Potentiation of acid-sensing ion channel activity by peripheral group I metabotropic glutamate receptor signaling

Xiong Gan, Jing Wu, Cuixia Ren, Chun-Yu Qiu, Yan-Kun Li, Wang-Ping Hu∗

Abstract

Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluRmediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration–response curve upwards, with a 47.3 ± 7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLC, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia.

Keywords:
Group I metabotropic glutamate receptor
Acid-sensing ion channel Proton-gated current
Neuronal excitability
Pain
Dorsal root ganglion neuron

1. Introduction

Glutamate, a dominant excitatory neurotransmitter of central nervous system, has been known as one of the inflammatory mediators released into the peripheral tissue from damaged cells, inflammatory cells, afferent nerve terminals or glia [1–3]. The released glutamate contributes to generation and potentiation of inflammatory pain through activating ionotropic and/or metabotropic glutamate receptors (mGluRs) on peripheral unmyelinated sensory afferents [3–5]. Group I mGluRs, consisting of mGluR1 and mGluR5 (mGluR1/5), have been shown to play an important role in peripheral pain sensitization [4,6]. Several lines of evidence indicate that mGluR1/5 is expressed in small dorsal root ganglion (DRG) neurons and unmyelinated afferents in peripheral tissue [4,7,8]. Peripheral activation of mGluR1/5 with agonist DHPG causes mechanical allodynia and thermal sensitivity, while intraplantar blockade of mGluR1/5 reduces formalin-, complete Freund’s adjuvant-, and carrageenan-induced pain behaviors [4,8–10]. Local peripheral inhibition of mGluR5 alone reduces also mechanical hypersensitivity in models of carrageenan-induced inflammatory pain and colorectal visceral pain [8,11,12]. Group I mGluRs have been shown to play a role in mediating pain and hyperalgesia under inflammatory, neuropathic and post-operative conditions [4,13,14]. The mechanism(s) underlying this group I mGluRs, especially mGluR5, action on primary afferents mainly focus on sensitization of transient receptor potential vanilloid 1 (TRPV1) [9,15–17]. In the peripheral terminals of sensory neurons, mGluR5 has been shown to mediate synthesis of prostaglandins via the phospholipase C (PLC) cascade, and then directly activate or sensitize neighboring TRPV1 [15]. However, it is still not clear whether other ion channels are involved in group I mGluRs −mediated peripheral sensitization.
During inflammation, tissue injury, ischemic stroke and surgical trauma, proton is released and decreases extracellular pH level [18,19]. For example, local extracellular pH falls to 5.4 recovery after washout of DHPG. Representative currents were recorded for more than 60 min in a DRG neuron tested. DHPG was pre-applied to external solution for 1 min C. The graph shows DHPG increased the peak amplitude of proton-gated currents in a concentration-dependent manner with an EC50 of 1.88 × 10− M. DHPG was re-treated 8 for 60s. DRG neurons with membrane potential clamped at −60 mV. Each point represents the mean ± SEM of 7–9 neurons. in acute inflammation and 6.3 or lower in severe ischemia [20,21]. In humans, subcutaneous administration of acidic solutions causes pain [22,23]. The released proton activates acid-sensing ion channels (ASICs) expressed in both DRG cell bodies and sensory terminals, which contribute to proton-evoked pain signaling [24,25]. Studies show that ASICs, rather than TRPV1, mediate pain sensation induced by peripheral proton [19,26,27]. Increasing evidence has shown that ASICs play an important role in various pain conditions such as inflammatory pain, postoperative pain and migraine [28–30]. Herein, we report that group I mGluRs activation of nociceptive DRG neurons sensitizes ASICs and contributes to acidosis-evoked pain.

2. Methods

2.1. Isolation of DRG neurons

The experimental protocol was approved by the animal research ethics committee of Hubei University of Science and Technology. All procedures conformed to international guidelines on the ethical use of animals, and every effort was made to minimize the number of animals used and their sufferings. Five- to six-week old Sprague-Dawley male rats were anesthetized with ethyl ether and then decapitated. The DRGs were taken out and transferred immediately into Dulbecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4. After the removal of the surrounding connective tissues, the DRGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 ml of DMEM in which trypsin (type II-S, Sigma) 0.5 mg/ml, collagenase (type I-A, Sigma) 1.0 mg/ml and DNase (type IV, Sigma) 0.1 mg/ml had been dissolved, and incubated at 35◦C in a shaking water bath for 25–30 min. Soybean trypsin inhibitor (type II-S, Sigma) 1.25 mg/ml was then added to stop trypsin digestion. Dissociated neurons were placed into a 35-mm Petri dish and kept for at least another 60 min before electrophysiological recordings. The neurons selected for electrophysiological experiment were 15–35 m in diameter.

