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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC May 10, 2011.
Published in final edited form as:
PMCID: PMC3021322
NIHMSID: NIHMS251187

TRPA1 Contributes to Cold Hypersensitivity

Abstract

TRPA1 is a non-selective cation channel expressed by nociceptors. While it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions where reactive oxygen species and pro-inflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but not Trpa1-/- mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain.

Introduction

TRPA1 is a calcium-permeable, non-selective cation channel of the Transient Receptor Potential (TRP) superfamily expressed by sensory neurons in the dorsal root, trigeminal, and nodose ganglia (Story et al., 2003; Nagata et al., 2005). It is structurally and functionally unique, distinguished by a large N-terminal cytoplasmic domain containing more than a dozen cysteine residues and 18 predicted ankyrin repeats (Jaquemar et al., 1999). Covalent modification of these cysteines by reactive compounds is sufficient to activate TRPA1 (Hinman et al., 2006; MacPherson et al. 2007b).

This unique mechanism of activation positions TRPA1 as a broad irritancy receptor for endogenous and exogenous reactive compounds. Indeed, TRPA1 is activated by a range of environmental irritants, including mustard oil, acrolein, formalin, and reactive oxygen species (Bang and Hwang, 2009). Furthermore, mice lacking functional TRPA1 exhibit diminished pain behaviors in response to mustard oil and formalin (MacPherson et al., 2007a; Bautista et al., 2006; Kwan et al., 2006; Bessac et al., 2008; McNamara et al., 2007).

In addition to exogenous agonists, TRPA1 responds to a variety of endogenous reactive compounds associated with pathological pain conditions, including 4-hydroxynonenal (4-HNE), cyclopentane prostaglandins, and reactive oxygen species such as hydrogen peroxide (Trevisani et al., 2007; MacPherson et al. 2007a; Taylor-Clark et al., 2008; Materazzi et al., 2008; Cruz-Orenga et al., 2008; Andersson et al., 2008; Bessac et al., 2008). These findings, combined with the observation that TRPA1 antagonists are analgesic in rodent models of pain, suggests that TRPA1 plays an important role in some pathological pain states (McNamara et al.; 2007; Petrus et al. 2007; Eid et al., 2008).

Despite considerable study of the role of TRPA1 in cold pain, ambiguity remains (reviewed in Kwan and Corey, 2009). Some groups report that noxious cold directly activates TRPA1 (Story et al., 2003; Sawada et al., 2007; Karashima et al., 2009), while others suggest that cold-induced elevation of intracellular calcium indirectly causes this activation (Zurborg et al., 2007). Studies employing two independently made TRPA1 knock-out animals yield conflicting results, leaving the controversy unsettled (Kwan et al., 2006; Bautista et al., 2006; Karashima et al., 2009; Gentry et al., 2010).

We investigated the effect of cold on TRPA1 gating using electrophysiological recordings of both heterologously expressed rat TRPA1 and native TRPA1 from acutely isolated DRG neurons. Temperature reductions activated recombinant TRPA1 in a calcium-independent fashion, though to a much lesser degree than treatment with chemical agonists. However, even mild cooling substantially potentiated TRPA1 activation by reactive compounds.

We also examined the role of TRPA1 in cold sensation in vivo using behavioral assays. Cold responsiveness in wild type but not Trpa1-/- mice was profoundly increased by subcutaneous injection of 4-HNE at a non-nocifensive concentration. In rats, selectively antagonizing TRPA1 with HC-030031 reduced cold allodynia following nerve injury or inflammation, but had no effect on acute cold sensation. From these data, we postulate that TRPA1 is a critical mediator of cold hypersensitivity in pathological conditions. TRPA1 is thus a potential therapeutic target for pathological conditions accompanied by cold hypersensitivity.

Methods

Cells

Full-length rat TRPA1 cDNA was subcloned into pCDNA5/TO (Invitrogen, Carlsbad, CA). Stable clonal 293-T-REx cell lines (Invitrogen) were selected in 100 μg/ml hygromycin and grown according to the manufacturer's instructions.

Adult (9-12 week) rat DRG were dissected and incubated at 37°C in 3 μg/mL Collagenase Type I (Worthington Biochemicals) in PBS for 30 minutes. The collagenase solution was removed and the ganglia were incubated for 30 minutes at 37°C in 0.05% trypsin (Gibco) in order to dissociate the cells. The DRG preparation was resuspended and triturated in DMEM/F12 medium containing 10% FBS 0.5% penicillin-streptomycin and 0.5% glutamine. Neurons were cultured on poly-D-lysine (Sigma Aldrich) coated cover slips in this media. DRG neurons were used for electrophysiological recordings 16-24 hours after plating.

