5,6-EET is released upon neuronal activity and induces mechanical pain hypersensitivity via TRPA1 on central afferent terminals - PubMed (original) (raw)

. 2012 May 2;32(18):6364-72.

doi: 10.1523/JNEUROSCI.5793-11.2012.

Chul-Kyu Park, Carlo Angioni, Dong Dong Zhang, Christian von Hehn, Enrique J Cobos, Nader Ghasemlou, Zhen-Zhong Xu, Vigneswara Kumaran, Ruirui Lu, Andrew Grant, Michael J M Fischer, Achim Schmidtko, Peter Reeh, Ru-Rong Ji, Clifford J Woolf, Gerd Geisslinger, Klaus Scholich, Christian Brenneis

Affiliations

5,6-EET is released upon neuronal activity and induces mechanical pain hypersensitivity via TRPA1 on central afferent terminals

Marco Sisignano et al. J Neurosci. 2012.

Abstract

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

5,6-EET concentrations in DRG tissue and release from sensory neurons upon activation. A, EET synthesis after nociceptive activation. 5,6-EET concentrations in the paw, L4–L6 DRGs, and the dorsal horn of L4–L6 spinal cords were measured 30 min after intraplantar injection of capsaicin (2 μg/25 μl) or vehicle. EET levels were determined from tissue extracts by LC-MS/MS. Shown is the average ± SEM form tissues of 10 animals per group. B, C, Levels of AA and 5,6-EET from cell lysates and supernatants of cultured DRG neurons. Neuron-enriched cultures from DRGs were incubated with A23187 (2 μ

m

) for 2 h. Then EETs and AA were extracted from cell lysates (B) or supernatants (C) and quantified by LC-MS/MS analysis. Data shown represent the average ± SEM from five culture dishes. D, E, Tissues from the plantar side of the paw (D), and L4–6 DRGs (E) were dissected 30 min or 6 h after intraplantar injection of 20 μl of CFA or vehicle. EET levels were determined by LC-MS/MS. Shown is the average ± SEM form tissues of six animals per group. *p ≤ 0.05, **p ≤ 0.01; Student's t test.

Figure 2.

Figure 2.

5,6-EET induces calcium influx in sensory neurons. A, In vitro stability of 5,6-EET was tested under all buffer conditions (pH, temperature) used in this study. 5,6-EET (1 μ

m

) was dissolved in each buffer and was incubated for up to 6 h. 5,6-EET concentrations were measured from extracted buffers by LC-MS/MS. B, Stimulation of adult DRG neurons with 5,6-EET (100 n

m

, 10 s), but not its metabolite 5,6-DHET (1 μ

m

, 10 s), induced a transient and reversible calcium flux. Neurons were identified by responses to KCl (40 m

m

, 10 s). Shown is a representative trace. C, 5,6-EET dose dependently activates a maximal of 11% of DRG neurons. Cells were stimulated with different 5,6-EET concentrations or acetonitrile (ACN) as vehicle control (n = 5–6 experiments). D, Effects of COX inhibitors on 5,6-EET-mediated calcium flux in wild-type DRGs. Cultured DRG neurons were treated with 1 μ

m

indomethacin, 1 μ

m

celecoxib, or vehicle (0.1% DMSO, v/v) 1 h before stimulation with 5,6-EET (250 n

m

) and KCl (40 m

m

, 30 s each). Shown are representative traces. E, Statistical analysis of peak amplitudes from DRG neurons stimulated with 5,6-EET, as in D (n = 6 experiments each). NB, Neurobasal; RT, room temperature.

Figure 3.

Figure 3.

5,6-EET induces calcium influx in DRG neurons by activation of TRP channels. A, 5,6-EET-induced calcium influx is blocked by Ca2+-free EGTA buffer. Ca2+-free EGTA buffer was washed in 5 min before a second 5,6-EET stimulation (100 n

m

, 10 s). Shown are representative traces of control (black) and Ca2+-free treated DRG neurons (gray). B, Average values of peak amplitudes normalized to the control (first 5,6-EET stimulation) peaks of traces from cells as stimulated in A (n = 5–6 experiments). C, 5,6-EET-induced calcium influx is blocked by RR. Five micrometers of RR were washed in for 5 min before DRG neurons were again stimulated with 5,6-EET (100 n

m

, 10 s). Shown are representative traces of control (black) and RR (5 μ

m

; gray) treated DRG neurons. D, Average values of peak amplitudes normalized to the control (first 5,6-EET stimulation) peaks of traces as in C (n = 6–7 experiments). **p < 0.01; Student's t test.

Figure 4.

Figure 4.

5,6-EET-induced calcium influx in sensory neurons is mediated by TRPA1. A, 5,6-EET-induced calcium influx does not depend on TRPV4. 5,6-EET (300 n

m

, 10 s)-induced calcium fluxes in DRG neurons from wild-type and TRPV4−/− mice were compared. Shown is a representative trace. B, Statistical analysis of neurons as shown in A (n = 150–200 cells). C, Effect of the selective TRPA1-antagonist HC-030031 (20 μ

m

) on 5,6-EET (250 n

m

)-induced calcium flux. Shown is a representative trace. D, Statistical analysis of the effects of the TRPV1 (AMG9810, 1 μ

m

) and TRPA1 (HC-030031, 20 μ

m

) antagonists on 5,6-EET-induced peak amplitudes (n = 5–6 experiments). E, DRG neurons from TRPA1−/− mice (gray) respond less to AITC (100 μ

m

, 30 s) and not to 5,6-EET (250 n

m

, 30 s) but do respond similarly to capsaicin (250 n

m

, 30 s) and KCl (40 m

m

, 30 s). Shown are representative traces. F, Statistical analysis of the peak amplitudes from recordings of wild-type and TRPA1-deficient DRG neurons after 5,6-EET stimulation (250 n

m

). Shown is the average ± SEM from six to eight experiments. G, Percentage of responding cells stimulated as shown in E from wild-type and TRPA1-deficient DRG neurons. Shown is the average ± SEM from six to eight experiments. **p < 0.01; Student's t test. caps., Capsaicin; wt, wild-type.

