P2X₃ and TRPV1 functionally interact and mediate sensitization of trigeminal sensory neurons

Abstract

Musculoskeletal pain conditions, particularly those associated with temporomandibular disorders (TMD) affect a large percentage of the population. Identifying mechanisms underlying hyperalgesia could contribute to the development of new treatment strategies for the management of TMD and other muscle pain conditions. In this study, we provide evidence of functional interactions between two ligand-gated channels, P2X₃ and transient receptor potential V1 (TRPV1), in trigeminal sensory neurons, and propose that the interactions serve as an underlying mechanism for the development of mechanical hyperalgesia. Mechanical sensitivity of the masseter muscle was assessed in lightly anesthetized rats via an electronic anesthesiometer (Ro et al., 2009). Direct intramuscular injection of a selective P2X₃ agonist, alpha,beta-methylene adenosine triphosphate (αβmeATP), induced a dose- and time-dependent hyperalgesia. Mechanical sensitivity in the contralateral muscle was unaffected suggesting local P2X₃ mediate hyperalgesia. Anesthetizing the overlying skin had no effect on αβmeATP-induced hyperalgesia confirming the contribution of P2X₃ from the muscle. Importantly, the αβmeATP-induced hyperalgesia was prevented by pretreatment of the muscle with a TRPV1 antagonist, AMG9810. P2X₃ was co-expressed with TRPV1 in the masseter muscle afferents confirming the possibility for intracellular interactions. Additionally, in a subpopulation of P2Xv/TRPV1 positive neurons, capsaicin-induced Ca(2+) transients were significantly amplified following P2X₃ activation. Finally, activation of P2X₃ induced phosphorylation of serine, but not threonine, residues in TRPV1 in trigeminal ganglia cultures. Significant phosphorylation was observed at 15 min, the time point at which behavioral hyperalgesia was prominent. Previously, activation of either P2X₃ or TRPV1 had been independently implicated in the development of mechanical hyperalgesia. Our data propose P2X₃ and TRPV1 interact in a facilitatory manner, which could contribute to the peripheral sensitization known to underlie masseter hyperalgesia.

Figure 1

αβmeATP induces mechanical hyperalgesia in the masseter muscle. (A) The line graphs depict P2X₃ agonist αβmeATP-induced changes in mechanical thresholds in the rat masseter muscle. (B) The bar graphs show the overall effects of different doses of αβmeATP on mechanical sensitivity, as shown by Area Under the Curve. (C) The overall effect of intramuscular αβmeATP on masseter sensitivity was examined with topical application of Benzocaine (20%). (D) The overall effect of αβmeATP (750 µg) was compared between the injected and the contralateral muscles. (E) The specificity of αβmeATP was examined with A-317491, a selective P2X₃-receptor antagonist. A-317491 was injected into the masseter 5 min prior to αβmeATP. BL indicates baseline 15 min prior to αβmeATP injection. Black arrow indicates αβmeATP injection at 0 min. +, * indicates significant (P < 0.05) group and time effects, respectively.

Figure 2

αβmeATP-induced masseter hyperalgesia involves TRPV1. (A) Line graph shows changes in masseter mechanical thresholds induced by αβmeATP in the presence of the TRPV1 antagonist, AMG9810. (B) Bar graph shows the overall magnitude of drug effect as measured by Area Under Curve (AUC). BL: baseline 15 min prior to αβmeATP injection. Gray arrow: vehicle or antagonist injection 5 min prior to αβmeATP injection. Black arrow: αβmeATP injection at 0 min. (C) Mechanical thresholds were measured at the indicated time points following the injection of AMG9810 (100 nmol) or vehicle. (n = 6/group). +, * indicates significant (P < 0.05) group and time effects, respectively.

Figure 3

The effect of AMG9810 on αβmeATP-evoked Ca²⁺ responses. (A) Example traces showing αβmeATP-evoked responses in the presence of vehicle or AMG9810. (B) Box-and-whisker plot demonstrating the magnitude of αβmeATP-evoked responses in the presence or absence of AMG9810 (1 µM). The number of neurons that responded to αβmeATP as a proportion of KCl responders is displayed within the boxes. There were no statistical differences in either the amplitudes or proportion of αβmeATP-evoked responses.

Figure 4

The somata of masseter afferents labeled by retrograde transport of Fast Blue (FB) were assessed for TRPV1 and P2X₃ immunoreactivity. (A) Micrographs illustrating TRPV1 and P2X₃ expression in TG neurons, an arrow indicates the FB positive neuron. (B) Soma size distribution of TRPV1 and P2X₃ positive masseter afferents in TG based on cell body area (µm²), small (<400µm²), medium (400–1000 µm²), and large (>1000 µm²). The expression profile of TRPV1 and P2X₃ was assessed in 400 masseter afferents. Scale bar=20µm

Figure 5

The effects of αβmeATP on capsaicin-induced Ca²⁺ responses in TG neurons. Representative traces show Fura ratio (FR) from TG neurons in (A) αβmeATP and (B) vehicle treated groups. Each color trace represents responses from one neuron. (C) Averaged changes in Fura response (ΔFR) of the first and second 10 nM capsaicin application in αβmeATP and vehicle-treated groups. This protocol was tested on 12 cultures generated from 12 rats. + denotes significant effect at P < 0.05 and * indicates P < 0.05 following post-hoc analysis.

Figure 6

The αβmeATP effect is mediated by P2X₃. (A) The number of responses and (B) the magnitude of responses evoked by 50 µM αβmeATP in the presence of the selective P2X₃ antagonist, A-317491. This protocol was tested on 5 cultures generated from 5 rats. The data were analyzed with Kruskal-Wallis One-Way Anova. + indicates significant effect at P < 0.05.

Figure 7

The effect of αβmeATP on TRPV1 phosphorylation in TG cultures. (A,C) Immunoblots for p-Ser and p-Thr following IP using an anti-TRPV1 antibody (upper). Immunoblots using anti-TRPV1 antibody from the same sample in the upper panel (lower). The samples were collected at the indicated time points following αβmeATP (50 µM) treatment. (B,D) Averaged relative optical densities (p-Ser/TRPV1 and p-Thr/TRPV1) are also shown. * denotes significant effect at P < 0.05. n = 6 for each time point.

Figure 8

The effect of PMA on serine phosphorylation, and the effect of αβmeATP on TRPV1 expression in TG cultures. (A) (upper) Immunoblots for p-Ser following IP using an anti-TRPV1 antibody. Samples were either naïve, treated with 1µM PMA or vehicle for 15 min. (lower) Immunoblots of the same sample using an anti-TRPV1 antibody. (B) Representative western blot for TRPV1 (upper) and GAPDH (lower) from the same sample. The samples were collected at the indicated time points following αβmeATP (50 µM) treatment. (C) Averaged relative optical density (TRPV1/GAPDH). n = 3 for each time point.