Electrophysiology

All currents were recorded in whole-cell configuration using EPC-9 and EPC-10 amplifiers and Patchmaster software (HEKA). Patch pipettes had a resistance of 1.5-3 MΩ and 60-75% of the series resistance was compensated. Our standard pipette solution consisted of 140mM CsAsp, 10 mM EGTA, 10 mM HEPES, 2.27 mM MgCl2, 1.91 mM CaCl2, 4 mM MgATP, and 0.1-0.3 mM Na2GTP, with pH adjusted to 7.2 with CsOH. In the experiments shown in Figure 2 panels B and C a different pipette solution was used containing 145 mM CsCl, 10 mM HEPES, 10 mM EGTA and 1 mM MgCl2 (pH 7.2 adjusted with CsOH). The standard bath solution contained 150 mM NaCl, 10 mM HEPES, 10 mM glucose, 4.5 mM KCl, 1 mM EGTA, 3 mM MgCl2, with pH adjusted to 7.4 with NaOH. In the recordings from primary dorsal root ganglia neurons shown in Figure 4 and in the indicated recordings from recombinant TRPA1 (Figure 2B and Supplemental Figures 3 and 4), 2 mM CaCl2 was added in place of EGTA and the concentration of MgCl2 was reduced to 1 mM.

Figure 2
Rapid Cooling Potentiates TRPA1 Currents After Activation by Agonist
Figure 4
Cooling Potentiates AITC or 4-HNE-Activated Currents in Isolated DRG Neurons

Data were collected either by continuous recordings at -60 mV or by applying voltage ramps from a holding potential of 0 mV every 4 s. Continuous recordings were collected at 400 Hz and digitally filtered off-line at 10 Hz for presentation. Voltage ramps were applied from -100 mV to 100 mV over the course of 400 ms, and data were collected at 10 kHz and filtered at 2.9 kHz. Inward and outward currents were analyzed from the ramps at -80 and 80 mV, respectively. Liquid junction potential correction was not used.

Solutions were switched using a gravity-fed continuous focal perfusion system. To achieve rapid temperature changes, two temperature control and perfusion systems were employed simultaneously. For temperatures ≥ 22°C, a Warner Instruments bipolar temperature controller (TC-344B) and inline heater (SHM-8) were used. For temperatures below 22°C a Warner Instruments temperature controller (CL-100) and thermal cooling module (TCM-1) were used. Final temperatures were confirmed using a thermistor (Voltcraft K101), with temperatures at the recorded cell estimated to be within +/- 2°C of those reported. While it was impossible to have a small enough thermistor in place to record the actual rate of temperature change during the experiments, we did record the rate of temperature change during a simulated experiment (i.e. without a cell present). These data are presented in Supplemental Figure 1, predicting that during recordings, the cell experiences each temperature change in less than 15 seconds

Chemicals

AITC, acrolein and CFA were purchased from Sigma-Aldrich (St. Louis, MO). 4-HNE was obtained from both Alexis Biochemicals (San Diego, CA) and EMD Chemicals (Gibbstown, NJ). HC-030031 was obtained from ChemBridge (San Diego, CA). All reagents were prepared fresh daily.

In vivo studies

All experiments involving rats were conducted in accordance with approved IACUC protocols from the relevant institutions. In addition all experiments were performed in accordance with the Society for Neuroscience Policies on the Use of Animals and Humans in Neuroscience Research. A 15 mg/mL homogenous suspension of HC-030031 was achieved by homogenization in 0.5% 4000-centipoises methyl cellulose. Male Sprague Dawley rats (Charles River) received a single intraperitoneal (IP) injection of 150 mg/kg HC-030031 or vehicle (0.5% methyl cellulose) 60 minutes prior to testing. Dose solution concentration was confirmed by analysis with LC/MS/MS.

4-HNE induced thermal hyperalgesia

All experiments involving mice were performed blind with respect to the genotype. Behavioral analysis was conducted on male and female mice 8-12 weeks old. TRPA1 mutant strain mice were obtained from David P. Corey and backcrossed to C57BL6/J for 5 generations. Animals were acclimated to their testing environment for at least 20 minutes prior to all experiments. Student's T test was used for all statistical calculations.

