Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia.

Abstract

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 HXB3, 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 IC50s: 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.

• Authors: Ann M Gregus, Darren S Dumlao, Spencer C Wei, Paul C Norris, Laura C Catella, Flore G Meyerstein, Matthew W Buczynski, Joanne J Steinauer, Bethany L Fitzsimmons, Tony L Yaksh, Edward A Dennis
• Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology (FASEB J) Vol. 27 Issue 5 Pg. 1939-49 (May 2013) ISSN: 1530-6860 [Electronic] United States
• PMID: 23382512 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• 12-15-lipoxygenase
• Lipoxygenase Inhibitors
• Arachidonate 12-Lipoxygenase
• Arachidonate 15-Lipoxygenase
• Intramolecular Oxidoreductases
• hepoxilin synthase

Topics

• Animals
• Arachidonate 12-Lipoxygenase (drug effects, metabolism)
• Arachidonate 15-Lipoxygenase (drug effects, metabolism)
• HEK293 Cells
• Humans
• Hyperalgesia (etiology)
• Intramolecular Oxidoreductases (metabolism)
• Lipoxygenase Inhibitors (pharmacology)
• Male
• Rats