The primary hypothesis driving our investigation described here is that pre-treatment with 15d-PGJ2 protects against TRPA1-mediated nociception and hypersensitivity via inhibition of TRPA1 signaling in nociceptive neurons. Our behavioral data correspond to our observations in DRG sensory neurons. Our results obtained using the acute AITC model suggest that 15d-PGJ2 is sufficient to inhibit acute TRPA1-dependent nociception. Moreover, our findings support that a TRPA1-dependent mechanism contributes to the ability of 15d-PGJ2 to reduce established inflammatory mechanical hypersensitivity. Finally, we have probed the utility of 15d-PGJ2 (a TRPA1 agonist) as a potential therapeutic and find that analgesic effects are maintained over the course of days with repeated dosing.
All TRPA1 agonists identified to date induce nociceptive behavior in rodents or burning sensations in humans (for review) [31–33]. Similarly, we and others have shown that 15d-PGJ2 (10–47.4 μg/paw) causes robust nocifensive behavior in WT, but not TRPA1−/−, mice [6, 7]. These data suggest that 15d-PGJ2 (at these concentrations) induces nocifensive behavior that is specific to TRPA1 activation. In contrast, several studies have identified potent anti-nociceptive properties of 15d-PGJ2. These studies do not report nocifensive behavior in response to intrathecal (100 μg/site), intraganglionar (100 ng/site), ipl. (30–300 ng/paw) or temporomandibular joint (10–100 ng/site) injection of 15d-PGJ2. Concentrations of 15d-PGJ2 shown to inhibit nociception in these studies are typically lower than those we have tested here (474 ng-47.4 μg/paw) [6, 9, 19, 20].
In our view, it remains unclear whether the high concentrations of endogenous ligands we and others have tested for their nociceptive- and anti-nociceptive effects are physiologically relevant to pain modulation or to TRPA1 modulation in vivo. For example, inflammatory exudate concentrations of 15d-PGJ2 rise coincident to the resolution of the inflammatory state. Levels detected range between ~0.5 to 5 ng/ml—orders of magnitude lower than those we and others have potentially introduced by ipl. injection [34, 35]. There is currently no in vitro data on the time course or kinetics of occupancy of TRPA1 receptors by 15d-PGJ2. We are actively attempting to investigate this question and resolve the putative differences in agonist modulation at the channel level. We also currently have no method of ascertaining whether we are actively inhibiting TRPA1 channels at sub-cutaneous nociceptive fibers at the concentration(s) and dosing paradigm(s) we have utilized. However, 15d-PGJ2 is not broken down further enzymatically or non-enzymatically. Though it is a reactive electrophile, 15d-PGJ2 is highly stable in aqueous phase; when aqueous solutions are spiked with radioactively labeled 15d-PGJ2, the compound is 100% recoverable after 48 hours .
This prostaglandin metabolite also has anti-inflammatory and anti-nociceptive effects that are TRPA1-independent. For example, Napimoga et al. (2008) reported the anti-nociceptive property of 15d-PGJ2 is mediated in part by PPARγ and peripheral opioid receptors, as antagonists targeting both receptors inhibited effects of 15d-PGJ2 against PGE2-induced “hypernociception.” This study also examined the anti-nociceptive effect of 15d-PGJ2 in the formalin model of temporomandibular joint inflammation and ipl. delivery of carrageenan, finding that it attenuated mechanical “hypernociception” in both models. They hypothesize that increases in the macrophage population in the periphery contributed to the enhancement the anti-nociceptive effects of 15d-PGJ2
. Whereas we are examining its action in strictly the pain neuraxis involving primary TRPA1-expressing peripheral nociceptors, perhaps the effects of 15d-PGJ2 on macrophages and/or opioids are downstream of signaling through primary afferents expressing TRPA1. For instance, activation of TRPA1-expressing nociceptors could result in their release of factors that contribute to an immune response (i.e. 15d-PGJ2 release) or endogenous opioid signaling.