2.2. Electrophysiological recordings

Whole-cell patch clamp and voltage-clamp recordings were carried out at room temperature (22–25◦C) using a MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments, CA, USA). Recording pipettes were pulled using a Sutter P-97 puller (Sutter Instruments, CA, USA). The micropipettes were filled with internal solution containing (mM): KCl 140, MgCl2 2.5, HEPES 10, EGTA 11 and ATP 5; its pH was adjusted to 7.2 with KOH and osmolarity was adjusted to 310 mOsm/L with sucrose. Cells were bathed in an external solution containing (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 2, HEPES 10, d-glucose 10; its osmolarity was adjusted to 330 mOsm/l with sucrose and its pH to 7.4. The resistance of the recording pipette was in the range of 3–6 M. A small patch of membrane underneath the tip of the pipette was aspirated to form a giga seal and then a negative pressure was applied to rupture it, thus establishing a whole-cell configuration. The adjustment of capacitance compensation and series resistance compensation was done before recording the membrane currents. The membrane voltage was maintained at −60 mV in all voltage-clamp experiments unless otherwise specified. Current-clamp recordings were obtained by switching to current-clamp mode after a stable whole-cell configuration was formed in voltage-clamp mode. Only cells with a stable resting membrane potential (more negative than −50 mV) were used in the study. Signals were sampled at 10–50 kHz and filtered at 2–10 kHz, and the data were stored in compatible PC computer for off-online analysis using the pCLAMP 10 acquisition software (Axon Instruments, CA, USA).

2.3. Drug application

Drugs were purchased from Sigma and used in the experiments include: hydrochloric acid, (R,S)-3,5-dihydroxyphenylglycine (DHPG), 2-methyl-6- (phenylethynyl)-pyridine hydrochloride (MPEP), YM-298198, capsazepine and tetrodotoxin (TTX). All drugs were dissolved daily in the external solution just before use and held in a linear array of fused silica tubes (o.d./i.d. = 500 m/200 m) connected to a series of independent reservoirs. The application pipette tips were positioned ∼30 m away from the recorded neurons. The application of each drug was driven by gravity and controlled by the corresponding valve, and rapid solution exchange could be achieved within about 100 ms by shifting the tubes horizontally with a PC-controlled micromanipulator. Cells were constantly bathed in normal external solution flowing from one tube connected to a larger reservoir between drug applications. In some experiments where GDP–S (Sigma), U-73122(Sigma), GF109203X (RBI), BAPTA-AM (Sigma) and FSC-231 (Gether Lab, University of Copenhagen, DK-2200Copenhagen, Denmark) were applied for intracellular dialysis through recording re-patch pipettes, they were dissolved in the internal solution before use. And re-patch recording is performed as described in our previous study [31]. To functionally characterize ASIC activity, we used capsazepine (10 M) to block TRPV1 in this study [32,33].

2.4. Nociceptive behavior induced by acetic acid in rats

Rats were placed in a 30 × 30 × 30 cm Plexiglas chamber and allowed to habituate for at least 30 min before nociceptive behavior experiments. Separate groups of rats were pretreated with 20 l capsazepine (100 M) together with vehicle, different dosages of DHPH, MPEP and YM-298198 in ipsilateral hindpaw before injection of acetic acid. After 5 min, the other experimenters subcutaneously administered acetic acid solution (0.6%, 20 l) into the dorsal face of the hind paw using a 30 gauge needle connected to a 100 l Hamilton syringe. Nociceptive behavior (that is, number and time of flinches) was counted over a 5 min period starting immediately after the injection [26,34].

2.5. Data analysis

Data were statistically compared using the Student’s t-test or analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Statistical analysis of concentration-response data was performed using nonlinear curve-fitting program ALLFIT. Data are expressed as mean ± SEM.