A clear Plexiglas chamber containing a Peltier-cooled cold plate, temperature controller and heat sink (TECA, Chicago, IL) was used to assess cold responses. Two independent temperature probes were used to confirm/monitor the surface temperature of the plate. Prior to assays, mice were acclimated in an equivalent chamber at room temperature for 20 minutes. 10 μL of 5 mM 4-HNE (50 nMoles) was injected subcutaneously in both hind paws. Mice were returned to their acclimation chambers for an additional five minutes before being placed on a 0° C cold plate. Nocifensive behavior was then assessed by counting the latency to the first jump, or the onset of brisk hindpaw lifts or flicking/licking of the hindpaws during a 5 minute period.

Thermal hyperalgesia data was conducted using a Plantar IR Analgesia Meter (Hargreaves's Apparatus) from Ugo Basile. Mice were acclimated in a Plexiglas container for one hour and averages of 3 exposures per paw, to IR intensity 20, were recorded.

Environmental Cold and CFA-induced cold allodynia

On the day prior to injection of CFA, the rats were pretested/acclimated—that is, submitted to the testing procedure. Paw Withdrawal Latency (PWL) for each naïve animal was determined. Briefly, each animal was carefully placed on a PE34 Cold Plate Analgesia Meter (IITC Life Science, Woodland Hills, CA) set to 5°C and the built in timer activated. The PWL was determined by observing the rat's behavioral response, i.e. a sustained period of stimulus avoidance (lifting/withholding) or a period of licking/biting. Simultaneous PWL readings for both hind paws were recorded. Following each reading, the cold plate was wiped clean of urine and feces and confirmed to be at 5°C before a subsequent animal was tested.

The following day, 100 μL of neat CFA was injected under the skin of the plantar surface of the left hind paw under brief gaseous anesthesia. Two days later, the rats were again tested to confirm hyperalgesia: only animals that had a 40% difference in PWL between the injected and un-injected paw were included in the study.

Animals were then randomized so each group had similar mean PWLs. Animals were dosed with either vehicle or HC-030031 under blinded conditions (the operator of the cold-plate was unaware as to what each animal had received). Following the 45 minutes post-treatment time, the post-dose PWL readings were taken. Upon completion of the PWL readings, the animals were sacrificed.

Since we saw no effect of HC-030031 on the withdrawal latency of the uninjured rat paw to 5° C, we also asked whether colder temperatures would unmask a potential role for TRPA1 in the detection of intense environmental cold, and exposed the rats to a cold plate at -5° C. In addition to measuring the PWL, we also counted the number of seconds of pain behavior over the course of five minutes. A cut-off time of five minutes was set to limit any possible tissue damage resulting from excessive exposure to the cold. At this time point there was no tissue injury detectable upon visual inspection.

Noxious cold detection

Animals were dosed with either vehicle or HC-030031 under blinded conditions (the operator of the cold-plate was unaware as to what each animal had received). 60 minutes post-treatment reovery each animal was carefully placed on a PE34 Cold Plate Analgesia Meter (IITC Life Science, Woodland Hills, CA) set to -5°C and the built in timer activated. The number of total responses during the 5 minute observation period was recorded as well as the latency to first response. Following each reading, the cold plate was wiped clean of urine and feces and confirmed to be at -5°C before a subsequent animal was tested. Upon completion of the PWL readings, the animals were sacrificed.

SNI –induced cold allodynia

Baseline cold sensitivity was assessed on a 10°C cold plate and animals were randomized into vehicle and drug treated groups. SNI surgery was performed as previously described (Decosterd and Woolf, 2000). Briefly, an axotomy and ligation of the tibial and common peroneal nerves was performed, leaving the sural nerve intact. The common peroneal and the tibial nerves were tight-ligated with 5.0 silk and sectioned distal to the ligation, removing 2-4 mm of the distal nerve stump. Muscle and skin were closed in two layers. Seven days after surgery, paw withdrawal latency was assessed again (pre-Rx). Animals were then subsequently injected intraperitoneally with HC-030031 or vehicle. One-hour post compound administration (post-Rx), paw withdrawal latency was again determined.

Methods for mechanical allodynia testing

Mechanical hyperalgesia data was conducted with a Dynamic Plantar Aesthesiometer from UGO Basile. Mice were acclimated for 60 minutes to the testing environment and an average of 4 exposures per paw were recorded. Baseline von Frey threshold or paw withdrawal latency was measured first and again at 15 minutes post injection of 4HNE (as above).