Differential behavioral and cellular effects of TRPA1 agonists
Previously published studies characterizing single-dose AITC-induced nocifensive responses in mice have utilized injections of 0.1-0.75% (10–75 mM, ipl.), while most behavioral desensitization studies using repeated AITC stimuli have utilized ≤ 20 mM. (e.g. [3, 26, 27]). Therefore, we compared behavioral desensitization to repeated AITC- and 15d-PGJ2-stimuli at two concentrations within this range. Using our dosing protocol, nocifensive responses to AITC did not desensitize. In contrast, 15d-PGJ2 evoked a significant desensitization to itself and AITC. Unlike previous studies using multi-dose protocols, we have administered agonists 1 h apart, which might account for the differences between our results and those of other studies, which report desensitization [26, 29, 37] or sensitization  by AITC. We hypothesize that the difference in the effects of 15d-PGJ2 and AITC on heterologous and homologous inhibition might result via differential acute and pharmacological desensitization of TRPA1 in vivo produced by the two agonists at various concentrations. Furthermore, topically applied and injected AITC causes an inflammatory response, including plasma extravasation and tissue edema, while there is no evidence supporting that 15d-PGJ2 promotes such responses [38, 39].
To our knowledge, this is the first study to closely examine bidirectional cross-inhibition of DRG neurons by exposure to two different TRPA1 agonists. The mechanisms underlying the different effects of AITC and 15d-PGJ2 remain unclear (see also below). Since Ca2+ imaging does not directly measure desensitization, electrophysiological and further cellular studies are warranted to directly measure the mechanisms of desensitization, as well as desensitization kinetics, stimulus- and concentration-dependence. Key to our conclusions regarding inhibition of TRPA1 signaling by 15d-PGJ2, we have attempted to make our stimulus and recording approach(es) as systematic as possible in order to reveal differential modulation of TRPA1 by 15d-PGJ2 vs. AITC. First, our review of the available literature indicated to us that ~100 μM concentrations of 15d-PGJ2 and AITC are saturating in calcium imaging analyses of cultured sensory neurons [7, 40]. We have confirmed these findings using our DRG culture and stimulus protocols. Similar to previously published EC50 data derived from heterologous expression studies of TRPA1 channels, we found 15d-PGJ2 and AITC to be of similar potency in their activation of TRPA1-expressing DRG neurons [6–8, 11, 40].
In DRG neurons, we observed strong homologous desensitization of AITC responses and cross-desensitization of 15d-PGJ2 in response to a pre-pulse of 100 μM AITC. Similarly, 100 μM 15d-PGJ2 inhibited subsequent responses to itself and to 100 μM AITC. Our results agree with those of Taylor-Clark et al. who reported that pre-treatment of trigeminal sensory neurons with 100 μM AITC significantly diminished subsequent responses to both 100 μM AITC and 15d-PGJ2
. While the majority of our studies examined the inhibitory efficacy of 100 μM 15d-PGJ2, we also investigated cross- and homologous desensitization using 50 μM concentrations of these compounds. We found that 50 μM AITC no longer exhibited these properties, whereas 15d-PGJ2 did at this lower concentration. Our data evince different magnitudes of homologous and heterologous inhibition evoked by these two compounds, each with steep time- and concentration-dependence, again the mechanisms of which remain uncertain.
A number of reports have demonstrated that similar to heat- and capsaicin-activated TRPV1, TRPA1 exhibits strong desensitization and tachyphylaxis , [29, 40]
. However, a more recent study partially refutes these data, showing that TRPA1 also undergoes sensitization and trafficking to the plasma membrane in response to AITC and inflammatory mediators in vitro and in vivo
. On balance, the results of our current study and those of previous studies reveal that sensitization/desensitization could be dependent on many variables, including agonist concentration, stimulus sequence, stimulus duration and inter-stimulus interval.
