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. 2015 Jan;16(1):67-76.
doi: 10.1016/j.jpain.2014.10.008. Epub 2014 Nov 1.

Peripheral group I metabotropic glutamate receptor activation leads to muscle mechanical hyperalgesia through TRPV1 phosphorylation in the rat

Man-Kyo Chung  1 , Jongseok Lee  1 , John Joseph  1 , Jami Saloman  1 , Jin Y Ro  2
Affiliations

Peripheral group I metabotropic glutamate receptor activation leads to muscle mechanical hyperalgesia through TRPV1 phosphorylation in the rat

Man-Kyo Chung et al. J Pain. 2015 Jan.

Abstract

Elevated glutamate levels within injured muscle play important roles in muscle pain and hyperalgesia. In this study, we hypothesized that protein kinase C (PKC)-dependent TRPV1 phosphorylation contributes to the muscle mechanical hyperalgesia following activation of Group I metabotropic glutamate receptors (mGlu1/5). Mechanical hyperalgesia induced by (R,S)-3,5-dihydroxyphenylglycine (DHPG), an mGlu1/5 agonist, in the masseter muscle was attenuated by AMG9810, a specific TRPV1 antagonist. AMG9810 also suppressed mechanical hyperalgesia evoked by pharmacologic activation of PKC. DHPG-induced mechanical hyperalgesia was suppressed by pretreatment with a decoy peptide that disrupted interactions between TRPV1 and A-kinase-anchoring protein (AKAP), which facilitates phosphorylation of TRPV1. In dissociated trigeminal ganglia, DHPG upregulated serine phosphorylation of TRPV1 (S800), during which DHPG-induced mechanical hyperalgesia was prominent. The TRPV1 phosphorylation at S800 was suppressed by a PKC inhibitor. Electrophysiologic measurements in trigeminal ganglion neurons demonstrated that TRPV1 sensitivity was enhanced by pretreatment with DHPG, and this was prevented by a PKC inhibitor, but not by a protein kinase A inhibitor. These results suggest that mGlu1/5 activation in masseter afferents invokes phosphorylation of TRPV1 serine residues including S800, and that phosphorylation-induced sensitization of TRPV1 is involved in masseter mechanical hyperalgesia. These data support a role of TRPV1 as an integrator of glutamate receptor signaling in muscle nociceptors.

Perspective: This article demonstrates that activation of mGlu1/5 leads to phosphorylation of a specific TRPV1 residue via PKC and AKAP150 in trigeminal sensory neurons and that functional interactions between glutamate receptors and TRPV1 mediate mechanical hyperalgesia in the muscle tissue.

Keywords: Peripheral; muscle pain; sensory neurons; trigeminal.