3. Results

3.1. DHPG enhanced ASIC currents in rat dorsal root ganglia neurons

Proton-gated currents were recorded in acutely isolated DRG neurons (15–35 m in diameter). In order to purify the ASIC current from the proton-gated currents, capsazepine (10 M) was added to external solution to block proton-induced TRPV1 activation [32,33]. In the presence of capsazepine, a rapid fall of extracellular pH from 7.4 to 6.0 for 5 s produced an inward current (IpH6.0) in 82.5% (165/200) of DRG neurons. The IpH6.0 could be completely blocked by 100 M of amiloride, a broad-spectrum ASIC channel blocker (Fig. 1A). They were thus considered to be ASIC currents in the presence of capsazepine to block proton-induced TRPV1 activation. Most (77.6%, 128/165) of these proton-evoked currents were characterized by a large transient current followed by fast
A2-5 show that DHPG potentiation of proton-gated currents was blocked by intracellular dialysis of non-hydrolyzable GDP analog GDP–S (500 M), PLC inhibitor U-73122 (10 M), PKC inhibitor GF109203X (2 M), or PICK1 (protein interacting with C-kinase 1) inhibitor FSC-231 (50 M) by the re-patched recording pipettes. Proton-gated currents were elicited by application of pH 6.0 for 5-s durations. DHPG (10−7 M) was pre-applied to external solution for 1min. B. The bar graph shows the percentage increases in the IpH6.0 induced by DHPG (10− M) with recording pipettes filled with the normal internal solution, GDP–S, U-73122,GF109203X or FSC-231 containing 7 internal solution in the second patch experiments. **P < 0.01, post hoc Bonferroni’s test, compared with normal internal solution. n = 8 in each column.
inactivation and then a small sustained current with no or very slow inactivation [35]. As shown in Fig. 1A, these characterized currents were also completely blocked by 3 M of APETx2, a specific ASIC3 homomeric and heteromeric channel blocker [35]. We further considered them to be ASIC3-like currents and observed the ASIC3-like currents in the following study.
In some neurons sensitive to acid stimuli (56.3%, 72/128), we observed that the proton-evoked currents were enhanced by the pre-application of DHPG, a selective group I mGluR agonist, for 1 min (Fig. 1B and C). We first investigated whether the potentiation of ASIC currents was dependent upon the concentration of DHPG. Fig. 1B shows that the amplitude of IpH6.0 increased as concentration of re-treated DHPG increased from 3×10−9M to 3×10−7M in a representative DRG neurons. And the DHPG evoked enhancing effect was reversible in washout experiments. Fig. 1C shows the concentration–response curve for DHPG in the potentiation of proton-gated currents. DHPG had a maximum effect (58.6 ± 6.3%, n=8) at a concentration of 3×10−7M. The half-maximal response (EC50) value of the concentration–response curve for DHPG was 18.81 ± 1.63 nM. The results indicated that DHPG concentrationdependently enhanced the ASIC current in rat DRG neurons.
We then investigated whether the potentiation of DHPG was dependent upon pH. Fig. 2A shows the enhancing effects of DHPG (10−7M) pretreatment for 1min on currents evoked by three different pHs. Fig. 2B shows the concentration–response curve to protons in the presence and absence of DHPG (10−7M). First, pre-treatment of DHPG shifted the concentration–response curve to protons upwards, as indicated by an increase of 47.3 ± 7.0% in the maximal current response to protons when DHPG was pre-applied. However, the slopes or Hill coefficients of those two curves were essentially similar (n = 1.42 ± 0.16 in the absence of DHPG versus n = 1.37 ± 0.20 in the presence of DHPG; P > 0.1, Bonferroni’s post hoc test). Second, the pH for half-maximal activation (pH0.5) values of both curves had no statistical difference (pH0.5 of 6.02 ± 0.04 without DHPG pretreatment versus pH0.5 of 6.06 ± 0.05 with DHPG pretreatment; P > 0.1, Bonferroni’s post hoc test). Third, the threshold pH values of both curves had no significant difference in the presence and absence of DHPG.

3.2. Group I mGluR1/5 involved in DHPG potentiation of ASIC currents

We further verify whether the potentiation of ASIC currents by DHPG, a selective group I mGluR agonist, was mediated by mGluRs. Firstly, we observed the effect of MPEP, a selective mGluR5 antagonist, on the DHPG-induced potentiation of proton-gated currents. The amplitude of IpH6.0 increased 52.7 ± 7.0% after pretreatmentwithDHPG(10−7M)alonefor1minineightDRGneurons
Bonferroni’s post hoc test, n = 8; Fig. 3). Secondly, we found that YM-298198, a selective mGluR1 antagonist, partly blocked the potentiation of DHPG. The amplitude of IpH6.0 increased 30.1 ± 7.9% when DHPG was pretreated with 10−6M YM-298198 (P=0.057, Voltage-clamp recording of current induced by a pH 6.0 acid stimulus. Holding potential was −60 mV. Right panel: Current-clamp recording (I = 0 pA) of the membrane depolarization evoked by the pH 6.0 acid stimulus from the same neuron as the left panel. The treatment of DHPG (10−7 M) for 1min also increased the acidosis-induced by the membrane depolarization in the neuron tested in the presence of capsazepine (10 M) and TTX (1 M) to block proton-induced TRPV1 activation and Na+ channel-mediated action potentials, respectively. C and D. Bar graphs show the effects of DHPG on the number of spikes and membrane potential depolarization produced by pH 6.0. The acidosis-evoked depolarization and spikes recovered to control condition after washout of DHPG. *P < 0.05, paired t-test, compared with pH alone, n = 8 in each column. compared with DHPG alone, Bonferroni’s post hoc test, n = 8; Fig. 3). Finally, we examined the effect of the simultaneous blockade of mGluR5 and mGluR1 by co-administration of MPEP and YM298198 on the potentiation of DHPG. DHPG only produced an increase of 7.0 ± 3.9% on ASIC currents after co-administration of 10−6 M MPEP and 10−6 M YM-298198 (P<0.01, compared with DHPG alone, Bonferroni’s post hoc test, n = 8; Fig. 3). The results indicated that group I mGlu1/5 receptor involved in DHPG potentiation of ASIC currents.

