Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1

Abstract

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.

Fig. 1.

Cyclopentenone PGs and isoprostane increase [Ca²⁺]_i in mouse DRG neurons. (A) Concentration-response curves of cyclopentenone PGs or isoprostane and PGE₂ in DRG neurons of Swiss mice (n = 521 neurons). (B) Representative traces of [Ca²⁺]_i evoked by 15-d-PGJ₂ (30 μM) or PGE₂ (30 μM), followed by capsaicin (CPS, 1 μM) and potassium (KCl, 50 mM). A calcium response was induced by 15-d-PGJ₂ in DRG neurons from TRPA1^+/+ but not from TRPA1^−/− mice. PGE₂, capsaicin or potassium evoked a similar response in neurons from TRPA1^+/+ or TRPA1^−/− mice. (C) Pooled data of [Ca²⁺]_i evoked by cinnamaldehyde (CNM, 30 μM), capsaicin (1 μM), 15-d-PGJ₂, PGA₁, PGA₂, 8-iso-PGA₂, PGD₂, and PGE₂ (all 30 μM) in DRG neurons from TRPA1^+/+ (gray or orange columns) or TRPA1^−/− (filled or red columns) mice. Among CPS-sensitive neurons from wild-type animals, a subpopulation of CNM-responsive cells was observed. In DRG neurons from TRPA1-deficient mice CNM, 15-d-PGJ₂, PGA₁, PGA₂, or 8-iso-PGA₂ did not cause any response. PGE₂ evoked a small response in DRG neurons from TRPA1^+/+ or TRPA1^−/− mice. PGD₂ failed to cause any response. The robust response evoked by capsaicin was similar in neurons from TRPA1^+/+ and TRPA1^−/− mice. Each column represents the mean ± SEM of n ≥ 23 cells; *, P < 0.01 compared with TRPA1^−/− DRG neurons.

Fig. 2.

Cyclopentenone PGs and isoprostane release neuropeptides in the dorsal spinal cord. Release of CGRP-like immunoreactivity (LI) (upper panel) and SP-LI (bottom panel) from slices of rat spinal cord after exposure to cyclopentenone PGs or isoprostane (30 μM). Desensitization by pretreatment with capsaicin (CPS), or omission and chelation of extracellular Ca²⁺ (1 mM EDTA, −Ca) abolished the evoked neuropeptide release. Each column represents mean ± SEM; n ≥ 4 experiments per condition. *, P < 0.05 compared with cyclopentenone agonist alone.

Fig. 3.

Cyclopentenone PGs and isoprostane evoke c-fos expression in L4–L5 spinal cord lumbar sections. Photomicrographs (A) of transverse sections of the lumbar spinal cord and pooled data (B) showing the effects of intraplantar injection of cinnamaldehyde (CNM, 30 nmol) capsaicin (CPS, 0.1 nmol), cyclopentenone prostaglandins or isoprostane (15 nmol each) on the expression of c-fos-like immunoreactivity (LI) in TRPA1^+/+ (gray or orange columns) or TRPA1^−/− (filled or red columns) mice 90 min after injection in the hind paw (4–5 animals for each treatment). Each photomicrograph is a representative example of one section (L4–L5) of dorsal horn ipsilateral to the injection. CNM, 15-d-PGJ₂, PGE₂, PGA₁, PGA₂, and 8-iso-PGA₂ and CPS increased c-fos-LI in TRPA1^+/+ mice. In TRPA1^−/− mice the effects of CPS and PGE₂ were maintained, whereas the effects of CNM, 15-d-PGJ₂, PGE₂, PGA₁, PGA₂ were abolished. The vehicle induced a moderate increase in c-fos-LI expression, identical in TRPA1^+/+ and TRPA1^−/− mice. Each column represents the mean ± SEM of c-fos immunoreactive cells; n ≥ 10 slices per condition in L4–L5 segments; *, P < 0.05 compared with TRPA1^+/+ mice.

Fig. 4.

TRPA1 mediates cyclopentenone PG- and isoprostane-induced nocifensive behavior in mice. Agonists were injected into the hind paws of TRPA1^+/+ (empty circles or gray and orange columns) or TRPA1^−/− (filled circles or filled and red columns) mice, and time spent licking or lifting after the injection was recorded for 15 min. Insets show the total time spent licking or lifting over the 15 min of observation. 15-d-PGJ₂, PGA₁, PGA₂, or 8-iso-PGA₂ (all 15 nmol) caused an early and transient nociceptive response in TRPA^+/+ mice, an effect that was completely absent in TRPA1^−/− littermates. In TRPA^+/+ mice, the TRPA1 agonist cinnamaldehyde (30 nmol) and the TRPV1 agonist capsaicin (0.1 nmol) produced a response with a time course similar to that of cyclopentenone compounds. Whereas the response to capsaicin was maintained, that to cinnamaldehyde was abolished in TRPA1^−/− mice. PGD₂ (15 nmol) did not evoke any measurable nociceptive behavior either in TRPA1^+/+ or in TRPA1^−/− mice. PGE₂ (15 nmol) produced a slowly developing and protracted nociceptive behavior that was similar in TRPA1^+/+ and TRPA1^−/− mice. Error bars indicate mean ± SEM of 6–9 animals per trial; *, P < 0.05 vs. TRPA1^+/+ mice.