SPECIFICITY of P2X3 AGONIST AND TRPV1 ANTAGONIST IN HEK293 CELLS

Patients with temporomandibular joint disorders suffer from hyperalgesia of the masticatory muscles.  Injury or inflammation result in the release of inflammatory mediators which bind to receptors such as P2X₃ (homomers or heteromers) and TRPV1. Objectives:  Previously collected behavioral data show that intra-masseter activation of P2X₃ results in mechanical hyperalgesia which is prevented by pre-treatment of the muscle with a specific TRPV1 antagonist. This suggests P2X₃ functionally interacts with TRPV1. The purpose of this experiment is to test the specificity of the drugs used in the behavioral studies. Specifically, this study was designed to confirm αβmeATP, a P2X₃ agonist, directly activates P2X₃, while AMG9810, a TRPV1 antagonist, directly acts on TRPV1.  Methods: Ca²⁺ imaging was performed on dissociated trigeminal ganglia and HEK293 cells transiently transfected with TRPV1 or P2X₃ cDNA.  Results: αβmeATP (50μM) did not evoke a Ca²⁺ response in HEK293 cells transfected with TRPV1, however, the TRPV1 agonist, capsaicin (1 μM), evoked a robust Ca²⁺ influx. As predicted, αβmeATP (50μM) evoked Ca²⁺ responses in HEK293 cells expressing P2X₃ and capsaicin (1 μM) did not. In the presence of the TRPV1 antagonist, AMG9810 (1 μM), αβmeATP (50μM) still evoked Ca²⁺ responses. However, at this time we do not have vehicle data to compare the number or amplitude of responses. Conclusions: Further studies are needed to confirm the mechanism of action of AMG9810. However, αβmeATP and capsaicin specifically activate their respective receptors. This data supports our behavioral studies which showed a functional interaction between P2X₃ and TRPV1 in trigeminal sensory neurons.