3.3. Intracellular mechanisms underling the potentiation of proton-gated currents by DHPG

Group I mGluRs are most commonly associated with the Gq/11 subtype of G-protein, which activates PLC and leads to a cascade of events including the activation of protein kinase C (PKC) [6]. We further explore intracellular signal transduction mechanisms underlying potentiation of ASIC currents by DHPG using a re-patch technique. We examined neurons whether response to DHPG under normal internal solution condition before re-patch recording, only the neurons which IpH6.0 can be enhanced by DHPG exceeding 10.0% were used for intracellular dialysis. As shown in Fig. 4A1 and B, DHPG (10−7 M) caused still a 50.6±6.3% enhancing effect on IpH6.0 after re-patch with normal internal solution on the same neurons. In contrast, 30 min after non-hydrolyzable GDP analog GDP--S (500 M), PLC inhibitor U-73122 (10 M), or PKC inhibitor GF109203X (2 M) was applied internally to DRG neurons through recording re-patch pipettes, pre-application of DHPG (10−7 M) increased I to 11.2 ± 5.7%, 11.4 ±2.1% and 15.2±3.3%, separately (P < 0.01, post hoc Bonferroni’s test, compared with normal internal solution, n = 8; Fig. 4A2-4 and B). Moreover, DHPG (10−7 M) only caused a 14.3 ± 4.2% enhanced effect on I after FSC-231 (50 M), a protein interacting with C-kinase 1 (PICK1) inhibitor, was applied internally to DRG neurons (P < 0.01, post hoc Bonferroni’s test, n = 8; Fig. 4A5 and B). To verify whether the involvement of Ca2+ dependent mechanism, BAPTA-AM (500M) was infused into DRG neurons through recording re-patch pipettes. DHPG (10–7 M) increased IpH6.0 to 47.2 ± 6.3%, similar to 50.6 ± 6.3% enhancing effect on IpH6.0 after re-patch with normal internal solution (P > 0. 1, post hoc Bonferroni’s test, n = 8). Further, we found that DHPG (10−7 M) had also a 49.2 ± 6.7% enhancing effect on I in 8 neurons with intracellular solution containing low concentration of EGTA (0.1 mM). Re-patch and intracellular dialysis did not significantly alter the amplitude of IpH6.0 (data no shown). These data collectively indicated that the potentiation of IpH induced by DHPG was dependent upon a GPCR, PLC, PKC and PICK1 signal pathway.

3.4. DHPG augmented proton-induced membrane excitability of rat DRG neurons

In the presence of capsazepine (10 M), excitability of rat DRG neurons was detected under current-clamp model. As shown in Intraplantar injection of acetic acid evoked a flinch/shaking response in the presence of 100 M capsazepine. The pretreatment of DHPG dose-dependently (0.2–20 mol) increased flinching behavior induced by acetic acid. The effect of DHPG (20 mol) was blocked by co-treatment of 40 mol MPEP and 40 mol YM-298198, also by treatment of 40 mol MPEP alone. Flinching shaking of paw was recorded as the number (A) and time (B) of flinches during first 5 min after injection of acetic acid. *P < 0.05, **P < 0.01, Bonferroni’s post hoc test, compared with white column; −P < 0.01, Bonferroni’s post hoc test, compared with DHPG (20 mol) alone column. n = 10 rats in each column.
We then observed the membrane potential of rat DGR neurons in the presence of capsazepine (10 M) and TTX (1 M) to block proton-induced TRPV1 activation and Na+ channel-mediated action potentials, respectively. As shown in Fig. 5B, a pH 6.0 acid stimulus induced a depolarization of the resting membrane potential under current-clamp recording (I = 0 pA) conditions in a DRG neuron. In this particular cell, pretreatment of 10−7 M DHPG enhanced the depolarization evoked by pH6.0 acidic stimuli (Fig. 5B). In eight neurons tested, the magnitude of membrane potential depolarization (Vm) was 18.76 ± 2.18 mV after exposure to pH 6.0. In contrast, the Vm evoked by acidosis of pH 6.0 was 28.20 ± 2.59 mV with pretreatment of DHPG (paired t-test, P < 0.05, n = (Fig. 5D). The Vm recovered to control condition after a washout of DHPG. These results indicated that DHPG reversibly increased proton-induced membrane excitability of rat DRG neurons.

3.5. DHPG exacerbated acid-induced nocifensive behaviors in rats

Intraplantar injection of acetic acid elicits an intense flinch/shaking response in rats, and the flinch response is mediated by ASICs and mainly occurred during 0–5 min after injection of acetic acid [26,34]. We demonstrated that DHPG potentiated ASIC activity in vitro. To ascertain whether DHPG facilitates pain-related behaviors through interacting with ASICs in vivo, we injected DHPG and acetic acid into the rat hind paws and measured the total the number and time that the animal spent lift and licking the injected paw during a 5 min period. As shown in Fig. 6, intraplantar injection of acetic acid solution (0.6%, 20 l) in the presence of the TRPV1 inhibitor capsazepine (100 M) caused an intense flinch/shaking response in rats. And the acidinduced nocifensive behaviors was potently blocked by treatment with 20 l amiloride (200 M), a broad-spectrum ASIC channel blocker, or APETx2 (20 M), a blocker of ASIC3 homomeric and heteromeric channels (data no shown). Thus, the acidosis-evoked pain may be mediated by ASIC3. We found that pre-administration of DHPG dose-dependently exacerbated the acid-induced nocifensive behaviors. The acetic acid-induced the number and time of flinches was significantly greater in rats treated with mediumand high-dose (2 and 20 nmol) of DHPG than that observed in rats injected with acetic acid alone (Bonferroni’s post hoc test, p < 0.05 and p < 0.01, n = 10). However, the low dose (0.2 nmol) of DHPG had no effect on the acid-induced nocifensive behaviors (Bonferroni’s post hoc test, p > 0.05, n = 10). In addition, the exacerbating effect of 20 nmol DHPG on acid-induced nocifensive behaviors was blocked by co-administration of 40 nmol MPEP and 40 nmol YM298198, even by administration of 40 nmol MPEP alone (Bonferroni’s post hoc test, P < 0.01, compared with 20 nmol DHPG alone, n = 10). These results indicated that DHPG contributed to acid-induced nocifensive behaviors at the periphery through Group I mGluR1/5 in rats.

