4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

Abstract

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous alpha,beta-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Fig. 1.

HNE activates the cloned TRPA1 channel. (A) HEK293 cells expressing rat TRPA1 after tetracycline induction (TRPA1+TET; red trace) were challenged with HNE (100 μM), and responses was assessed by calcium imaging. Uninduced (TRPA1-TET; black trace) or vector-transfected control cells (VC+TET; black trace) showed no significant response. (B) Dose–response analysis of HNE-evoked calcium responses in tetracycline-induced (TRPA1+TET; red), uninduced (TRPA1-TET) or vector-transfected control (VC+TET) HEK293 cells. Analysis was performed by using a multiwell fluorescence plate reader. Each well contained 30,000 cells; n = 4 wells per agonist concentration. (C) Representative voltage-clamp recording (+70 mV holding potential; Left) from Xenopus oocytes expressing human TRPA1 channel, showing robust activation by HNE and block by ruthenium red (RR). Asterisks indicate times at which voltage ramps were acquired to generate current–voltage relationships (Right) for APB (black) and HNE (green). (D and E) Oocytes expressing TRPA1–3C or TRPA1–3C/K-Q mutant channels were challenged with 2-aminoethyl diphenylborinate (APB; 200 μM; black), HNE (200 μM; green), or ruthenium red (RR; 10 μM; red), as indicated.

Fig. 2.

TRPA1 mediates dose-dependent HNE-evoked responses in cultured sensory neurons. (A) Dose–response analysis (Left) for HNE-evoked responses in cultured rat DRG neurons as assessed by calcium imaging. Response to capsaicin (Cap) is shown for comparison. Effects of several antagonists, including ruthenium red (RR, 1 μM), (+) camphor (Cam, 1 mM), and gentamycin (GM, 100 μM) on HNE-evoked responses were assessed (Right). The TRPV1 antagonist, capsezapine (CPZ, 10 μM), had no effect on HNE-evoked responses. Error bars represent SEM; n ≥ 22 cells; *, P < 0.05, Bonferroni's test vs. vehicle (Veh). (B) Trigeminal neurons from TRPA1^+/+ (Left) and TRPA1^−/− (Right) mice were exposed to HNE (100 μM), followed by capsaicin (Cap, 1 μM) and then high potassium (KCl; 100 mM), and responses were assessed by calcium imaging. Among Cap-sensitive neurons from wild-type animals, a subpopulation of HNE-responsive cells was observed. No HNE-sensitive cells were detected from TRPA1-deficient mice. Each trace represents an average of 15 responsive cells; n ≥ 399 neurons examined in ≥3 independent cultures per genotype.

Fig. 3.

HNE mediates neurogenic inflammation by triggering release of neuropeptides and promoting plasma extravasation. (A and B) Release of substance P or CGRP peptides from rat dorsal spinal cord (A) or esophagus (B) was detected after exposure of tissue slices to HNE (50 or 100 μM, as indicated) for 20 min. Desensitization (Des) by pretreatment with capsaicin (10 μM; 20 min before HNE application), or chelation of extracellular Ca²⁺ (−Ca) by addition of 2 mM EGTA to the bath abolished HNE-evoked neuropeptide release. Error bars represent ±SEM; n ≥ 4 slices per condition. *, P < 0.05, Bonferroni's test versus HNE alone. (C) Injection of HNE into the rat hind paw leads to dose-dependent plasma extravasation as determined by measuring accumulation of circulating Evan's blue dye. The tachykinin NK1 receptor antagonist SR140333 reduced HNE-evoked plasma extravasation. Error bars represent SEM; n ≥ 4 measurements per condition. *, P < 0.05 versus vehicle (Veh) control; #, P < 0.05 versus HNE (150 nmol/30 μl/paw) alone, Bonferroni's test.

Fig. 4.

TRPA1 is both necessary and sufficient to mediate nocifensive behavior in rodents. (A) Injection of HNE (15–240 nmol/20 μl) into the hind paw of Swiss mice induced pain-related behavior (licking and lifting of affected paw) in a dose-dependent manner. (B Left) (+) camphor (Cam, 125 nmol/20 μl), gentamycin (GM, 250 nmol/20 μl), but not capsazepine (CZP, 1.0 nmol/20 μl) reduced effect of HNE (60 nmol/20 μl). In contrast, these agents had no effect on nociceptive behavior induced by capsaicin (Cap, 0.1 nmol/20 μl), which was blocked by capsazepine (B Right). (C Left) Injection of HNE (150 nmol/50 μl; red trace) into the rat hind paw induced tactile allodynia. Coinjection with (+) camphor (250 nmol/50 μl; green trace) or gentamycin (50 nmol/50 μl; blue trace) reduced the effect of HNE. In contrast, coinjection with capsazepine (0.5 nmol/50 μl; yellow trace) had no blocking effect. No effect was observed upon injection of vehicle alone (Veh, 0.9% saline, 20 μl; black trace). (C Right) Injection of capsaicin (Cap, 20 nmol/50 μl; red trace) also produced tactile allodynia that was unaffected by camphor or gentamycin (green and blue trace, respectively) but attenuated by capsazepine (orange trace). Error bars represent mean ± SEM; n ≥ 6 animals per condition. *, P < 0.05 versus HNE or capsaicin alone. (D) Hind paws of wild-type (+/+) or TRPA1-deficient (−/−) littermate mice were injected with HNE (120 nmol in 20 μl of 0.9% saline). Total time spent licking or lifting the injected paw was recorded over a period of 5 min. Error bars indicate average responses ± SEM (n = 6 animals per trial; *, P < 0.0002, ANOVA single-factor analysis). All measurements were carried out blind to genotype.

Fig. 5.

Inflammatory agents regulate TRPA1 and TRPV1 through direct and indirect mechanisms. Tissue injury, ischemia, or cellular stress generates an array of proalgesic and proinflammatory agents, collectively referred to as the “inflammatory soup.” This includes extracellular protons (H⁺), bradykinin (BK), and nerve growth factor (NGF), as well as reactive oxygen species (ROS) that convert polyunsaturated fatty acids into reactive carbonyl species, such as 4-hydroxy-2-nonenal (HNE). Some factors, such as HNE and protons, activate TRPA1 or TRPV1 directly, whereas others, such as BK and NGF, modulate channel gating indirectly by binding to cognate receptors (BR and TRKA, respectively) to activate cellular signaling cascades, most notably those downstream of phospholipase C (PLC). Thus, TRPA1 and TRPV1 function as polymodal signal integrators capable of detecting chemically diverse products of cell and tissue injury. In doing so, these channels promote pain hypersensitivity by depolarizing the primary afferent nerve fiber and/or lowering thermal or mechanical activation thresholds.