Statistics

For all experiments, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

Results

Noxious cold activates heterologously expressed TRPA1 channels

In an effort to understand the effect of temperature on TRPA1 gating, we first attempted to recapitulate previous findings showing that noxious cold activates TRPA1 (Story et al., 2003; Sawada et al., 2007; Karashima et al., 2009; Wang et al., 2008). We used whole-cell patch-clamp recordings from HEK cells stably transfected with rat TRPA1. Since Ca2+ permeating through the channel exerts complicated effects, both activating the current and accelerating its run-down, (Zuborg et al., 2007; Doerner et al., 2007), these hetrologous expression experiments were conducted in the absence of external Ca2+. To eliminate the possibility of nonspecific effects due to prolonged exposure to low temperatures, we used a perfusion system permitting rapid temperature changes, with a lag time of <15 seconds to completion of a temperature switch (see Supplemental Figure 1). At 25°C, small outwardly rectifying TRPA1 currents were observed during voltage ramps from -100 to 100 mV (Figure 1A, B). Reducing the temperature from 25°C to 10°C resulted in a clear potentiation of both the inward and outward currents. Once the temperature was increased back to 25°C, the current rapidly returned to pre-cooling levels. Importantly, the TRPA1-specific blocker HC-030031 virtually abolished the cold-induced current, strongly indicating that the currents were TRPA1-mediated.

Figure 1
Cooling Activates TRPA1 Currents

To rule out the possibility that residual intracellular calcium contributes to the activation of TRPA1 by cooling, we examined TRPA1 currents in the absence of intracellular calcium. To accomplish this, we released internal calcium stores with inositol 1,4,5-trisphosphate and chelated any remaining free calcium with BAPTA. Under these conditions, cooling still activated TRPA1 currents (data not shown). These results demonstrate that the cold-mediated activation of TRPA1 current is Ca2+-independent, as previously reported (Sawada et al., 2007; Karashima et al., 2009).

We next compared the cold-induced current increase with that generated, within the same cells, by addition of a sub-saturating concentration of the agonist AITC (Figure 1C). Lowering the temperature to 10°C increased the basal inward current about 10-fold, to ~100 pA. In contrast, addition of AITC increased the basal current ~300-fold, resulting in nearly 3,000 pA of inward current. In summary, while noxious cold clearly activates TRPA1, the amplitude of TRPA1 current activated by AITC is very much larger (Figure 1D).

Effect of temperature changes on agonist-evoked TRPA1 currents

Although the TRPA1 currents evoked by noxious cold alone are small, it is possible that noxious cold in combination with other activators of the channel could have a more substantial effect. We therefore studied the impact of cold on TRPA1 currents already activated by the addition of agonists. TRPA1 currents activated by 25 μM AITC at 25°C were strongly potentiated when the temperature was lowered to 10°C (Figure 2A). This potentiation was reversible, and completely blocked by HC-030031, indicating that it was TRPA1-mediated.

To determine whether the strong, cooling-induced potentiation of TRPA1 was limited to AITC-induced currents, we tested two other agonists of TRPA1. Currents activated by acrolein, a potent pulmonary irritant, were also strongly potentiated by cold (Supplemental Figure 2A). We also examined 4-Hydroxynonenal (4-HNE), a putative endogenous activator of TRPA1. Unlike AITC, 4-HNE irreversibly activated TRPA1. Thus, after removal of 4-HNE, the level of current remained stable for a prolonged period of time. Cooling from 25° C to 10° C substantially potentiated these persistent currents (Supplemental Figure 2B).

The cold-induced potentiation of TRPA1 currents in the presence of agonists conflicts with a previous report showing that AITC-activated TRPA1 currents decrease when the temperature is lowered from 30°C to 10°C (Karashima et al., 2009). Karashima et al demonstrated that in the presence of 5 μM AITC, slowly cooling cells heterologously expressing TRPA1 from 30°C to 10°C resulted in decreased currents. They noted that while the overall current amplitude decreased in this paradigm, the open probability of TRPA1 was increased by cooling. When we repeated the Karashima protocol by adding Ca2+ to the external solution and slowly cooling the cells from 30°C to 10°C over the course of five minutes, we saw results very similar to those previously reported (Supplemental Figure 3). We hypothesize that the discrepancy in the effect of cooling observed in these two protocols is due to significant calcium induced desensitization of TRPA1 observed with the very slow rate of cooling. Indeed, cold-induced potentiation of TRPA1 current was also observed in the presence of extracellular Ca2+ when the temperature was rapidly decreased (>10°C in 3 seconds, Figure 2B). We found that cold also augmented TRPA1 currents with the slower rate of cooling (~4°C/min.) used by Karashima et al. when extracellular calcium was absent (Figure 2C).

