Petrus M et al. (2007), A role of TRPA1 in mechanical hyperalgesia is r...

XB-ART-37564

Mol Pain 2007 Dec 17;3:40. doi: 10.1186/1744-8069-3-40.

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

Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, Jegla T, Patapoutian A.

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Mechanical hyperalgesia is a clinically-relevant form of pain sensitization that develops through largely unknown mechanisms. TRPA1, a Transient Receptor Potential ion channel, is a sensor of pungent chemicals that may play a role in acute noxious mechanosensation and cold thermosensation. We have developed a specific small molecule TRPA1 inhibitor (AP18) that can reduce cinnameldehyde-induced nociception in vivo. Interestingly, AP18 is capable of reversing CFA-induced mechanical hyperalgesia in mice. Although TRPA1-deficient mice develop normal CFA-induced hyperalgeisa, AP18 is ineffective in the knockout mice, consistent with an on-target mechanism. Therefore, TRPA1 plays a role in sensitization of nociception, and that compensation in TRPA1-deficient mice masks this requirement.

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Species referenced: Xenopus
Genes referenced: bdkrb2 tbx2 trpa1 trpm8 trpv1 trpv2 trpv3 trpv4

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	Figure 1. AP18 blocks TRPA1 activation. (A) Chemical structures of the AP18 (upper) and cinnamaldehyde (lower). (B) Dose-response relationships for block of the calcium influx by AP18 into CHO cells expressing mouse and human TRPA1 elicited by 50 μM cinnamaldehyde (left panel). Calcium influx was measured using a standard FLIPR assay, data points are the average of four wells (~8,000 cells/well) and error bars show standard error. Values are normalized to the maximal response (observed in the absence of AP18). (C) Ratiometric Ca2+ imaging of average of ~50 mTRPA1-expressing CHO cells in response to 1 mM Iodoacetamide (IA) and 100 μM of AP18 (black trace). Grey trace represents cells that were treated with IA alone. (D) Current-voltage relationship of TRPA1. Outward rectifying currents elicited by cinnamaldehyde (left panel) in inside-out macropatches derived from TRPA1-expressing Xenopus oocytes were suppressed by AP18 coapplications (right panel).
	Figure 2. AP18 specifically blocks TRPA1. Calcium influx (FLIPR) in HEK cells transiently transfected with the indicated cDNAs in response to 200 nM capsaicin (TRPV1), 150 μM 2-APB (TRPV2), 30 μM 2-APB (TRPV3), 20 μM 4alpha-PDD (TRPV4), 20 μM menthol (TRPM8), and 80 μM cinnamaldehyde (TRPA1). Values are given as a percentage of agonist response in the absence of AP18.
	Figure 3. AP18 suppresses acute nociceptive behaviours caused by cinnamaldehyde but not capsaicin. Time spent licking and flicking hindpaws injected with cinnamaldehyde (16.4 mM) or capsaicin (0.328 mM) was measured for five minutes and compared with animals co-injected with AP18 (1 mM). Numbers of cases for each experiment from the left is 8, 8, 6 and 6, respectively (*p < 0.05, two-tailed Student's t-test).
	Figure 4. TRPA1 is involved in CFA-induced mechanical hyperalgesia. (A) AP18 reverses CFA-induced mechanical hyperalgseia in wildtype mice (n = 8). Red symbols represent the paws treated with CFA. 24 hours later, mechanical sensitivity was assayed again (after CFA timepoint), and the same paws were injected with either AP18 (red circles) or vehicle (red triangle). Mechanical sensitivity was again monitored during the next four hours. Blue symbols represent the control uninjected paws. (B) AP18 reverses mechanical hyperalgesia in wildtype (red) but not TRPA1-deficient (green) littermates (n = 12, females). Same protocol as (A), data from CFA- and AP18-injected paws shown. Arrows indicate time of AP18 (or vehicle) injections. *p < 0.001, p < 0.01, *p < 0.05, two-tailed Student's t-test.
	Figure 5. AP18 does not reverse CFA-induced heat hyperalgesia. Same designation of symbols as in Figure 4A is used. Arrow indicates time of AP18 (or vehicle) injections. Paw-withdrawal latencies to infrared 20 setting are measured and averaged (n = 8).
	Figure 6. Acute mechanosensory phenotype in TRPA1-deficient mice. Frequency of paw withdrawal to fixed forces applied to the hindpaw is measured. 13 WT and 11 TRPA1-deficient male mice were tested. Briefly, hindpaws were exposed to the force actuator starting with the greatest force. Each force was applied eight times at ~1/s. The actual g-force values used differ between this study and Kwan et al. (2006) probably due to the use of different instrumentation [16].
	Figure 7. Bradykinin (BK)-induced mechanical hyperalgesia is partially blocked by AP18. Same designation of symbols as in Figure 4A is used; however, BK (instead of CFA) is co-injected with AP18 in wildtype mice, and mechanical sensitivity is measured within 15 minutes (n = 12). Arrow indicates time of AP18 (or vehicle) injections. **p < 0.001, p < 0.05, two-tailed Student's t-test.
	Figure 8. AP18 partially reverses cold hyperalgesia in rats treated with CFA. Same designation of symbols as Figure 4A is used. Number of flicks, licks, paw raises are counted for 10 minutes and averaged on a 5°C cold plate (n = 8). Arrow indicates time of AP18 (or vehicle) injections. *p < 0.05, two-tailed Student's t-test.

References [+] :

Alessandri-Haber, A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators. 2006, Pubmed