title: 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: Department of Anesthesiology, University of California-San Diego, La Jolla, CA 92093, USA.
authors: Ann M
authors: AM
authors: Gregus
authors: Darren S
authors: DS
authors: Dumlao
authors: Spencer C
authors: SC
authors: Wei
authors: Paul C
authors: PC
authors: Norris
authors: Laura C
authors: LC
authors: Catella
authors: Flore G
authors: FG
authors: Meyerstein
authors: Matthew W
authors: MW
authors: Buczynski
authors: Joanne J
authors: JJ
authors: Steinauer
authors: Bethany L
authors: BL
authors: Fitzsimmons
authors: Tony L
authors: TL
authors: Yaksh
authors: Edward A
authors: EA
authors: Dennis
mesh_terms: D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000187:drug effects / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006930:Hyperalgesia / Q000209:etiology*; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008297:Male; D051381:Rats
chemical_list: C475064:12-15-lipoxygenase; D016859:Lipoxygenase Inhibitors; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D019746:Intramolecular Oxidoreductases; C473385:hepoxilin synthase
references: J Cell Biochem. 2012 Jul;113(7):2279-89
references: 22573549
references: Nat Med. 2010 Mar;16(3):313-8
references: 20154695
references: J Biol Chem. 2004 Jul 9;279(28):29023-30
references: 15123652
references: J Histochem Cytochem. 2000 Oct;48(10):1411-9
references: 10990494
references: J Immunol. 2009 Sep 1;183(5):3383-9
references: 19675173
references: Prostaglandins Other Lipid Mediat. 2009 Dec;90(3-4):98-104
references: 19804839
references: Nat Med. 2010 May;16(5):592-7, 1p following 597
references: 20383154
references: Biochemistry. 2008 Jul 8;47(27):7295-303
references: 18547056
references: J Invest Dermatol. 2013 Jan;133(1):172-80
references: 22832496
references: Prostaglandins. 1983 Dec;26(6):955-72
references: 6084855
references: Pain. 2010 Jun;149(3):514-521
references: 20392563
references: Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):10150-5
references: 12097645
references: Physiol Behav. 1976 Dec;17(6):1031-6
references: 14677603
references: J Biol Chem. 1998 Dec 11;273(50):33540-7
references: 9837935
references: Methods Mol Med. 2004;99:109-21
references: 15131333
references: Pain. 2010 Nov;151(2):394-403
references: 20739123
references: Cytogenet Cell Genet. 1998;81(1):79-82
references: 9691181
references: J Cell Biol. 2007 Apr 9;177(1):173-82
references: 17403930
references: Br J Pharmacol. 2009 Oct;158(4):972-81
references: 19422397
references: Proc Natl Acad Sci U S A. 2004 May 11;101(19):7421-6
references: 15123795
references: Eur J Pharmacol. 2003 Mar 28;465(1-2):171-81
references: 12650847
references: J Neurosci Methods. 1994 Jul;53(1):55-63
references: 7990513
references: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9162-7
references: 12881489
references: Eur J Pharmacol. 2004 Apr 26;491(1):21-30
references: 15102529
references: Eur J Pharmacol. 2008 Dec 14;600(1-3):18-25
references: 18930721
references: Biochem Biophys Res Commun. 1993 May 14;192(3):1023-9
references: 8507177
references: Prog Lipid Res. 2011 Jan;50(1):115-31
references: 20970452
references: J Exp Med. 2007 Feb 19;204(2):245-52
references: 17242163
references: FEBS Lett. 1999 Mar 5;446(1):142-8
references: 10100631
references: J Neurochem. 2010 Aug;114(4):981-93
references: 20492349
references: Mol Pain. 2005 Sep 28;1:29
references: 16191203
references: Arch Biochem Biophys. 2010 Nov 15;503(2):161-74
references: 20801095
references: J Invest Dermatol. 2007 Aug;127(8):1893-7
references: 17429434
references: J Biol Chem. 1994 May 13;269(19):13979-87
references: 8188678
references: J Pharmacol Exp Ther. 2003 May;305(2):638-45
references: 12606597
references: J Med Chem. 1991 Apr;34(4):1503-5
references: 2016727
references: Neurosci Lett. 1995 Apr 21;189(3):159-62
references: 7542757
references: J Pharmacol Exp Ther. 2011 Jul;338(1):205-13
references: 21447614
references: Eur J Neurosci. 2004 Oct;20(8):2049-58
references: 15450084
references: Mol Cell Biol. 2010 Aug;30(16):4077-91
references: 20530198
references: Biochem Biophys Res Commun. 1990 Sep 28;171(3):944-8
references: 2171511
references: J Biol Chem. 2000 Jan 14;275(2):1287-93
references: 10625675
references: J Lipid Res. 2009 Jun;50(6):1015-38
references: 19244215
references: Biochem Biophys Res Commun. 1982 Apr 14;105(3):1090-5
references: 6807310
references: Methods. 2001 Dec;25(4):402-8
references: 11846609
references: Am J Pathol. 2010 Sep;177(3):1436-47
references: 20724598
references: J Med Chem. 2010 Oct 28;53(20):7392-404
references: 20866075
references: Mol Cells. 2009 Apr 30;27(4):417-22
references: 19390822
references: J Med Chem. 2011 Aug 11;54(15):5485-97
references: 21739938
references: Exp Brain Res. 2003 Feb;148(4):482-7
references: 12582831
references: Curr Pharm Des. 1999 Jan;5(1):11-20
references: 10066881
references: Allergy. 1986 Sep;41(7):493-8
references: 3024521
references: Prog Lipid Res. 2006 Jul;45(4):334-56
references: 16678271
references: Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6721-6
references: 22493235
references: Arch Biochem Biophys. 2006 Nov 15;455(2):188-96
references: 17045234
references: Genomics. 2001 May 1;73(3):323-30
references: 11350124
references: Prostaglandins Other Lipid Mediat. 2001 Jan;63(3):93-107
references: 11204741
references: Br J Pharmacol. 2010 Aug;160(7):1754-64
references: 20649577
references: J Biol Chem. 1996 Sep 20;271(38):23338-44
references: 8798535
references: Biochem J. 2000 Jun 1;348 Pt 2:329-35
references: 10816426