Although cooling cells from 30°C or 25°C down to 10°C clearly potentiated agonist-activated currents, we wondered whether potentiation of agonist-activated TRPA1 currents could be achieved by lowering the temperature within a more physiologically relevant range. Figure 3A demonstrates that cooling from 30°C to 20°C in the presence of 5 μM AITC potentiated TRPA1 currents by ~4-fold. Even when the AITC concentration was increased to 50 μM, this mild cooling enhanced the TRPA1 current by >2-fold (Figure 3B). In contrast, at 500 μM AITC, (a near-saturating concentration), cooling resulted in a small current decrease (Figure 3C), suggesting that the magnitude of cold potentiation decreases as higher levels of TRPA1 activation are achieved.

Figure 3
Magnitude of Cooling-Induced TRPA1 Potentiation is Dependent on the temperature and AITC Concentration

That mild cooling potentiates TRPA1 in the presence of agonists was also observed under more physiological recording conditions. We performed recordings starting at 35°C in the presence of 2 mM external Ca2+. In these conditions, cooling to 20°C in the presence of sub-saturating concentrations of AITC also led to a clear increase in current (supplemental Figure 4A, B) Overall, these results demonstrate that physiologically relevant fast cooling potentiates TRPA1 in the presence of an agonist.

Effect of cold on agonist-evoked native TRPA1 currents in rat DRG neurons

To examine whether cooling elicited similar effects on native TRPA1 currents, we tested the effect of cooling on acutely isolated adult rat dorsal root ganglia (DRG) neurons. To more accurately simulate the normal physiological environment, 2mM Ca2+ was included in the extracellular buffer in all these experiments. In multiple TRPA1-expressing neurons, currents activated by perfusion with either AITC (Figure 4A) or 4-HNE (Figure 4B) were reversibly potentiated 3-fold by cooling from 25°C to 10°C. Cold-potentiated currents were blocked by HC-030031, demonstrating that they were mediated by TRPA1. The cooling-induced current increase in the presence of agonists is quantified in Figure 4C. Note that in the absence of a chemical agonist, temperature reduction did not significantly alter baseline current (Figure 4A, B), indicating that the effect of cold on native TRPA1 is likely minimal in the absence of an agonist. However, cooling dramatically potentiates agonist-evoked TRPA1 currents in DRG neurons as it did after heterologous expression.

In vivo effects of 4-HNE on cold hypersensitivity

Since 4-HNE sensitized native TRPA1 currents to cold in vitro, we explored whether the same effects could be observed in vivo at the behavioral level. Cold-induced pain is often measured by monitoring nocifensive behaviors such as jumping or licking/flicking of the hindpaw after placement on a cold plate. Reports on the acute cold sensitivity differences between wild-type (WT) and TRPA1 null mice have been inconsistent (Bautista et al., 2006; Kwan et al., 2006; Karashima et al., 2009), perhaps because of procedural differences. We chose to examine the effect of 4-HNE on cold-elicited behavior using a 0°C cold plate to avoid confounding effects of cold sensing with tissue damage, which may occur at lower temperatures.

In contrast to previous reports (Trevisani et al., 2007), we found intraplantar injection of 5 mM 4-HNE was insufficient to elicit pain behavior at room temperature (data not shown). We examined whether this concentration of 4-HNE induced cold hypersensitivity. Indeed, injection of 4-HNE dramatically reduced the onset of nocifensive behaviors in response to a 0°C cold plate (16 ± 3.4 vs. 126 ± 44 seconds; p < 0.05, Figure 5A). The 4-HNE-induced cold sensitivity depended on TRPA1, as the effects were greatly diminished in TRPA1-deficient mice compared to wildtype littermates (p<0.05, Figure 5B). In the absence of 4-HNE injection, no significant difference in onset of cold-evoked behavior was observed between wild type mice and TRPA1-deficient littermates (Supplemental Figure 5). This demonstrates the prominent role of TRPA1 in cold nociception in the presence of TRPA1 agonists, and agrees well with our electrophysiological findings.

Figure 5
5 mM 4-HNE Induces TRPA1-Dependent Cold Allodynia

Injection of 5 mM 4-HNE also induced heat hyperalgesia and mechanical allodynia as detected by the Hargreave's plantar radiant heat test and assessment with von Frey filaments (Supplemental Figure 6). This increased sensitivity to heat and mechanical stimuli was observed in both Trpa1-/- and Trpa1+/+ mice, indicating that while 4-HNE-induced cold allodynia is dependent on functional TRPA1, the heat hyperalgesia and mechanical allodynia are not. Therefore, at least at these concentrations, 4-HNE can also affect nociceptive behavior through TRPA1-independent mechanisms. These results differ from previous reports suggesting that the mechanical allodynia caused by 4-HNE depends on expression of functional TRPA1 (Trevisani et al., 2007). Experimental differences may contribute to this apparent conflict. First, the concentration of 4-HNE used in our study (50 nmols) is much lower than that used by Trevisani et al (150 nmols). Second, we used a genetic disruption of TRPA1 to study its function, whereas the earlier results depend on gentamycin and camphor, which are weak and poorly selective inhibitors of TRPA1.

