PAR-2 agonists activate trigeminal nociceptors and induce functional competence in the delta opioid receptor

The role of protease activated receptor-2 (PAR-2) activation in trigeminal nociception and in induction of functional competence in the delta opioid receptor (DOR) is not known. In this study, we evaluated whether agonists of PAR-2 activate the capsaicin-sensitive subclass of trigeminal nociceptors in a PLC-PKC-dependent manner and induce functional competence in the DOR. Adult male rat trigeminal ganglion (TG) cultured neurons were treated with the PAR-2 agonist (SL-NH₂) or an enzyme activator of PAR (trypsin) and the activation of TG nociceptors was assessed using three independent methods: neuropeptide release, calcium influx, and whole cell patch-clamp. The specificity of SL-NH₂ and trypsin responses was evaluated using TG cultures transfected with siRNA against PAR-2. The in vivo role of PAR-2 activation was determined measuring SL-NH₂ and trypsin-evoked nocifensive behavior and increase in blood flow. Trigeminal neurons were treated with SL-NH₂/vehicle and then the DOR agonist to determine DOR inhibition of evoked neuropeptide release and cAMP accumulation. The results showed that SL-NH₂ (100 μM) and trypsin (1-600 nM) activate TG nociceptors, which is partly reversible by the PKC inhibitor bisindolylmaleimide (500 nM) and by ruthenium red (10 μM). In cultures treated with siRNA against PAR-2, both SL-NH₂ and trypsin responses were significantly diminished. Both SL-NH₂ and trypsin evoke nocifensive behavior and increases in blood flow in an orofacial pain model. Application of SL-NH₂ rapidly produced functional competence of DOR for inhibiting nociceptor function. In inflamed tissue, endogenous proteases may activate TG nociceptors and generate pain. Moreover, activation of PAR-2 can also induce functional competence in DOR.