4. Discussion

The present study provided electrophysiological and behavioral evidence that group I mGluRs on primary sensory neurons can sensitize ASICs in rats. DHPG increased the amplitude of ASIC currents and acidosis-evoked membrane excitability in dissociated rat DRG neurons. We found that mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. Moreover, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats.
It has been known that extracellular protons generate currents in a subset of primary afferents [36]. The current study showed a rapid drop in the extracellular pH from 7.4 to 6.0 evoked an inward current in the presence of capsazepine to block the activation of TRPV1 channels in native DRG neurons. Most of these acid currents can be characterized by a large transient current followed by fast inactivation and then a small sustained current with no or very slow inactivation [35]. Our current and previous studies had identified that the characterized acidosis-evoked currents are mainly mediated by ASIC3, since they could be completely blocked by broad-spectrum ASIC channel blocker amiloride and ASIC3 channels blocker APETx2 [37]. They were thus considered to be ASIC3-like currents, although precise ASIC subunits need to be identified. Seven ASIC subunits, which are encoded by at least four genes, have been cloned in mammals [27]. Among all ASIC subunits, ASIC3 subunit is a critical pH sensor predominantly expressed in nociceptors, although they besides ASIC4 subunit are also present in DRG neurons All ASICs besides ASIC4 are present in DRG neurons [24–26,38].
The current study demonstrated that DHPG, a selective group I mGluR agonist, exerted an enhancing effect on ASIC currents in rat DRG neurons in a concentration-dependent manner. DHPG sensitized ASICs by shifting the proton concentration–response curve upward and increasing the maximum response without changing the EC50 values. Blockade of DHPG potentiation of ASIC activity by both mGluR5-selective antagonist MPEP and mGluR1selective antagonist YM-298198 showed that DHPG activates group I mGluRs. Immunohistochemical studies demonstrate that group I mGluR1/5 are expressed on small DRG neurons and unmyelinated cutanous nerve fibers in mouse and rat [4,8,10]. However, mGluR5 seemed to play a key role in DHPG potentiation of ASIC functional activity compared with relative contribution of peripheral mGluR1/5. Although mGluR1 antagonist YM-298198 alone partly blocked the DHPG potentiation of ASIC currents, mGluR5 antagonist MPEP caused a significant inhibition of DHPG potentiation. Further, intraplantar injection of MPEP alone completely blocked exacerbating effect of DHPG on acid-induced nocifensive behaviors.
The current study showed that DHPG potentiation of ASIC currents was blocked by intracellular dialysis of GDP--S, indicating that G-protein was involved in the intracellular mechanisms of this potentiation. Group I mGluRs primarily couple the Gq/11 subtype of G-protein family, which activates PLC [6]. Lack of the potentiating effect in cells treated with PLC inhibitor U-73122 indicated a PLC-dependent pathway is involved in the mGluRs-mediated ASIC sensitization. Hu et al. demonstrated that mGluR5 activation in the peripheral terminals of sensory neurons potentiates TRPV1 function by PLC pathway and activation of PKA subsequent to prostanoid production, not by activation of PKC [15]. In contrast to the TRPV1-regulating mechanism of mGluR5, we found that PKC inhibitor GF109203X also prevented the potentiation of ASIC currents by mGluRs activation. The observation suggested that activation of PKC contributed to sensitization of ASICs. In oocytes co-expressing TRPV1 and mGluR5, the activation of mGluR5 causes a rapid TRPV1 membrane translocation and potentiates TRPV1 currents via a PKC-dependent signaling [39]. Peripheral mGluR5 also mediates mechanical hypersensitivity via protein kinase C dependent mechanisms [40]. Evidence indicate ASICs can be regulated by a variety of intracellular protein kinases [41]. Our previous studies show that