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. 2009 Nov 3;106(44):18820-4.
doi: 10.1073/pnas.0905415106. Epub 2009 Oct 20.

Activation of TRPV1 in the Spinal Cord by Oxidized Linoleic Acid Metabolites Contributes to Inflammatory Hyperalgesia

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Free PMC article

Activation of TRPV1 in the Spinal Cord by Oxidized Linoleic Acid Metabolites Contributes to Inflammatory Hyperalgesia

Amol M Patwardhan et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Transient receptor potential vanilloid 1 (TRPV1) plays a major role in hyperalgesia and allodynia and is expressed both in the peripheral and central nervous systems (CNS). However, few studies have evaluated mechanisms by which CNS TRPV1 mediates hyperalgesia and allodynia after injury. We hypothesized that activation of spinal cord systems releases endogenous TRPV1 agonists that evoke the development of mechanical allodynia by this receptor. Using in vitro superfusion, the depolarization of spinal cord triggered the release of oxidized linoleic acid metabolites, such as 9-hydroxyoctadecadienoic acid (9-HODE) that potently activated spinal TRPV1, leading to the development of mechanical allodynia. Subsequent calcium imaging and electrophysiology studies demonstrated that synthetic oxidized linoleic acid metabolites, including 9-HODE, 13-HODE, and 9 and 13-oxoODE, comprise a family of endogenous TRPV1 agonists. In vivo studies demonstrated that intrathecal application of these oxidized linoleic acid metabolites rapidly evokes mechanical allodynia. Finally, intrathecal neutralization of 9- and 13-HODE by antibodies blocks CFA-evoked mechanical allodynia. These data collectively reveal a mechanism by which an endogenous family of lipids activates TRPV1 in the spinal cord, leading to the development of inflammatory hyperalgesia. These findings may integrate many pain disorders and provide an approach for developing analgesic drugs.

Conflict of interest statement

Conflict of interest statement: The University of Texas has claimed intellectual property on this discovery.

Figures

Fig. 1.
Fig. 1.
Depolarization of the spinal cord releases endogenous TRPV1 agonist(s) (A) Freshly isolated rat spinal cords (9) were washed for 50 min in Hanks buffer and a basal sample (20 min in Hanks, 37 °C, Non- Depol- SC) was collected. Spinal cords were then depolarized with a buffer containing 50 mM potassium (20 min, 37 °C, Depol- SC). Both supernatants were passed through C18 SepPak columns and washed with water/0.05 TFA to remove salts. The substances adsorbed onto the column were eluted with 90% acetonitrile/0.05% TFA. The eluates were dried down under a flow of nitrogen and reconstituted in Hanks buffer. In a fura-2 calcium imaging set up, a representative tracer demonstrates the effect of the application of reconstituted eluates on a TG neuron from WT mice that also responded to capsaicin (100 nM). Ratiometric data are shown. (B) A representative tracer demonstrating the effect of the same eluate (from depolarized spinal cords) applied to a neuron from TRPV1 KO mice. The positive control for neuronal viability was stimulation with 50 mM potassium. (C) Comparison of calcium accumulation evoked by eluate from depolarized spinal cords in TG neurons from WT versus TRPV1 KO mice (n = 48 for WT and 43 for KO neurons,***, = P < 0.001, t-test). (D) Effect of pretreatment with either vehicle or the TRPV1 antagonist, AMG 9810 (1 μM, 3 min) on calcium accumulation in rat TG neurons evoked by eluate from depolarized spinal cords (n = 15 for vehicle and 41 for AMG ***, = P < 0.001, t-test). (E) A representative tracer demonstrating the effect of the application of eluate from depolarized spinal cords in CHO cells expressing rat TRPV1. Capsaicin (100 nM) was used as a positive control for TRPV1 expression. (F) Comparison of calcium accumulation evoked by applying eluates collective from nondepolarized and depolarized spinal cords to CHO cells expressing TRPV1 (n = 57 for nondepol and 47 for depol, ***, = P < 0.001, t-test).
Fig. 2.
Fig. 2.
Depolarized spinal cord eluate contains elevated levels of oxidized linoleic acid metabolites that are TRPV1 agonists. (A) ELISA demonstrating the 9-HODE contents in eluates from nondepolarized and depolarized spinal cords (n = 3 separate samples, *, = P < 0.05, t-test). (B) Calcium imaging experiment demonstrating the effect of application of synthetic 9-HODE (100 μM, 1 min) to a rat TG neuron that was pretreated with Vehicle. Capsaicin (100 nM) served as a positive control for the presence of TRPV1 in that neuron. (C) Calcium imaging experiment demonstrating the effect of pre and cotreatment of AMG 9810 (1 μM, 3 min) on stimulatory effects of synthetic 9-HODE (100 μM). Neuronal viability was assessed by response to a buffer containing 50 mM potassium. (D) In a calcium imaging set up, the effect of application of synthetic 13-HODE (100 μM) on a rat TG neuron that also responded to capsaicin (100 nM). (E) Comparison of calcium accumulation evoked by various linoleic acid metabolites in rat TG neurons and the effect of pre and cotreatment with AMG 9810 (1 μM, 3 min) on their activity (n = 43–121 cells per condition).
Fig. 3.
Fig. 3.
Oxidized linoleic acid metabolites activate recombinant rat TRPV1. (A) A representative tracer demonstrating the I-V plot obtained after 9-HODE (100 μM) application on CHO cells expressing TRPV1. (B, C, and D) Similar tracers obtained for 13-HODE, 9-oxoODE and 13-oxoODE (100 μM each) respectively. (E) Comparison of the current density evoked by various oxidized linoleic acid metabolites at −60 mV.
Fig. 4.
Fig. 4.
Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites plays a role in CFA-evoked behavioral responses. (A) After habituation, rats were injected with vehicle, AMG 9810 (50 μg), Cap (5 μg) or Cap with AMG (intrathecal, 30 μL volume) under light anesthesia and responses to Von Frey filaments were observed over 1 h post injection. The data are presented as 50% paw withdrawal threshold and analyzed using two way ANOVA with Bonferroni post hoc test (*, = P < 0.05, **, = P < 0.01, ***, = P < 0.001, compared to the vehicle group, n = 6–7 per group). All behavioral studies were conducted by blinded observers. (B) In a similar behavioral set up, comparison of allodynia evoked by intrathecal application of synthetic 9-HODE (5 μg) with or without AMG 9810 (50 μg). The data were analyzed using two way ANOVA with Bonferroni post hoc test (**, = P < 0.01, n = 6–7 per group). (C) After obtaining basal paw withdrawal thresholds, animals were injected with CFA unilaterally (ipsi, 100 μL). The post-CFA withdrawal thresholds in both paws were obtained after 24 h. Then the animals were injected either with vehicle or antibodies against 9- and 13-HODE (30 μg each) and withdrawal thresholds were obtained in both ipsilateral and contralateral paws 60 min post antibody combination injection (***, = P < 0.001, two way ANOVA with Bonferroni post hoc test, n = 5 per group).

Comment in

  • Fat location defines sensation.
    Oh U, Wood JN. Oh U, et al. Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18435-6. doi: 10.1073/pnas.0910766106. Epub 2009 Oct 28. Proc Natl Acad Sci U S A. 2009. PMID: 19864623 Free PMC article. No abstract available.

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