Characterization and inhibition of 15-lipoxygenase in human monocytes: comparison with soybean 15-lipoxygenase.
These studies characterized 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 10(4) to 7 x 10(5) HMC/assay, from 2 to 160 microM 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-benzyloxy-phenyl 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 (microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gleason
M M
MM
Rhône-Poulenc Rorer Central Research, Collegeville, Pennsylvania 19426, USA.
Rojas
C J
CJ
Learn
K S
KS
Perrone
M H
MH
Bilder
G E
GE
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D015847:Interleukin-4 / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors; D009000:Monocytes / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology*
D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; D015847:Interleukin-4; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001093:Arachidonate 15-Lipoxygenase