PKC activator PMA also produces a similar enhancing effect on ASIC currents in DRG neurons [31]. In addition, potentiation of ASIC currents by mGluR signaling may depend upon an interaction with PICK1, since the potentiation also reversed by intracellular dialysis of FSC-231, a small molecule inhibitor of the PICK1. PICK-1 was previously reported to interact directly with the PDZ-binding domain sequences of ASIC1a, ASIC2a, and ASIC2b [42]. The interaction participates in the PKC-mediated phosphorylation of ASIC2b and potentiates the PKC regulation of ASIC current in ASIC3+2b heteromeric channels [43]. However, ASIC3 seems to not interact with PICK1, which was essential for the potentiation of ASIC3-like currents by DHPG, One possible explanation was that the present ASIC3-like currents were mediated by ASIC3/2 heteromers, but not ASIC3 homomers. Together, group I mGluRs primarily couple the Gq/11 subtype of G-protein family, which activates PLC. PLC hydrolyzes phospholipid phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) to generate diacylglycerol (DAG) and inositol 1,4,5triphosphate (IP3), resulting in activation of PKC and release of Ca2+ from intracellular stores. Activation of mGluR1/5 by DHPG potentiated ASIC activity via this signal transduction pathways, while inhibition of Gq/11 proteins, PLC, PKC and PICK1 signaling by GDP--S, U-73122, GF109203X and FSC-231 thereby blocked the potentiation of ASIC activity by mGluR1/5 activation. Recently, we found that the activation of metabotropic P2Y UTP receptors also enhances ASIC activity via above signal transduction pathways in DRG neurons [44]. Muscarinic M1 receptor also belongs to Gq/11 subtype of G-protein family. However, the activation of M1 receptors is found to inhibit ASIC1a mediated currents in CHO cells and interneurons of hippocampus CA1 and striatum [45]. The inhibitory effects of M1 receptor on ASICs are subunit specific, since it has no effect on ASIC2a, ASIC3 and ASIC1a-containing heteromeric channels [45]. The current study showed that Gq/11 coupled mGluR1/5 potentiated ASIC3-like currents. Thus, similar GPCRs may have different effects on different ASIC subunits.
Extracellular acid stimuli open ASICs and mainly induce sodium influx, which can depolarize membrane potentials to the threshold of excitability and result in bursts of action potentials [46]. The current study showed that acid-evoke depolarization and action potentials were enhanced by DHPG. The increased acid-evoked neuronal excitability appeared to correlate with DHPG potentiation of ASIC currents in voltage clamp experiments. Moreover, behavioral studies showed that DHPG dose-dependently exacerbated acid-induced nocifensive behaviors through activating mGluR1/5 in rats. Obviously, the electrophysiological data corroborated the behavioral studies and vice versa. Our results indicated that activation of mGluR1/5 by DHPG could potentiate ASIC currents via an intracellular cascade and then increase the number of action potentials in primary sensory neurons, resulting in amplification of proton-induced pain.
The potentiation of ASICs activity by activation of mGluR1/5 is likely to be of physiological relevance in pathological condition. Protons are released from damaged cells and the de-granulation of mast cells during issue injury and inflammation, and extracellular pH values can drop to 5.4 [20,21]. In response to inflammation, glutamate is also released from a variety of sources, including damaged cells, mast cells, glia, and primary afferents themselves [1–3]. It has been shown that glutamate is released intradermally after formalin injection in to the hind paw [2]. During tissue injury and inflammation, once both proton and glutamate release together, they could initiate and/or sensitize nociceptive process through activating their cognate receptors expressed in peripheral neuronal terminals. Further, our results demonstrated that the activation of mGluR1/5 signaling by released glutamate could further sensitize ASICs, which exacerbated proton-evoked pain.

