Abstract
Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.
Highlights
From the Department of Biochemistry, The George Washington University, Schoolof Medicine and Health Sciences, Washington, D.C. 20037
The lipoxygenase pathway initially forms15-HPETE which is nonsteroidal anti-inflammatory drug ibuprofen inhib- either reduced to 15-HETE or meited production of the cyclooxygenase product throm- tabolized to other products including those of the 15-series boxane B, (160 = 65 FM), whereas the lipoxygenase leukotrienes (8, 9). 15-HPETE has been reported to inhibit product 12-HETE was not appreciably affected even vascular prostacyclin synthesis (10) and platelet aggregation at 5 mM ibuprofen
The5-lipoxygenase of human (11).Other studies indicate that both 15-HPETE and 15
Summary
Lation of the enzyme, whereas aspirin andindometha- Blood was obtained from human donors who had not taken any cin resulted in an average 1.5- and 2-fold enhance- aspirin-like compounds during the preceding 2 weeks. After the addition of 1 ation of arachidonicacid via the cyclooxygenase pathway leads to prostaglandinst,hromboxanesa, ndprostacyclin, whereaslipoxygenase-catalyzed metabolism producespolyunsaturated hydroxy fatty acids and leukotrienes( 1 , Z ) These volume of ice-coldTris/EDTA/NaCl buffer, pH 7.4, the platelet pellet was isolated by centrifugation at 1500 X g for 10min at 4 “C.Platelets were resuspended in Krebs-Henseleit buffer at a concentration of 3.3 X 10’ cells/ml. ’The abbreviations used are: PMN, polymorphonuclear leukocytes; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid LT, leukotriene; NSAID, nonsteroidal anti-inflammatory drug; SP-HPLC, straight phase high pressure liquid chromatography; RIA, radioimmunoassay. The radioactive component of the glyceride and phospholipid fractions wasidentified as [14C]arachidonic acidby alkaline deacylation (28) Both the glyceride and phospholipid subclasses were separated by TLC in Solvent A and chloroform/methanol/concentrated ammonium hydroxide/water (6535:2.5:2.5), respectively.
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