Cold hypersensitivity in pathological conditions

Since endogenous TRPA1 agonists such as reactive prostaglandins, reactive oxygen species and 4-HNE are produced during inflammatory responses, we investigated the role of TRPA1 in cold responses during the localized inflammation elicited by injection of CFA into the hindpaw in the rat. We measured paw withdrawal latency upon exposure to a 5°C cold plate at baseline, after CFA injection, and after treatment with HC-030031 or vehicle. 48 hours after intraplantar injection with CFA, the paw withdrawal latency had decreased compared to withdrawal latencies at baseline (Figure 6A 89.7 ± 16.6 seconds goes to 33.7 ± 9.9 seconds) similar to that previously reported (Allchorne et al., 2005). Treatment with HC-030031 (150 mg/kg IP) increased the paw withdrawal latency from 36.3 ± 2.8 seconds to 58.9 ± 5.3 seconds (n=7, Figure 6B). Similar results have been demonstrated with locally injected AP-18, another TRPA1 antagonist (Petrus et al., 2007). Vehicle treatment had no effect on the cold evoked paw withdrawal latency (Figure 6A). The observed increase in paw withdrawal latency by with HC-030031 in CFA treated animals likely represents a specific effecton the cold allodynia, since the paw withdrawal latency of the uninflamed (contralateral) paw remained unaffected (87.8 ± 3.8 seconds after treatment with HC-030031 compared to 84.8 ± 1.8 seconds, Supplemental Figure 7). Furthermore, animals treated with HC-030031 at this dose did not show any impairment in an accelerating rotarod assay (McNamara et al., 2007). The selectivity profile of HC-030031 (McNamara et al., 2007; Eid et al, 2008), further supports the observation that the observed analgesic effects are mediated through TRPA1.

Figure 6
HC-030031 Ameliorates CFA-Induced Cold Allodynia

Since we saw no effect of HC-030031 on the withdrawal latency of the uninjured paw to 5° C, we asked whether colder temperatures would unmask a potential role for TRPA1 in the detection of environmental cold. In naïve rats, we injected HC-030031 (150 mg/kg IP) one hour prior to determining paw withdrawal latency on a cold plate held at -5°C. Despite achieving similar plasma exposure as in the previous study (8±1 μg/mL), HC-030031 did not alter the paw withdrawal latency compared to vehicle-treated animals and had no effect on the number of elicited pain behaviors observed over 5 minutes (Figure 7A; n=16, and Figure 7B; average plasma level of 7 ± 4 μg/mL; n=8).

Figure 7
HC-030031 Has No Effect on the Detection of Noxious Cold in the Absence of CFA Injection

Finally, we investigated whether TRPA1 is involved in the cold hypersensitivity that comprises a prominent part of the neuropathic pain phenotype in the spared-nerve injury model. At the pre-injury baseline, vehicle-treated and HC-030031-treated rats showed similar paw withdrawal latencies when placed on a 10° C cold plate (137.6 ± 6.2 compared to 148.3 ± 1.7 seconds; Figure 8B, C). To induce nerve injury, we ligated the tibial and common peroneal nerve, resulting in greatly reduced paw withdrawal latencies after 7 days similar to previous reports (Allchorne et al., 2005). Treatment with HC-030031 significantly increased paw withdrawal latency, to 96.8 ± 12.1 seconds (150 mg/kg IP; 2-way ANOVA, p<0.001, Figure 8A, C), while there was no significant change in the paw withdrawal latency of animals treated with the vehicle (63.7 ± 13.1 seconds, Figure 8A, B). Plasma levels of HC-030031 at the time of sacrifice were similar to those seen in previous experiments (11 ± 1 μg/mL). We conclude that TRPA1 is a significant contributor to cold allodynia in both neuropathic and inflammatory pain states.