5. Conclusions

Our results indicated group I mGluRs activation of nociceptive DRG neurons enhanced ASICs function and contributed to acidosis-evoked pain, which revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons. Conflicts of interest

References

[1] V. Neugebauer, S.M. Carlton, Peripheral metabotropic glutamate receptors as drug targets for pain relief, Expert Opin. Ther. Targets 6 (2002) 349–361.
[2] K. Omote, T. Kawamata, M. Kawamata, A. Namiki, Formalin-induced release of excitatory amino acids in the skin of the rat hindpaw, Brain Res. 787 (1998) 161–164.
[3] N.B. Lawand, T. McNearney, K.N. Westlund, Amino acid release into the knee joint: key role in nociception and inflammation, Pain 86 (2000) 69–74.
[4] G. Bhave, F. Karim, S.M. Carlton, Gereau RWt peripheral group i metabotropic glutamate receptors modulate nociception in mice, Nat. Neurosci. 4 (2001) 417–423.
[5] P.J. Conn, J.P. Pin, Pharmacology and functions of metabotropic glutamate receptors, Annu. Rev. Pharmacol. Toxicol. 37 (1997) 205–237.
[6] B.J. Kolber, Mglurs head to toe in pain, Prog. Mol. Biol. Transl. Sci. 131 (2015) 281–324.
[7] S.M. Carlton, G.L. Hargett, Colocalization of metabotropic glutamate receptors in rat dorsal root ganglion cells, J. Comp. Neurol. 501 (2007) 780–789.
[8] K. Walker, A. Reeve, M. Bowes, J. Winter, G. Wotherspoon, A. Davis, P. Schmid, F. Gasparini, R. Kuhn, L. Urban, Mglu5 receptors and nociceptive function ii. Mglu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia, Neuropharmacology 40 (2001) 10–19.
[9] M.K. Chung, J. Lee, J. Joseph, J. Saloman, J.Y. Ro, Peripheral group i metabotropic glutamate receptor activation leads to muscle mechanical hyperalgesia through trpv1 phosphorylation in the rat, J. Pain 16 (2015) 67–76.
[10] S. Zhou, S. Komak, J. Du, S.M. Carlton, Metabotropic glutamate 1alpha receptors on peripheral primary afferent fibers: their role in nociception, Brain Res. 913 (2001) 18–26.
[11] C.Z. Zhu, S.G. Wilson, J.P. Mikusa, C.T. Wismer, D.M. Gauvin, J.J. Lynch 3rd, C.L. Wade, M.W. Decker, P. Honore, Assessing the role of metabotropic glutamate receptor 5 in multiple nociceptive modalities, Eur. J. Pharmacol. 506 (2004) 107–118.
[12] E. Lindstrom, M. Brusberg, P.A. Hughes, C.M. Martin, S.M. Brierley, B.D. Phillis, R. Martinsson, C. Abrahamsson, H. Larsson, V. Martinez, L.A. Blackshaw, Involvement of metabotropic glutamate 5 receptor Capsazepine in visceral pain, Pain 137 (2008) 295–305.
[13] C.Z. Zhu, G. Hsieh, O. Ei-Kouhen, S.G. Wilson, J.P. Mikusa, P.R. Hollingsworth, R. Chang, R.B. Moreland, J. Brioni, M.W. Decker, P. Honore, Role of central and peripheral mglur5 receptors in post-operative pain in rats, Pain 114 (2005) 195–202.
[14] A. Dogrul, M.H. Ossipov, J. Lai, T.P. Malan Jr., F. Porreca, Peripheral and spinal antihyperalgesic activity of sib-1757, a metabotropic glutamate receptor (mglur(5)) antagonist, in experimental neuropathic pain in rats, Neurosci.Lett. 292 (2000) 115–118.
[15] H.J. Hu, G. Bhave, R.W. Gereau 4th, Prostaglandin and protein kinase a-dependent modulation of vanilloid receptor function by metabotropic glutamate receptor 5: potential mechanism for thermal hyperalgesia, J.Neurosci. 22 (2002) 7444–7452.
[16] K. Szteyn, M.P. Rowan, R. Gomez, J. Du, S.M. Carlton, N.A. Jeske, A-kinase anchoring protein 79/150 coordinates metabotropic glutamate receptor sensitization of peripheral sensory neurons, Pain 156 (2015) 2364–2372.
[17] T. Masuoka, T. Nakamura, M. Kudo, J. Yoshida, Y. Takaoka, N. Kato, T. Ishibashi, N. Imaizumi, M. Nishio, Biphasic modulation by mglu5 receptors of trpv1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity, Br. J. Pharmacol. 172 (2015) 1020–1033.
[18] A.I. Basbaum, D.M. Bautista, G. Scherrer, D. Julius, Cellular and molecular mechanisms of pain, Cell 139 (2009) 267–284.
[19] J.A. Wemmie, R.J. Taugher, C.J. Kreple, Acid-sensing ion channels in pain and disease, Nat. Rev. Neurosci. 14 (2013) 461–471.
[20] H.J. Kweon, B.C. Suh, Acid-sensing ion channels (asics): therapeutic targets for neurological diseases and their regulation, BMB Rep. 46 (2013) 295–304.
[21] K.H. Steen, P.W. Reeh, F. Anton, H.O. Handwerker, Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro, J. Neurosci. 12 (1992) 86–95.
[22] K.H. Steen, U. Issberner, P.W. Reeh, Pain due to experimental acidosis in human skin: evidence for non-adapting nociceptor excitation, Neurosci. Lett.199 (1995) 29–32.
[23] S. Ugawa, T. Ueda, Y. Ishida, M. Nishigaki, Y. Shibata, S. Shimada, Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors, J. Clin. Investig. 110 (2002) 1185–1190.
