Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain
Yi Dai, Shenglan Wang, Makoto Tominaga, Satoshi Yamamoto, Tetsuo Fukuoka, Tomohiro Higashi, Kimiko Kobayashi, Koichi Obata, Hiroki Yamanaka, Koichi Noguchi
Yi Dai, Shenglan Wang, Makoto Tominaga, Satoshi Yamamoto, Tetsuo Fukuoka, Tomohiro Higashi, Kimiko Kobayashi, Koichi Obata, Hiroki Yamanaka, Koichi Noguchi
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Research Article Neuroscience

Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain

Abstract

Proinflammatory agents trypsin and mast cell tryptase cleave and activate PAR2, which is expressed on sensory nerves to cause neurogenic inflammation. Transient receptor potential A1 (TRPA1) is an excitatory ion channel on primary sensory nerves of pain pathway. Here, we show that a functional interaction of PAR2 and TRPA1 in dorsal root ganglion (DRG) neurons could contribute to the sensation of inflammatory pain. Frequent colocalization of TRPA1 with PAR2 was found in rat DRG neurons. PAR2 activation increased the TRPA1 currents evoked by its agonists in HEK293 cells transfected with TRPA1, as well as DRG neurons. Application of phospholipase C (PLC) inhibitors or phosphatidylinositol-4,5-bisphosphate (PIP2) suppressed this potentiation. Decrease of plasma membrane PIP2 levels through antibody sequestration or PLC-mediated hydrolysis mimicked the potentiating effects of PAR2 activation at the cellular level. Thus, the increased TRPA1 sensitivity may have been due to activation of PLC, which releases the inhibition of TRPA1 from plasma membrane PIP2. These results identify for the first time to our knowledge a sensitization mechanism of TRPA1 and a novel mechanism through which trypsin or tryptase released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

Authors

Yi Dai, Shenglan Wang, Makoto Tominaga, Satoshi Yamamoto, Tetsuo Fukuoka, Tomohiro Higashi, Kimiko Kobayashi, Koichi Obata, Hiroki Yamanaka, Koichi Noguchi

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Figure 3

PLC activation, but not its downstream products, sensitized AITC- and cinnamaldehyde-activated currents in transfected HEK cells expressing hTRPA1.

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PLC activation, but not its downstream products, sensitized AITC- and ci...
Cells were perfused with AITC or cinnamaldehyde solution for 20 seconds in all experiments. (A) SL-NH2–mediated (100 μM) potentiation of AITC-activated currents is a PLC-dependent event. AITC (100 μM) was reapplied 60 seconds after exposure to bath solution with or without SL-NH2 or LR-NH2. Currents were normalized to values first induced by AITC application in the absence of SL-NH2 or LR-NH2. In some experiments, the bath solution was perfused with either a PLC inhibitor — ET-18-OCH3 (ET; 2 mM) or U73122 (2 mM) — or a PKC inhibitor, GF (0.5 mM or 10 mM) 120 seconds before SL-NH2 reapplication. Numbers in parentheses indicate cells tested. *P < 0.05 versus control group; #P < 0.05 versus LR-NH2; ΧP < 0.05 versus SL-NH2; unpaired Student’s t test. (B) A representative trace of increase of AITC-activated current by a PLC activator, m-3M3FBS. Cells were perfused for 90 seconds with solution containing m-3M3FBS (10 μM) before reapplication of the AITC. (C and D) Effects of PLC activation and its downstream products on the AITC- (30 μM) (C) or cinnamaldehyde-activated (500 μM) (D) current. Cells were perfused for 90 seconds with solution containing activators before reapplication of the AITC or cinnamaldehyde; m-3M3FBS at 10 μM; OAG, a cell-permeable analog of DAG at 200 μM; and the PKC activator PMA at 1 μM were used. Currents were normalized to the values evoked initially by AITC or cinnamaldehyde in the absence of the additives. Cells in the control group were perfused with bath solution without the additives before reapplication of agonists; *P < 0.05, **P < 0.005 versus control group; unpaired Student’s t test. Note that OAG and PMA did not significantly potentiate the hTRPA1 currents. Numbers in parentheses indicate cells tested. Vh was –60 mV in all experiments.