Characterization and inhibition of 15-lipoxygenase in human monocytes;
comparison with soybean 15-lipoxygenase.
Gleason, Marie M., Camilo J. Rojas, Keith S. Learn, Mark H. Perrone, and
Glenda E. Bilder.
Marie M. Gleason, PhD., Cardiovascular Biology, NW4, Rhone-Poulenc Rorer
Central Research, 500 Arcola Road, Collegeville, PA USA 19426, telephone:
(610) 454-5091, fax: (610) 454-8740
APStracts 2:0039C, 1995.
These studies characterize a method to measure 15-lipoxygenase (15-LO)
activity in human monocytes (HMC) exposed to interleukin-4 (IL4) and compare
the activity to that of soybean 15-LO (15-SLO). 15-LO activity was
quantitated by measuring [14C]15-hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic
acid (15-HETE) production from the substrate [14C]arachidonic acid (AA)
following HPLC or TLC separation. 15-HETE production by human monocytes was
significantly elevated following continuous exposure to a single dose
(10ng/ml) of IL4 for 4 days, was maximal at 5 days and remained elevated at 6
days. At 6 days 15-LO activity in IL4-treated HMC was increased significantly
(2.81+/-0.70 fmol 15-HETE/cell), compared to 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 50K to 700K HMC/assay, from 2[mu]M to 160[mu]M AA and during 5
to 10 minutes incubation with AA. ETYA, ethyl 2-cyano-3-(3',4'-
dihydroxyphenyl) propenoate (a caffeic acid analog), NDGA, N-methyl-4-
benzyloxyphenyl-aceto -hydroxamate (RG 6866), esculetin, and 4 novel
lipoxygenase inhibitors; a phenylcyanomethylene analog (RP 27493) and 3
benzoxadiazine analogs (RP 64835, RP 65047 and RP 65208), inhibited HMC 15-LO
with IC50 values ([mu]M) 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 15-SLO,
respectively. These data define conditions for optimal production of 15-HETE
by IL4-treated HMC. Known and novel lipoxygenase inhibitors inhibit IL-4
treated HMC 15-LO and 15-SLO suggesting that IL-4 treated HMC provide an
appropriate system to study human 15-LO activity.
Received 27 July 1994; accepted in final form 22 November 1994
APS Manuscript Number C0438-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1994 The American Physiological Society.
Published in APStracts on 27 February 1995.