A25 COLONIC PROTEASES EVOKE SUSTAINED PAIN SIGNALING VIA A NOVEL ENDOSOMAL PATHWAY IN NEURONS

Proteases are increased in IBS patient colonic tissue and have been shown to activate and sensitize nociceptive (pain-sensing) dorsal root ganglia (DRG) neurons. We found that activation of protease activated receptor 2 (PAR2) on DRG neurons leads to prolonged pain signaling (> 1h). Serine proteases (e.g. trypsin) are major mediators of this response and we identified that trypsin signals through novel pathways that are distinct from cysteine proteases (cathepsin S, elastase). Here we found that trypsin signals by PAR2 endocytosis trafficking to endosomes to induce sustained signaling. It is unknown whether other tissue proteases also act through PAR2 to elicit sustained signaling and if a specific endosomal antagonist can block the effect of proteases in IBS tissues. 1) to determine if sustained signaling by both serine and cysteine proteases are blocked by the selective PAR2 antagonist I-343 and 2) to determine whether PAR2 in endosomes is a therapeutic target using a novel lipidated PAR2 antagonist that targets endosomes. DRG neurons from C57BL/6 mice were pre-incubated with trypsin (10 min; 50 nM), elastase (30 min; 390 nM) or cathepsin-S (60 min; 500 nM) and then washed out. Changes in neuronal excitability (rheobase, amount of current to elicit an action potential) was measured using patch clamp recordings, immediately (T=0 min) or after a sustained period (T=30 min). The role of PAR2 was evaluated with PAR2 antagonist I-343 (10 μM) and to evaluate the role of PAR2 in endosomes, we used a lipidated I-343 (MIPS15479). Supernatants containing representative proteases of colonic biopsies were obtained from diarrhea-predominant IBS patients or controls (HC). DRG neurons were pre-incubated with MIPS15479 (30 μM), washed and allowed to recover in antagonist-free medium for 120 min before applying the protease agonists or IBS supernatant. Two-way ANOVA and Tukey’s post hoc tests were used to analyze the data. The PAR2 antagonist blocked the sustained (T=30 min) excitability evoked by trypsin (37.5%, P<0.05) and cathepsin S (54%, P<0.001) whereas the antagonist had no effect on the sustained actions of elastase. In contrast, the immediate excitability (T= 0 min) evoke by all three proteases was completed blocked by the antagonist. IBS-D supernatants also evoked sustained excitability of DRG neurons (decrease 43% in rheobase compared to HC; P <0.01) and this effect was blocked by the lipidated PAR2 antagonist MIPS15479. Trypsin-mediated sustained excitability was also inhibited by MIPS15479, but the acute response was unaffected The lipidated PAR2 antagonist blocked the sustained but not immediate excitability evoked by trypsin, consistent with an endosomal action. This antagonist also blocked the sustained protease-PAR2 signaling mediated by IBS tissues, suggesting that this antagonist could be a novel therapeutic agent. CCC

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