-ETE, which may be an important regulator of the activity of neutrophils, especially if they become desensitized to LTB4.
Highlights
We recently identified a novel pathway for the me- a specificrecognitionmechanismfo5r -oxo-ETE, tabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic which may bean important regulatoorf the activityof acid (5-HETE) by human neutrophils, resulting in ox- neutrophils, especially if they become desensitized to idation of the5-hydroxygl rouptogive
Stimulation of human neutrophils leads to the release of arachidonic acid and the formation of two major products, leukotriene B4 (LTB4)l and5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) [1, 2]
The mechanism of action in biological activity [5]. 5(S)-HETE is a substrate for of 5-oxo-ETE on neutrophils appeared to be distinct the 20-hydroxylase [6], but, unlike LTB4i,s metabolized from that of leukotriene B4 (LTB4), since it was not to 5-oxo-6,8,11,14-eicosatetraenoiaccid (5-oxo-ETE) by anblocked by the LTB4 antagonist LY255283 at a con- other pathway which we have recently identified in human centration whichcompletely prevented the response to polymorphonuclear leukocytes ( 7 ) .This reaction iscatalyzed
Summary
Dihydroxy-6,8,11,13-eicosatetraenoicacid(5,15-diHETE) by the pathway responsible for the formation of 5-oxo-ETE, raised cytosolic calcium levels in human neutrophils, with an ECBoof about 15 nM. HETE, the precursoorf 5-oxo-ETE, had this effecmtetabolite of 6-trans-LTB4(8),which appeared tobe formed but was about 100 times less potent than the latter via a 5-0XO intermediate [9] It was not clear why a pathway substance. Desensitization experiments indicated that shouldexist for the metabolism of the nonenzymatically both5-oxo-15-hydroxy-ETEand5-HETEact by a formed6-trans isomers of LTB,, buttheidentification of mechanism similar to thaotf 5-oxo-ETE, but different 5(S)-HETE as thmeajor substrate suggested that metabolism from that of LTB4 In addition to their effects on cal- of this compound to products with greater leosrser biological cium levels, both 5-oxo-ETE and 5-oxo-15-hydroxy- activities could be its main function. The neutrophil counts were averaged for the five fields.Neutrophil chemotaxis was expressed as the leukotactic index, which was determined by adding the products of the numbers of cells a t each level times the distance from the top of the filter and dividing by the total number of cells in the filter, excluding the top layer [25]
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