Figure 8
Nerve Injury in Rats Induces Cold Allodynia that is Ameliorated by Blocking TRPA1

Discussion

TRPA1 was initially proposed to be a noxious cold receptor (Story et al. 2003), a hypothesis that was later contested (Jordt et al 2004; Zurborg et al. 2007; Sawada et al. 2007). Since then the role of TRPA1 in the detection of noxious cold has remained uncertain because genetic deletion of TRPA1 failed to resolve the controversy (Kwan et al., 2007; Bautista et al., 2007; Karashima et al., 2009; Fajardo et al., 2008; Madrid et al., 2009; Knowlton et al., 2010). Our results provide new evidence that supports a prominent role for TRPA1 in cold hypersensitivity rather than cold detection. Once TRPA1 has been activated by an agonist, we find both in vitro and in vivo that TRPA1 plays a substantial role in driving cold allodynia.

Effect of cold on TRPA1 current activation

Our results demonstrate that cold can activate TRPA1 in a heterologous expression system, but the current amplitude activated by cold alone is 1/30th of that activated by a submaximal concentration of AITC, the reactive chemical that underlies the pungency of mustard. Cooling in combination with exposure to AITC produces a much more substantial increase in TRPA1 current amplitude. This cooling-elicited current potentiation also occurs following activation by a presumed endogenous activator 4-HNE or the pulmonary irritant acrolein. Cooling induced current potentiation occurred across TRPA1 clones from different species (rat, mouse, human; DdC, unpublished data) and across a variety of recording conditions, in the presence or absence of extracellular Ca2+. Native TRPA1 currents in acutely isolated adult DRG neurons also showed marked sensitivity to cooling after stimulation with an agonist, and behavioral experiments with 4-HNE and cold are in agreement with our electrophysiological data.

The mechanism of cooling-induced potentiation of TRPA1 current could, in principle, be the result of effects on single channel conductance, open probability, the number of TRPA1 channels expressed in the membrane, or a combination of these (Schmidt et al., 2009). It has been shown that while cooling decreases the single channel conductance of TRPA1, it increases the open probability of this channel, even in excised inside-out patches (Sawada et al., 2007; Karashima et al., 2009). The simplest mechanism by which cooling might enhance agonist-activated current is by increasing the open probability of the channel, through either direct or indirect effects on channel gating. Ultimately, the interplay between changes in open probability and decrease in single channel conductance will determine net effect on current. This hypothesis is consistent with our observation that cooling has the greatest effect when agonist concentrations are low; at higher agonist concentrations, the open probability is already so high that any additional increase caused by cooling is insufficient to overcome the associated decrease in single channel conductance

In the absence of external Ca2+, the cold potentiation of agonist-induced TRPA1 currents is very clear regardless of the rate of cooling. In the presence of Ca2+, though, the situation is more complicated due to the dual effect of Ca2+, which causes both activation and desensitization of the channel. In fact, we found that while a very slow cooling rate (30° C to 10° C in about 5 min) leads to current decrease in the heterologous system, a faster cooling rate (30° C to 10° C in ~15 s) causes clear current potentiation. A possible explanation for this result is that during slow cooling, the combined effect of decreased single channel conductance and Ca2+-induced desensitization are dominant, inhibiting the current. Conversely, with faster cooling the combined agonist effects of cold and Ca2+ may be more prominent, causing current potentiation. Regardless of mechanism, we find that even a decrease in temperature in the innocuous range from 30°C to 20°C potentiates TRPA1 currents when low concentrations of AITC are present. Thus, although it is has been suggested that TRPA1 is only activated by noxious cold temperatures (Story et al., 2003), our results indicate even mild cooling can have profound effects on TRPA1 activation in vitro and in this way could contribute to cold allodynia (pain in response to a normally innocuous level of cooling).

