TY - JOUR
T1 - Characterization and inhibition of 15-lipoxygenase in human monocytes
T2 - Comparison with soybean 15-lipoxygenase
AU - Gleason, M. M.
AU - Rojas, C. J.
AU - Learn, K. S.
AU - Perrone, M. H.
AU - Bilder, G. E.
PY - 1995
Y1 - 1995
N2 - These studies characterize a method to measure 15-lipoxygenase (15-LO) activity in human monocytes (HMC) exposed to interleukin-4 (IL-4) and compare the activity with that of soybean 15-LO. 15-LO activity was quantitated by measuring 15-[14C]hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) production from the substrate [14C]arachidonic acid (AA) after high- performance liquid chromatographic or thin-layer chromatographic separation. 15-HETE production by HMC was significantly elevated after continuous exposure to a single dose (10 ng/ml) of IL-4 for 4 days, was maximal at 5 days, and remained elevated at 6 days. At 6 days 15-LO activity in IL-4- treated HMC was increased significantly (2.81 ± 0.70 fmol 15-HETE/cell) compared with background levels of 15-HETE in untreated HMC (0.098 ± 0.31 fmol 15-HETE/cell). 15-HETE production was linear in the range of 5 x 104 to 7 x 105 HMC/assay, from 2 to 160 μM AA, and during 5-10 min of incubation with AA. Ethyl 2-cyano-3-(3',4'-dihydroxyphenyl)propenoate (a caffeic acid analogue), N-methyl-4-benzyloxyphenyl acetohydroxamate (RG-6866), esculetin, and four novel lipoxygenase inhibitors, a phenylcyanomethylene analogue (RP- 27493) and three benzoxadiazine analogues (RP-64835, RP-65047, and RP- 65208), inhibited HMC 15-LO, with concentrations eliciting 50% of maximal inhibition (μM) of 0.21, 0.8, 6.3, 10, 22, 20, 6.3, 3, and 20 and 5.8, 5, 12, 1, 30, 0.8, 0.15, 0.05, and 2.8 for inhibition of soybean 15-LO, respectively. These data define conditions for optimal production of 15-HETE by IL-4-treated HMC. Known and novel lipoxygenase inhibitors inhibit IL-4- treated HMC 15-LO and soybean 15-LO, suggesting that IL-4-treated HMC provide an appropriate system to study human 15-LO activity.
AB - These studies characterize a method to measure 15-lipoxygenase (15-LO) activity in human monocytes (HMC) exposed to interleukin-4 (IL-4) and compare the activity with that of soybean 15-LO. 15-LO activity was quantitated by measuring 15-[14C]hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) production from the substrate [14C]arachidonic acid (AA) after high- performance liquid chromatographic or thin-layer chromatographic separation. 15-HETE production by HMC was significantly elevated after continuous exposure to a single dose (10 ng/ml) of IL-4 for 4 days, was maximal at 5 days, and remained elevated at 6 days. At 6 days 15-LO activity in IL-4- treated HMC was increased significantly (2.81 ± 0.70 fmol 15-HETE/cell) compared with background levels of 15-HETE in untreated HMC (0.098 ± 0.31 fmol 15-HETE/cell). 15-HETE production was linear in the range of 5 x 104 to 7 x 105 HMC/assay, from 2 to 160 μM AA, and during 5-10 min of incubation with AA. Ethyl 2-cyano-3-(3',4'-dihydroxyphenyl)propenoate (a caffeic acid analogue), N-methyl-4-benzyloxyphenyl acetohydroxamate (RG-6866), esculetin, and four novel lipoxygenase inhibitors, a phenylcyanomethylene analogue (RP- 27493) and three benzoxadiazine analogues (RP-64835, RP-65047, and RP- 65208), inhibited HMC 15-LO, with concentrations eliciting 50% of maximal inhibition (μM) of 0.21, 0.8, 6.3, 10, 22, 20, 6.3, 3, and 20 and 5.8, 5, 12, 1, 30, 0.8, 0.15, 0.05, and 2.8 for inhibition of soybean 15-LO, respectively. These data define conditions for optimal production of 15-HETE by IL-4-treated HMC. Known and novel lipoxygenase inhibitors inhibit IL-4- treated HMC 15-LO and soybean 15-LO, suggesting that IL-4-treated HMC provide an appropriate system to study human 15-LO activity.
KW - 15-HETE
KW - 15-lipoxygenase
KW - 5,8,11,14- eicosatetraynoic acid
KW - human monocytes
KW - interleukin-4
KW - nordihydroguaiaretic acid
UR - http://www.scopus.com/inward/record.url?scp=0029000373&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029000373&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.1995.268.5.c1301
DO - 10.1152/ajpcell.1995.268.5.c1301
M3 - Article
C2 - 7762624
AN - SCOPUS:0029000373
SN - 0363-6143
VL - 268
SP - C1301-C1307
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 5 37-5
ER -