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Figures

Figure 1
Figure 1. TRPV1 is involved in masseter mechanical hyperalgesia induced by dihydroxyphenylglycine (DHPG), a mGlu1/5 agonist
A. Mechanical threshold of rat masseter muscle following intramasseteric injection of 100 μl DHPG (1 μmol) under lightly anesthetized conditions. Five minutes before injection of DHPG, 10 μl of Vehicle (Veh, 5% DMSO in PBS), or 10 μl of 1 nmol or 100 nmol AMG9810 (a TRPV1 antagonist) was injected into the masseter muscle. Mechanical threshold values were normalized to the baseline (BL) in each animal. +p<0.05 in Two-way ANOVA with repeated measures; N=4 per group. B. Area under the curve (AUC) calculated from experiments in panel A. *p<0.05 in one-way ANOVA.
Figure 2
Figure 2. TRPV1 is involved in masseter hypersensitivity induced by phorbol myristate acetate (PMA), an activator of PKC but not by forskolin, an activator of PKA
A. Mechanical threshold of rat masseter muscle following masseteric injection of 30 μl PMA (300 nM in PBS) under lightly anesthetized condition. Five min before injection of PMA, 10 μl Vehicle (Veh, 5% DMSO in PBS) or 10 μl of 1 nmol or 100 nmol AMG9810 was injected into masseter muscle. +p<0.05 in Two-way ANOVA with repeated measures; N=6 in each group. B. Area under the curve (AUC) calculated from experiments in panel A. *p<0.05 in one-way ANOVA. C. Mechanical threshold of rat masseter muscle following masseteric injection of 20 μl forskolin (FSK; 20 μg in PBS) under lightly anesthetized condition. Five minutes before injection of FSK, 10 μl Vehicle (Veh, 5 % DMSO in PBS) or 10 μl of 100 nmol AMG9810 was injected into masseter muscle. D. Area under the curve (AUC) calculated from experiments in panel C.
Figure 3
Figure 3. Disruption of interactions between TRPV1 and AKAP150 attenuates DHPG-induced masseter hypersensitivity
A. Mechanical threshold of rat masseter muscle following intramasseteric injection of 100 μl DHPG (1 μmol in PBS) under lightly anesthetized conditions. Five minutes before injection of DHPG, 20 μl Vehicle (Veh, PBS), or 20 μl scramble peptide (30 μM), or 20 μl 736–745 peptide (10 μM or 30 μM) was injected into masseter muscle. Mechanical threshold values were normalized to the baseline (BL) in each animal. N=5 per group. B. Area under the curve (AUC) calculated from experiments in panel A. *p<0.05 in one-way ANOVA
Figure 4
Figure 4. DHPG enhances TRPV1 phosphorylation in a PKC-dependent manner in neurons dissociated from rat trigeminal ganglia (TG)
A–B. Effects of DHPG on phosphorylation of serine residues (panel A) or S800 (panel B) of TRPV1 in samples from rat TG culture. (Upper panels) Representative immunoblot of phospho-serine (panel A) or phospho-S800 of TRPV1 in biotinylated (surface) or GAPDH in corresponding total lysates (TL) (panel B) after indicated time following treatment of DHPG (200 μM). (Lower panels) Percent change in intensity ratio between phospho-serine (panel A) or phospho-S800 TRPV1 (panel B) and GAPDH in the same total lysate. DHPG-treated groups were normalized to non-treated group. *p<0.05 in one-way ANOVA; N=5 for each group. C–D. Inhibition by GF109203X of DHPG-induced increase of phospho-S800 TRPV1 expression. (Upper panels) Representative immunoblot of p-S800 of TRPV1 from TG neurons treated for 15 min with DHPG and 10 μM GF109203X, a PKC inhibitor (panel C) or vehicle (DMSO; panel D). (Lower panels) Percent change in intensity ratio between phospho-S800 and GAPDH in the same total lysate. The intensity of the DHPG-treated group was normalized to that of the non-treated group in the same blot. *p<0.05 in one-way ANOVA. N=5 for each group.
Figure 5
Figure 5. DHPG sensitizes TRPV1 in a PKC-dependent manner in TG neurons
A. Representative current traces evoked by consecutive application of capsaicin (200 nM) in rat TG neurons. Vehicle (Veh, PBS; upper) or DHPG (50 μM; lower) was applied for 10 minutes as indicated. Holding potential = −70 mV. Scale bars, 20 pA/pF - 2 min. B. Averaged normalized responses. Current density of the 1st and 2nd response from each neuron was normalized to the average of 1st response in the given group. *p<0.05, Student’s t-test. Numbers in parentheses represent the number of observations. C. Representative current traces evoked by consecutive application of capsaicin (300 nM) in rat TG neurons. DHPG (50 μM) was co-applied with vehicle (Veh, DMSO; upper), GF109203X (GF, 1 μM; middle) or KT5720 (KT, 1 μM; lower) for 10 minutes as indicated. Holding potential = −70 mV. Scale bars, 20 pA/pF - 2 min. D. Averaged normalized responses. NS, not significant; *p<0.05, Student’s t-test. Numbers in parentheses represent the number of observations.
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