Figure 5.

Figure 5.

Role of regulatory cysteine residues for 5,6-EET-induced TRPA1 activation. A, HEK-293 cells were transiently transfected with plasmids expressing (h)TRPA1 and stimulated with 5,6-EET (250 n

m

, 30 s) and carvacrol (250 μ

m

30 s). Shown is a representative trace. B, Statistical analysis of the peak amplitudes of recordings from cells stimulated as in A (n = 8 experiments). C, HEK-293 cells were transiently transfected with plasmids expressing the (h)TRPA1 3CK mutant and stimulated as in A. Shown is a representative trace. D, Statistical analysis of the peak amplitudes of recordings from cells as in C (n = 9 experiments). **p ≤ 0.01; Student's t test.

Figure 6.

Figure 6.

Peripheral TRPA1 activation by 5,6-EET causes acute pain and mechanical allodynia. A, Spontaneous pain induced by 5,6-EET. After intraplantar injection of 20 μl of 5,6-EET (5 μ

m

) or vehicle (acetonitrile, 1.6%, v/v), the licking time was monitored (n = 8 animals per group). B, Mechanical allodynia after 5,6-EET injection. Dynamic plantar test after intraplantar injections of 20 μl of 5,6-EET (5 μ

m

) or vehicle (n = 8–9 animals per group). C, Comparison of mechanical allodynia in wild-type and TRPA1-deficient mice after intraplantar injection of 20 μl of 5,6-EET (5 μ

m

) or vehicle (n = 8 animals per group). D, 5,6-EET does not sensitize responses to a radiant heat stimulus. Hargreaves test after intraplantar injections of 20 μl of 5,6-EET (5 μ

m

) or vehicle (n = 10 animals per group). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; two-way repeated-measures ANOVA followed by Bonferroni's post-test. i.plantar, Intraplantar; PWL, paw withdrawal latency.

Figure 7.

Figure 7.

5,6-EET enhances sEPSC frequency in lamina II neurons of spinal cord slices through TRPA1. A, B, sEPSC traces recorded in spinal cord slices of wild-type (A) and TRPA1-KO (B) mice. 1, Trace before EET treatment; 2, trace after EET treatment; 3, trace after AITC treatment (300 μ

m

). Note that EET- and AITC-induced sEPSC increases are abolished in TRPA1-KO mice. C, sEPSC frequency (top) and amplitude (bottom). Note that EET and AITC increase only the frequency but not the amplitude of sEPSCs. *p < 0.05, compared with pretreatment baseline (n = 5–10 cells). KO, Knock-out; wt, wild-type.

Figure 8.

Figure 8.

Intrathecal injections of 5,6-EET cause mechanical allodynia through the activation of TRPA1. A, 5,6-EET reduces mechanical thresholds when injected intrathecally. Five microliters of 5,6-EET (10 μ

m

) or vehicle (DMSO, 3.2%, v/v) were injected intrathecally, followed by determination of the mechanical pain thresholds at time points 15, 30, 45, 60, 90, 120, and 180 min after injection using a dynamic plantar aesthesiometer. Shown is the average ± SEM paw withdrawal latency from eight animals per group. B, Comparison of mechanical thresholds in wild-type and TRPA1-deficient mice after intrathecal injections of 10 μl of 5,6-EET (10 μ

m

) using a dynamic plantar aesthesiometer. Shown is the average ± SEM from eight animals per group. C, Comparison of thermal thresholds in wild-type mice after intrathecal 5,6-EET injection (10 μ

m

) at the same time points shown in A using a Hargreaves apparatus. Shown is the average ± SEM from six animals per group. *p ≤ 0.05, ***p ≤ 0.001; two-way ANOVA followed by Bonferroni's post-test. i.th, Intrathecally; PWL, paw withdrawal latency; WT, wild-type.

Similar articles

Cited by

References

    1. Alessandri-Haber N, Dina OA, Yeh JJ, Parada CA, Reichling DB, Levine JD. Transient receptor potential vanilloid 4 is essential in chemotherapy-induced neuropathic pain in the rat. J Neurosci. 2004;24:4444–4452. - PMC - PubMed
    1. Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, Yamoah EN, Basbaum AI, Julius D. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell. 2006;124:1269–1282. - PubMed
    1. Bessac BF, Sivula M, von Hehn CA, Escalera J, Cohn L, Jordt SE. TRPA1 is a major oxidant sensor in murine airway sensory neurons. J Clin Invest. 2008;118:1899–1910. - PMC - PubMed
    1. Brenneis C, Sisignano M, Coste O, Altenrath K, Fischer MJ, Angioni C, Fleming I, Brandes RP, Reeh PW, Woolf CJ, Geisslinger G, Scholich K. Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation. Mol Pain. 2011;7:78. - PMC - PubMed
    1. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288:306–313. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources