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Systematic analysis of rat 12/15‐lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia
Author(s) -
Gregus Ann M.,
Dumlao Darren S.,
Wei Spencer C.,
Norris Paul C.,
Catella Laura C.,
Meyerstein Flore G.,
Buczynski Matthew W.,
Steinauer Joanne J.,
Fitzsimmons Bethany L.,
Yaksh Tony L.,
Dennis Edward A.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.12-217414
Subject(s) - chemistry , hyperalgesia , inflammation , pharmacology , arachidonic acid , arachidonate 5 lipoxygenase , lipoxygenase , enzyme , spinal cord , proinflammatory cytokine , nociception , biochemistry , immunology , medicine , biology , receptor , neuroscience
Previously, we observed significant increases in spinal 12‐lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation‐induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15‐LOX enzymes in HEK‐293T cells and measured by LC‐MS/MS the formation of HXB 3 , 12‐HETE, 8‐HETE, and 15‐HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA‐861, CDC, baicalein, and PD146176) vs. vehicle‐treated and mock‐transfected controls. We detected the following primary intrinsic activities: 12‐LOX ( Alox12, Alox15 ), 15‐LOX ( Alox15b ), and HXS ( Alox12, Alox15 ). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12‐LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3 ) exhibited HXS without 12‐LOX activity when coexpressed with Alox12b or supplemented with 12‐HpETE. CDC potently inhibited HXS and 12‐LOX activity in vitro (relative IC 50 s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan‐evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3‐selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3‐mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12‐LOX/HXS inhibitors.—Gregus, A. M., Dumlao, D. S., Wei, S. C., Norris, P. C., Catella, L. C., Meyerstein, F. G., Buczynski, M. W., Steinauer, J. J., Fitzsimmons, B. L., Yaksh, T. L., Dennis, E. A. Systematic analysis of rat 12/15‐lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia. FASEB J. 27, 1939–1949 (2013). www.fasebj.org