TRPV1 and TRPA1 are co-expressed in DRG neurons with TRPA1 labeling a subset of TRPV1 neurons . Studies of the two channels in CHO cell overexpression and DRG neurons show that when co-expressed, TRPA1 undergoes heterologous and homologous desensitization via CAP (TRPV1 agonist) and AITC stimuli, respectively. A proposed mechanism of TRPA1 desensitization in this manner is through TRPV1-directed internalization [26, 29]. It is intriguing that AITC is able to inhibit subsequent behavioral and neuronal responses to CAP, but 15d-PGJ2 (at the concentrations we have tested) is not, thus further highlighting the differential effects of these two TRPA1 agonists. Similar to Taylor-Clark et al. but in contrast to the findings of other groups, we find that pre-treatment with a TRPA1 agonist (in our case, 15d-PGJ2) in sensory neurons does not inhibit subsequent responses to CAP . Correspondingly, we also did not observe behavioral desensitization to CAP as a result of 15d-PGJ2 pre-treatment, indicating that the inhibition is TRPA1 specific.
Although both compounds are electrophiles that activate TRPA1 via covalent modification of key cysteine (or other) residues, it is quite plausible that AITC and 15d-PGJ2 activate the channel differently, perhaps via differential modulation of required cysteines. For instance, there is indirect evidence that the cysteine required for 15d-PGJ2 activation in human TRPA1 is different than that required for activation by other compounds . Perhaps also the two compounds induce differential trafficking to the plasma membrane [see  that is dependent on concentration. Finally, intracellular signaling cascades upon TRPA1 activation could be ligand-dependent, but this has never been shown. These possibilities raise an interesting avenue of research and could be addressed in mutagenesis studies combined with approaches such mass spectroscopy.
TRPA1 and mechanosensitivity
We hypothesize that sufficient doses of 15d-PGJ2 effectively inhibit TRPA1 by desensitizing the channel. By applying this concept in our behavioral approaches to understanding the modulation of TRPA1 by 15d-PGJ2 in the CFA model of inflammatory hypersensitivity, we have essentially obtained the same results using a channel agonist as those studies which have utilized channel antagonists [21–23, 42]. While 15d-PGJ2 had no effect on naïve mechanical thresholds nor perturbed the development of mechanical hypersensitivity, it inhibited established inflammatory mechanical hypersensitivity in WT, but not TRPA1−/− mice. Although it appears that TRPA1 expression in peripheral mechanonociceptors is not required for the development of mechanical hypersensitivity, it is possible that sustained mechanical hypersensitivity results in part through an autocrine or paracrine positive feedback mechanism involving TRPA1. As TRPA1 is activated by products of cellular stress, factors released by mechanical damage could feed back on the channel. However, this possible signaling pathway does not resolve the conundrum of why TRPA1−/− mice develop and maintain mechanical hypersensitivity, but TRPA1 antagonists can inhibit inflammatory and neuropathic hypersensitivity via “on-target” mechanisms.
Conclusions and future directions
A goal of our studies was to determine whether 15d-PGJ2, or any strongly desensitizing TRPA1 agonist, could be useful as a pain therapeutic. For example, for a similar channel TRPV1, capsaicin is the active ingredient in over-the-counter and prescription topical treatments and provides effective relief of joint pain based on its ability to desensitize TRPV1-expressing fibers. Thus, TRPA1 agonists could serve a similar therapeutic role. Here we describe experiments in which we administered 15d-PGJ2, beginning 1 day after the induction of inflammation. Our dosing strategy repeatedly produced a ~2 h block of mechanical hypersensitivity as shown using the von Frey test. Our studies show the effectiveness of administering such a dose after, but not before, the development of inflammation or neuropathic injury. This is in accordance with the nature of pain treatment, which is given after a patient reports discomfort from inflammation or injury. With the route and concentration we tested in our multi-dose experiment (15 mM, ipl.), 15d-PGJ2 initially causes nocifensive responses, but appears to have no pro-inflammatory or neurotoxic effects on TRPA1-expressing neurons. Future studies will further test the efficacy of 15d-PGJ2 as a therapeutic by determining a dose or route of administration (e.g. oral or topical application) that causes minimal pain, while providing analgesic benefits.