A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition

Sensory neurons of the dorsal root ganglia (DRGs) can detect environmental changes through projections in the skin. Among these primary afferent fibers, nociceptors play an important role in sensing noxious levels of mechanical and thermal stimuli [1]. One of the basic properties of nociceptors is their sensitization in response to injury and inflammation. For example, an innocuous stimulus such as a warm shower can become painful after sunburn, and this is called allodynia. Indeed, cold or mechanical allodynia are serious medical problems for patients suffering from neuropathic pain [2, 3]. Hyperalgesia describes a similar condition, defined as an increased response to an already painful stimulus due to injury or inflammation.

TRPV1, a member of the Transient Receptor Potential family of cation channels, is activated by heat, low pH, and capsaicin [4]. TRPV1 is expressed in nociceptors, and is sensitized in response to a variety of signal transduction pathways activated during inflammation [5, 6]. TRPV1-deficient mice display negligible inflammatory heat hyperalgesia [7, 8]. Much less is known about molecules involved in mechanical hyperalgesia.

Originally characterized as a noxious cold-activated ion channel, TRPA1 is expressed in the same sensory neurons as TRPV1, and is activated directly by diverse reactive chemicals via covalent modification, and indirectly through G-protein coupled receptors [9‐12]. TRPA1 is required to sense these noxious reactive chemicals in vivo; however, whether TRPA1 is required to sense acute noxious cold and mechanical stimulus is not yet settled [13‐17]. Here, we examine the consequences of acute block of peripheral TRPA1 for pain transduction, focusing on mechanical hyperalgesia.

To test if acute block of TRPA1 can modulate pain sensation, we sought a specific, efficient TRPA1 inhibitor. Ruthenium red, gadolinium, menthol, and camphor can inhibit TRPA1 activation, but also interact with other channels, including other TRP channels[18]. Using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices) calcium-influx assay, we screened 43,648 small molecules for their ability to block cinnamaldehyde-activation of human TRPA1 in a Chinese Hamster Ovary (CHO) cell line. We further characterized one of these hits, AP18 ((Z)-4-(4-chlorophynyl)-3-methylbut-3-en-2-oxime – Maybridge, Cornwall, UK; Figure 1A). AP18 blocked activation of TRPA1 by 50 μM cinnamaldehyde with an IC₅₀ of 3.1 μM and 4.5 μM for human and mouse clones, respectively (Figure 1B). At concentrations up to 50 μM, AP18 was unable to appreciably block activation of TRPV1, TRPV2, TRPV3, TRPV4, or TRPM8 (Figure 2). AP18 reversibly blocked mouse TRPA1 responses to iodoacetamide (an irreversible cysteine-alkylating agent) in CHO cells assayed by ratiometric Ca²⁺ imaging (Figure 1C). AP18 also blocked cold- and mustard-oil-induced activation of mouse TRPA1 (data not shown). Moreover, AP18 blocked cinnamaldehyde-induced TRPA1 currents in excised patches from Xenopus oocytes (Figure 1D). In vivo, 1–10 mM of AP18 injected in hindpaw of mice did not cause any obvious behavioural responses (data not shown). Importantly, AP18 significantly blocked cinnamaldehdye-induced but not capsaicin-induced nociceptive events, demonstrating efficacy and specificity (Figure 3).

Sensitization of nociceptive neurons is the basis for many chronic pain states [1]. To investigate the role of TRPA1 in pain sensitization, we tested AP18 in mechanical hyperalgesia induced by complete Freund's adjuvant (CFA). Mechanical sensitivity was measured using an automated Von Frey apparatus which records the point at which the paw is withdrawn in response to a progressively-increasing force. AP18 but not vehicle solution injected in the paw 24 hours after CFA treatment almost completely reversed mechanical hyperalgesia (Figure 4A). AP18 did not reverse CFA-induced heat hyperalgesia (Figure 5). Acute nociceptive responses to both increasing mechanical force and infrared noxious heat applications to the paw without coincident inflammation were not affected by AP18 treatment (data not shown). Remarkably, AP18 did not reverse CFA-induced mechanical hyperalgesia in TRPA1-deficient mice, suggesting on-target mechanisms for the activity of AP18 (Figure 4B) [16, 17]. It is important to note that prior to AP18 treatment, TRPA1-deficient mice developed normal CFA-induced mechanical hyperalgesia (Figure 4B), suggesting that compensation in these mice masks the requirement for TRPA1 in mechanical sensitization (see discussion below). Also note that TRPA1-deficient mice do not show significantly increased acute mechanical threshold before CFA application in this assay (Figure 4B). However, we do observe the previously-reported deficit in acute mechanosensation in TRPA1-deficient mice at higher g-forces when using the protocol of Kwan et al.(2006) which assays frequency of paw withdrawal in response to a fixed (rather than increasing) force applied to the paw (Figure 6) [16]. In addition to the acute mechanosensory phenotype, TRPA1-deficient mice have strong deficits in bradykinin-induced inflammatory pain [16, 17]. As expected, AP18-treated mice showed reduced bradykinin-induced mechanical hyperalgesia compared to vehicle (Figure 7).

