TRPA1-dependent induction of acute pain behavior and mechanical hyperalgesia by OxPAPC in mice.

<p>(A) The acute nocifensive behavior of mice quantified within the first 5 min following injection of vehicle (Veh), OxPAPC or OxPAPC+HC-030031 injection into the hind paw, plotted in 1 min intervals. (B) Cumulated nocifensive behavior response time within the total 5 min recording period of animals in experiment Fig 5 (A). (C) Paw withdraw threshold (PWT) of mice measured by von Frey hair analysis. Tests were performed 1 hour after vehicle, OxPAPC or OxPAPC+HC-030031 injection into the hind paw. n = 7–8 mice/group, **p < 0.01 <i>vs</i>. Veh, ^##p < 0.01 <i>vs</i>. OxPAPC group.</p

Detection of OxPAPC in the inflamed plantar skin of mice after establishment of chronic pain in the CFA model.

<p>(A) Representative immunofluorescence images of EO6 antibody staining of OxPAPC in plantar tissue of mice from vehicle (Veh)-, CFA-treated groups and no 1^st antibody added control (No 1^st Ab Contl) group. Tissues were collected 7 days after CFA or vehicle injection. Areas staining positive for EO6 antibody are shown in green. Nuclei were labeled with DAPI (blue). (B) Summary of the % increase in fluorescence of EO6 staining as shown in (A). n = 5 mice/group, **p < 0.01 <i>vs</i>. No 1^st Ab Contl group, ^##p < 0.01 <i>vs</i>. Veh group, NS: no significance.</p

OxPAPC-induced TRPA1 activation is independent of EP2 and DP receptors in both HEK293 cells and native DRG neurons.

<p>(A) Summary of OxPAPC-induced Ca²⁺ responses in HEK293 cells expressing hTRPA1. Cells were first superfused with vehicle only (0.05% DMSO), AH6809 (10 μM), PF04418948 (20 nM) or ruthenium red (10 μM) for 5 min before recording started and then recorded in the continued presence of above treatments. Cells were challenged with OxPAPC (30 μM) and subsequently with mustard oil (MO, 70 μM) and ionomycin (1 μM). Responses of > 50 cells were averaged from each group. (B) Average peak amplitudes of OxPAPC-induced Ca²⁺ responses in HEK293 cells as shown in (A). (C) Percentages of mouse DRG neurons responding to OxPAPC in vehicle only, or in the presence of AH6809 (10 μM) or PF04418948 (20 nM). Neurons were pretreated with the antagonists for 5 min before recording and recorded in the continued presence of the treatments. Cells were challenged with OxPAPC (10 μM) for 100 s and subsequently with KCl (40 mM) for 40 s. n = 5–8 tests/group, averages from 100–200 neurons per group. (D) Maximal Fura-2 emission ratio amplitudes of OxPAPC-induced Ca²⁺ responses in mouse DRG neurons recorded in (C). **p < 0.01 <i>vs</i>. Veh, NS: no significance (p > 0.05).</p

Covalent modification sites in TRPA1 are essential for OxPAPC sensitivity.

<p>(A) Representative Ca²⁺ imaging traces showing responses of hTRPA1 and hTRPA1 mutant (3CK mutant)-expressing HEK293 cells following application of OxPAPC (10 μg/ml) and carvacrol (300 μM). (B) Summary of the Ca²⁺ responses induced by OxPAPC and mustard oil (MO, 70 μM). Increase in [Ca²⁺]_i is displayed as percentage of [Ca²⁺]_i activated by a saturating dose of carvacrol (300 μM). n = 5–6 tests/group. Each test contains up to 30 cells. **p < 0.01 <i>vs</i>. hTRPA1 group.</p