TRPA1 in the detection of environmental cold

In contrast to TRPV1 (Caterina et al., 2000) and TRPM8 (Dhaka et al., 2007; Colburn et al., 2007; Bautista et al., 2007), which are thermosensors that contribute to thermal preference in the absence of agonist, we observe only a limited role for TRPA1 in normal cold sensation. In our expression system, we saw a small tonic TRPA1 current at 25° C that was weakly potentiated by cold, whereas in acutely isolated rat AITC-responsive DRG neurons, we found that noxious cold (10° C) did not elicit a clear response from neurons. These results are very similar to recent findings looking at cold sensation in trigeminal neurons. Madrid and colleagues showed that only a very small percentage of AITC-responsive neurons were also cold responsive and that the percentage of cold sensitive neurons was not decreased by genetic deletion of TRPA1 (Madrid et al., 2009). In vivo, we observed no significant difference in cold sensitivity between WT and Trpa1-/- mice. This is in agreement with the study of Bautista et al 2006, even though we used one of the lines generated by Kwan and colleagues (Kwan et al., 2006). Similarly, when we turned to a selective antagonist of TRPA1, HC-030031, we saw no effects on either the number of pain behaviors or the latency to the first pain behavior of rats placed on a cold plate held at -5° C. There was also no difference in the latency to pain behaviors at 5° C. These data suggest that acute TRPA1 inhibition does not affect cold sensation in naïve animals. While Karashima and colleagues also failed to detect a difference in time to onset of pain behaviors on the cold plate, they did see a difference in the number of times the WT mice responded compared to Trpa1-/- animals. More recently, Bevan and colleagues developed a novel cold plate assay that measures paw withdrawal latencies of lightly restrained mice, making sure that there is full contact between the paws and the plate (Gentry et al., 2010). Using this method, they observe robust deficits in cold nociception in both TRPM8- and TRPA1-deficient mice. The discrepancy between the various cold nociception data sets is likely due to differences in methodologies and assay sensitivity. Finally, while our results indicate that TRPA1 may not contribute substantially to the detection of noxious cold in the DRG neurons, as also shown in trigeminal neurons (Madrid et al., 2009), a recent report suggests that TRPA1 mediates the detection of cold in nodose sensory neurons (Fajardo et al., 2008). Thus, it is possible that TRPA1's function differs in different neurons, perhaps as a result of subcellular milieu.

TRPA1 contributes to cold allodynia under pathological conditions

While we observed no difference in normal cold sensation between WT and Trpa1-/- animals, injection of the TRPA1 agonist 4-HNE led to a profound TRPA1-dependent cold hypersensitivity. These data agree with our findings in adult rat DRG neurons: 4-HNE-activated currents were strongly potentiated by lowering the temperature. To determine the role of TRPA1 in cold hypersensitivity in pathological pain states, we studied cold responses during acute local inflammation induced by intraplantar injection of CFA or after nerve injury induced by surgical ligation. In both of these paradigms, treating rats with the specific TRPA1 antagonist HC-030031 yielded similar results, significantly decreasing cold allodynia. In agreement with our results, recent reports indicate that CFA-induced cold hypersensitivity to tetrafluoroethane is also greatly reduced by treatment with HC-030031 (da Costa et al., 2010).

In contrast to previous reports that the role of TRPA1 in pathological pain conditions was due to an increase in expression levels, (Obata et al., 2005; Ji at al., 2008) we see no increase in TRPA1 transcript in the ipsilateral DRG after either SNI or CFA-injection (unpublished data). While this work does not directly address which TRPA1 activator or activators lead to the sensitization in these models of peripheral inflammatory and neuropathic pain, we posit that endogenous TRPA1 agonists present in pathological pain states potentiate the effects of cold. Taken together our in vitro and in vivo results suggest that blocking TRPA1 activity could reverse cold allodynia in pathological conditions.

Supplementary Material

Supp1

Acknowledgments

The authors would like to thank JoAnn Witek for tissue culture support and Jennifer Monsen for formulation work. We are grateful to Timothy Strassmaier, Xiaoguang Zhen, David Kimball and David Clapham for helpful comments on the manuscript. This work was supported in part by grants from the National Institutes of Health (grant NS039518 to C.J.W.)

Abbreviations used

TRP
Transient Receptor Potential
r
Rat
h
Human
AITC
allyl Isothiocyanate
4-HNE
4-hydroxy nonenal
DRG
dorsal root ganglia
CNS
central nervous system
IP
intraperitoneal

Footnotes

Conflict of interest statement: C.J.W. is serving on the scientific advisory board of Hydra Biosciences, Cambridge, MA. D.d.C., S.M., M.H., C.A.C., M.Z., M.D'A., N.D., N.J.H., J.A.C., C.M.F., M.M.M. are employees of Hydra Biosciences and receive options.

Author contributions: D.d.C. performed electrophysiological experiments, designed experiments, analyzed data, wrote the paper and supervised the project. S.M. performed electrophysiological experiments and analyzed data. M.H. performed electrophysiological experiments, analyzed data and wrote the paper. L.B.B. designed and performed SNI experiments. T.J.E. and M.J.P. performed mouse in vivo experiments. C.A.C. performed rat in vivo experiments. M.Z. performed rat in vivo experiments. M.D'A. performed analytical work. N.D. performed electrophysiological experiments and wrote the paper. G.J.B. performed SNI experiments. M.C. performed SNI experiments. N.J.H. wrote the paper. J.A.C. analyzed data and wrote the paper. C.M.F. analyzed data and wrote the paper. C.J.W. designed experiments, analyzed data, wrote the paper. A.P. designed experiments, analyzed data, wrote the paper. M.M.M. designed experiments, analyzed data, wrote the paper, and supervised the project.

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