TY - JOUR
T1 - COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes
AU - Tejera, Noemi
AU - Boeglin, William E.
AU - Suzuki, Takashi
AU - Schneider, Claus
PY - 2012/1
Y1 - 2012/1
N2 - Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-di-HETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5 S -HETE as a substrate for COX-2 forming 5 S,15 S -diHETE, 5 S,15 R -diHETE, and 5 S,11 R -di-HETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/106 cells (mean ± SEM, n = 6), reaching about half the level of LTB 4 (1.3 ± 0.5 ng/106 cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/106 cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/106 cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5 S,12 S -diHETE with the unusual 6 E,8 Z,10 E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A 4 -epoxide. Exogenous octadeuterated 5 S -HETE and 15 S -HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.
AB - Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-di-HETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5 S -HETE as a substrate for COX-2 forming 5 S,15 S -diHETE, 5 S,15 R -diHETE, and 5 S,11 R -di-HETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/106 cells (mean ± SEM, n = 6), reaching about half the level of LTB 4 (1.3 ± 0.5 ng/106 cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/106 cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/106 cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5 S,12 S -diHETE with the unusual 6 E,8 Z,10 E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A 4 -epoxide. Exogenous octadeuterated 5 S -HETE and 15 S -HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.
KW - 15-lipoxygenase
KW - 5-lipoxygenase
KW - Aspirin
KW - Leukotriene
KW - Prostaglandin
UR - http://www.scopus.com/inward/record.url?scp=84455192397&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84455192397&partnerID=8YFLogxK
U2 - 10.1194/jlr.M017822
DO - 10.1194/jlr.M017822
M3 - Article
C2 - 22068350
AN - SCOPUS:84455192397
SN - 0022-2275
VL - 53
SP - 87
EP - 94
JO - Journal of lipid research
JF - Journal of lipid research
IS - 1
ER -