Ca2+ mobilization with leukotriene A4 and epoxytetraenes in human neutrophils.
The biosynthesis of leukotrienes and lipoxins involves epoxide-containing intermediates which may be subject to several routes of transcellular metabolism. We have examined the capacity of leukotriene A4 (LTA4) and 15S-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid [5(6)-epoxytetraene] to stimulate the mobilization of free cytosolic calcium [( Ca2+]i) in human blood neutrophils. To gain insight into structure-activity relationships, a putative intermediate in lipoxin biosynthesis, 5S-hydroxy-14,15-oxido-6,10,12-trans-8-cis-eicosatetraenoic acid [14(15)-epoxytetraene], was prepared by total synthesis. When added to fura-2 loaded neutrophils, each of these compounds provoked a rapid and transient increase in [Ca2+]i (maximum by 8 sec) which returned to baseline within 60-90 sec. Ca2+ mobilization with LTA4 was dose dependent and, at 1 microM, the efficacies of LTA4 and LTB4 were quantitatively similar. The 5(6)-epoxytetraene and 14(15)-epoxytetraene were less potent than LTA4. Prior exposure of the cells to ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) (60 sec, 3 mM) did not diminish either the amplitude or the extent of [Ca2+]i elicited by LTA4. Methyl esters of LTA4 and 5(6)-epoxytetraene were less potent than their corresponding free acids, whereas the free acid of 14(15)-epoxytetraene and its methyl ester were quantitatively similar. Results from alcohol trapping studies showed that these epoxides were intact during the initial phase of Ca2+i mobilization (t0-10 sec) stimulated by LTA4, 5(6)-epoxytetraene, and 14(15)-epoxytetraene. In addition, the individual mixtures of products formed upon aqueous hydrolysis of each of the epoxides did not stimulate changes in [Ca2+]i. In each case, the products formed were identified by physical methods including reverse phase high pressure liquid chromatography, ultraviolet spectroscopy and gas liquid chromatography-mass spectrometry. These results indicate that, when added to human neutrophils, LTA4, 5(6)-epoxytetraene and 14(15)-epoxytetraene each stimulate a rapid mobilization of [Ca2+]i. Moreover, they suggest that intermediates in the biosynthesis of leukotrienes and lipoxins possess intrinsic activities that may serve to amplify cellular responses within their cell of origin or act on adjacent cells during their transcellular metabolism.
Luscinskas
F W
FW
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Nicolaou
K C
KC
Webber
S E
SE
Veale
C A
CA
Gimbrone
M A
MA
Serhan
C N
CN
D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D015777:Eicosanoids / Q000494:pharmacology; D000431:Ethanol; D016257:Fura-2; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D017572:Leukotriene A4; D007975:Leukotriene B4 / Q000494:pharmacology; D015289:Leukotrienes / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D011456:Prostaglandins B; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D001572:Benzofurans; D015777:Eicosanoids; D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D011456:Prostaglandins B; C062631:14(15)-epoxytetraene; D007975:Leukotriene B4; D000431:Ethanol; C050533:15-hydroxy-5,6-oxido-7,9,11,13-eicosatetraenoic acid; C042026:prostaglandin B2; D002118:Calcium; D016257:Fura-2