[24] D. Alvarez de la Rosa, P. Zhang, D. Shao, F. White, C.M. Canessa, Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2326–2331.
[25] C.J. Benson, J. Xie, J.A. Wemmie, M.P. Price, J.M. Henss, M.J. Welsh, P.M. Snyder, Heteromultimers of deg/enac subunits form h+-gated channels in mouse sensory neurons, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2338–2343.
[26] E. Deval, J. Noel, N. Lay, A. Alloui, S. Diochot, V. Friend, M. Jodar, M. Lazdunski,E. Lingueglia, Asic3, a sensor of acidic and primary inflammatory pain, EMBOJ. 27 (2008) 3047–3055.
[27] O. Krishtal, The asics: signaling molecules? Modulators? Trends Neurosci. 26 (2003) 477–483.
[28] E. Deval, J. Noel, X. Gasull, A. Delaunay, A. Alloui, V. Friend, A. Eschalier, M.Lazdunski, E. Lingueglia, Acid-sensing ion channels in postoperative pain, J.Neurosci. 31 (2011) 6059–6066.
[29] G. Dussor, Asics as therapeutic targets for migraine, Neuropharmacology 94 (2015) 64–71.
[30] E. Deval, E. Lingueglia, Acid-sensing ion channels and nociception in the peripheral and central nervous systems, Neuropharmacology 94 (2015) 49–57.
[31] C.Y. Qiu, Y.Q. Liu, F. Qiu, J. Wu, Q.Y. Zhou, W.P. Hu, Prokineticin 2 potentiates acid-sensing ion channel activity in rat dorsal root ganglion neurons, J.Neuroinflamm. 9 (2012) 108.
[32] N.R. Gavva, R. Tamir, Y. Qu, L. Klionsky, T.J. Zhang, D. Immke, J. Wang, D. Zhu, T.W. Vanderah, F. Porreca, E.M. Doherty, M.H. Norman, K.D. Wild, A.W.Bannon, J.C. Louis, J.J. Treanor, Amg 9810[(e)-3-(4-t-butylphenyl)-n-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl) acrylamide], a novel vanilloid receptor 1 (trpv1) antagonist with antihyperalgesic properties, J. Pharmacol. Exp. Ther. 313 (2005) 474–484.
[33] O. Poirot, T. Berta, I. Decosterd, S. Kellenberger, Distinct asic currents are expressed in rat putative nociceptors and are modulated by nerve injury, J.Physiol. 576 (2006) 215–234.
[34] M. Omori, M. Yokoyama, Y. Matsuoka, H. Kobayashi, S. Mizobuchi, Y. Itano, K. Morita, H. Ichikawa, Effects of selective spinal nerve ligation on acetic acid-induced nociceptive responses and asic3 immunoreactivity in the rat dorsal root ganglion, Brain Res. 1219 (2008) 26–31.
[35] X. Wang, W.G. Li, Y. Yu, X. Xiao, J. Cheng, W.Z. Zeng, Z. Peng, M. Xi Zhu, T.L. Xu, Serotonin facilitates peripheral pain sensitivity in a manner that depends on the nonproton ligand sensing domain of asic3 channel, J. Neurosci. 33 (2013) 4265–4279.
[36] O.A. Krishtal, V.I. Pidoplichko, A receptor for protons in the nerve cell membrane, Neuroscience 5 (1980) 2325–2327.
[37] F. Qiu, C.Y. Qiu, H. Cai, T.T. Liu, Z.W. Qu, Z. Yang, J.D. Li, Q.Y. Zhou, W.P. Hu, Oxytocin inhibits the activity of acid-sensing ion channels through the vasopressin, v1a receptor in primary sensory neurons, Br. J. Pharmacol. 171 (2014) 3065–3076.
[38] M.P. Price, S.L. McIlwrath, J. Xie, C. Cheng, J. Qiao, D.E. Tarr, K.A. Sluka, T.J. Brennan, G.R. Lewin, M.J. Welsh, The drasic cation channel contributes to the detection of cutaneous touch and acid stimuli in mice, Neuron 32 (2001) 1071–1083.
[39] C. Morenilla-Palao, R. Planells-Cases, N. Garcia-Sanz, A. Ferrer-Montiel, Regulated exocytosis contributes to protein kinase c potentiation of vanilloid receptor activity, J. Biol. Chem. 279 (2004) 25665–25672.
[40] J.S. Lee, J.Y. Ro, Peripheral metabotropic glutamate receptor 5 mediates mechanical hypersensitivity in craniofacial muscle via protein kinase c dependent mechanisms, Neuroscience 146 (2007) 375–383.
[41] X.M. Zha, Acid-sensing ion channels: trafficking and synaptic function, Mol. Brain 6 (2013) 1.
[42] A. Duggan, J. Garcia-Anoveros, D.P. Corey, The pdz domain protein pick1 and the sodium channel bnac1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons, J. Biol.Chem. 277 (2002) 5203–5208.
[43] E. Deval, M. Salinas, A. Baron, E. Lingueglia, M. Lazdunski, Asic2b-dependent regulation of asic3, an essential acid-sensing ion channel subunit in sensory neurons via the partner protein pick-1, J. Biol. Chem. 279 (2004) 19531–19539.
[44] C. Ren, X. Gan, J. Wu, C.Y. Qiu, W.P. Hu, Enhancement of acid-sensing ion channel activity by metabotropic p2y utp receptors in primary sensory neurons, Purinergic Signal. 12 (2015) 69–78.
[45] N.A. Dorofeeva, A.V. Karpushev, M.V. Nikolaev, K.V. Bolshakov, J.D. Stockand, A. Staruschenko, Muscarinic m1 modulation of acid-sensing ion channels, Neuroreport 20 (2009) 1386–1391.
[46] S. Kellenberger, L. Schild, International union of basic and clinical pharmacology Xci. Structure, function, and pharmacology of acid-sensing ion channels and the epithelial na+ channel, Pharmacol. Rev. 67 (2015) 1–35.