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. 2012 Apr 24;109(17):6721-6.
doi: 10.1073/pnas.1110460109. Epub 2012 Apr 9.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors

Ann M Gregus  1 , Suzanne Doolen, Darren S Dumlao, Matthew W Buczynski, Toshifumi Takasusuki, Bethany L Fitzsimmons, Xiao-Ying Hua, Bradley K Taylor, Edward A Dennis, Tony L Yaksh
Affiliations

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors

Ann M Gregus et al. Proc Natl Acad Sci U S A. .

Abstract

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Inhibition of spinal 12-LOX attenuates carrageenan-induced tactile allodynia and increased spinal formation of hepoxilins (HXB3). (A and B) IT pretreatment with (A) NDGA or (B) CDC dose-dependently attenuates the carrageenan-induced reduction in tactile thresholds. (C) Percent hyperalgesic index, or AUC, of tactile allodynia after IT pretreatment with vehicle (VEH) or maximum doses of NDGA, CDC, Baicalein (BAIC) or Zileuton (ZIL) reveals reduced nociceptive behavior after spinal inhibition of 12-LOX but not 5-LOX. (D) IT pretreatment with an antihyperalgesic dose of NDGA (60 μg) prevents the increase in spinal HXB3 4 h postcarrageenan. (E) Structures of HXA3 (Left) and HXB3 (Right). CONTRA, contralateral; CONTROL, naïve + IT VEH-treated; IPSI, ipsilateral; *P < 0.05, **P < 0.01, ***P < 0.001 vs. VEH 1; +P < 0.05, ++P < 0.01, +++P < 0.001 vs. VEH 2; n = 7–10.
Fig. 2.
Fig. 2.
Bioactive 12-LOX metabolites produce robust tactile allodynia after spinal delivery. (A–D) Dose–response depicting timecourses (Left) and AUC values (Right) reveal sustained reduction of tactile thresholds after a single IT administration of nanogram to microgram amounts of (A and B) HXA3 or (C and D) 12(S)-HpETE. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline vehicle; n = 5–6.
Fig. 3.
Fig. 3.
HXA3 produces direct activation of TRPV1 and TRPA1 in sensory neurons. (A) Representative micrographs of cultured rat sensory neurons before (Upper) and after (Lower) application of 1 μM HXA3. Arrowheads, Fura-2–loaded cells exhibiting Ca2+ mobilization in response to HXA3. (Scale bars, 50 μm.) (B) Representative traces of HXA3-evoked calcium responses in rat sensory neurons and attenuation by the TRPA1 antagonist HC030031 (Upper) or the TRPV1 antagonist AMG9810 (Lower). Peak Ca2+ responses represent mean change in fluorescence ratio (Δ340/380) ± SEM. (C) HXA3-evoked Ca2+-responses were reduced by HC030031 (10 μM) (VEH, 0.117 ± 0.01 vs. HC030031, 0.001 ± 0.02; ***P < = 0.001, n = 16 cells, 4 rats) and AMG 9810 (1 μM) (VEH, 0.159 ± 0.03 vs. AMG9810 0.061 ± 0.03; *P < 0.05, n = 5 cells, 3 rats). (D) HXA3 (1 μM) also increased calcium mobilization in HEK-TRPA1-tet [TRPA1(+) Induced, 0.077 ± 0.008 vs. TRPA1(−) Uninduced control, 0.020 ± 0.004; ***P < 0.001, n = 4 cells] and in CHO-TRPV1 cells [TRPV1(+) 0.070 ± 0.001 vs. TRPV1(−) CHO control, 0.012 ± 0.001; *P < 0.05, n = 3–5 cells]. (E) Representative traces of HXA3-evoked calcium responses in mouse sensory neurons from WT (Top) and attenuation in TRPA1 (Middle), or TRPV1 knockout (KO) (Bottom). (F) Percent of WT, TRPA1, or TRPV1 KO neurons in which 1 μM HXA3 increased free intracellular Ca2+ levels. (WT, 15.2 ± 4.9% n = 42 cells; TRPA1 KO, 0.0 ± 0.0%, n = 34 cells; TRPV1 KO, 1.1 ± 1.1%; n = 51 cells; *P < = 0.05, 3 mice per group). Cell viability was confirmed using 50 mM K+; functional TRPV1 or TRPA1 receptors were verified with 500 nM capsaicin or 5 μM icilin, respectively. VEH, artificial CSF.
Fig. 4.
Fig. 4.
HXA3-evoked tactile allodynia is mediated by spinal TRPV1 and TRPA1. (A) Timecourse and (B) corresponding AUC reveals significant attenuation of tactile allodynia following IT HXA3 (1 μg) by IT pretreatment with antihyperalgesic doses (10 μg) of antagonists of TRPA1 (HC030031) or TRPV1 (AMG9810). *P < 0.05, **P < 0.01 vs. VEH 3; n = 5–7.
Fig. 5.
Fig. 5.
Model of HXA3-mediated hyperalgesic effects at the spinal level. HXA3 is produced through 12-LOX either from arachidonic acid or via 12-HpETE. Cellular sources of HXA3 may represent DRG neurons or satellite cells, spinal neurons, or glia, and circulating leukocytes or platelets. Spinally generated HXA3 activates TRPV1 and TRPA1, resulting in calcium mobilization and release of SP from nociceptive afferents, internalization of NK1 receptors in dorsal horn, and ultimately tactile allodynia.
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