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

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 IC(50)s: CDC, similar to 0.5 and 0.8 mu M, 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