Whether TRPA1 is activated by noxious cold temperatures, and whether TRPA1-deficient mice show deficits in cold thermosensation is unsettled, and area of active research [9, 16, 17, 19‐22]. We have found it difficult to demonstrate noxious cold response in wildtype mice using temperatures as low as 0°C, either alone or following CFA injection (data not shown). Sprague Dawley rats, on the other hand, readily display cold hyperalgesia, and TRPA1 is implicated in this process [21]. CFA-induced cold hyperalgesia assayed on a 5°C plate is significantly reversed in rats treated with AP18, supporting a role of TRPA1 in cold nociception (Figure 8).

The consequence of acute pharmacological block of protein activity in adult animals sometimes deviates from the effects of germline ablation of the encoding gene. For example, it was recently shown that pharmacological inhibition of TRPV1 induces hyperthermia in wildtype mice in a TRPV1-dependent manner, although mice born genetically-deficient in TRPV1 do not display a hyperthermic phenotype [23, 24]. Using a small molecule inhibitor of TRPA1 we have demonstrated a novel role of TRPA1 in mechanical hyperalgesia. AP18 is ineffective in reversing CFA-induced mechanical hyperalgesia in TRPA1-deficient mice, suggesting an on-target mechanism for AP18. What compensatory mechanisms may account for the normal mechanical hyperalgesia observed in TRPA1-deficient mice? Upregulation of another protein that plays a similar role as TRPA1 (molecular compensation) is one explanation. TRPA1 is the only member of the TRPA family in mammals, and TRPV1, another nocisensor expressed in pain-sensing neurons, does not appear to be upregulated in TRPA1-deficient mice, although the two channels functionally interact [16, 17, 25]. TRPV4, a related ion channel, has been implicated in some forms of mechanical hyperalgesia and can be an intriguing candidate [26]. However, whether TRPV4 expression is required in skin cells or DRGs is unsettled [27]. Cellular compensation (i.e. a change in the composition of neuronal subtypes or in neuronal connectivity) is another potential explanation for the normal hyperalgesia observed in TRPA1-deficient mice. Furthermore, it is possible that TRPA1 is involved in the maintenance of mechanical hyperalgesia rather than its induction. In this case, normal development of mechanical hyperalgesia is expected in TRPA1-deficient mice, but compensation could contribute to maintenance of the condition.

TRPA1 is specifically expressed in nociceptors, its transcripts are upregulated in response to CFA in rats, and inflammatory signals sensitize the channel, consistent with TRPA1 being involved in hyperalgesia [21, 28, 29]. The mechanism by which TRPA1 contributes specifically to mechanosensation is less clear [30]. It is possible that mammalian TRPA1 is directly activated by mechanical forces, although no such data is currently present. Alternatively, mechanical damage could indirectly activate TRPA1 through release of reactive compounds or intracellular calcium (both of which can directly activate mammalian TRPA1) [11, 12, 31]. One other possibility is that TRPA1 is involved in modulation of mechanosensory signaling, and not directly involved with mechanotransduction. Interestingly, the ortholog of mammalian TRPA1 in C. elegans can be activated by pressure applied through a recording pipette when expressed in heterologous cells (although it is also not clear if cTRPA1 is directly or indirectly responding to this mechanical force) and plays an essential role in mechanosensory behaviour [32].

In conclusion, this study highlights a novel role of mouse TRPA1 in mechanical hyperalgesia, and underscores the importance of considering both acute pharmacological block and genetic ablations in assigning gene function.