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GPR171 Activation Modulates Nociceptor Functions, Alleviating Pathologic Pain

Modulation of the function of somatosensory neurons is an important analgesic strategy, requiring the proposal of novel molecular targets. Many G-protein-coupled receptors (GPRs) have been deorphanized, but the receptor locations, outcomes due to their activations, and their signal transductions remain to be elucidated, regarding the somatosensory nociceptor function. Here we report that GPR171, expressed in a nociceptor subpopulation, attenuated pain signals via Gi/o-coupled modulation of the activities of nociceptive ion channels when activated by its newly found ligands. Administration of its natural peptide ligand and a synthetic chemical ligand alleviated nociceptor-mediated acute pain aggravations and also relieved pathologic pain at nanomolar and micromolar ranges. This study suggests that functional alteration of the nociceptor neurons by GPR171 signaling results in pain alleviation and indicates that GPR171 is a promising molecular target for peripheral pain modulation.


Introduction
Nociceptor neurons in the dorsal root ganglia (DRG) are in charge of the initiation of pain-related signals in the body [1]. Upon activation by ligands, G-protein-coupled receptors (GPRs) modulate the excitability of nociceptors, negatively or positively tuning the strength of the initiated signals. While some GPRs, such as prostaglandin EP2 and EP4 receptors and bradykinin B2 receptors strongly facilitate nociceptor excitation, other GPRs, including µ-opioid receptors, GABA B receptors, and chemokine-like receptor 1 receptors, reduce excitation, highly contributing to relieving pain [2][3][4][5][6]. Better understanding of the identities of pain-relieving GPRs and the molecular mechanisms underlying the reduction in excitability may help to contribute to the development of novel painkilling strategies. GPR171 was initially cloned two decades ago but was recently deorphanized [7,8]. Here we examined its expression in the nociceptor population and hypothesized that GPR171 activation by its specific ligands may modulate nociceptor functions, consequently regulating pain phenotypes.
For CFA-induced inflammation, 10 µL CFA was injected into a hind paw (Supplementary Figure S8D). For determining changes in mechanical or thermal behaviors by 24 and 48 h CFA inflammation, mice were acclimated to the test environment for 30 min before performing the following assays. Hargreaves assay (using a Plantar Analgesia meter; IITC, Woodland Hills, CA, USA) for acute heat avoidance or thermal hyperalgesia, von Frey assay by up-and-down paradigm (using von Frey filaments; Stoelting, Dale Wood, IL, USA) for mechanical allodynia, and Randall-Selitto assay (using Analgesy-meter, UGO Basile, Italy) for flexion reflex to noxious pressures, were carried out as described previously [12][13][14][15][16] (Supplementary Figure S8C).
For neuropathic pain development, the CCI model was established as described previously [17] (Supplementary Figure S8D). Briefly, the mice were anesthetized with inhalation of 3% isoflurane in a mixture of N 2 O/O 2 gas. The full circumference of the sciatic nerve of the left hind limb was exposed and loosely tied three times with two silk sutures (7-0; Ailee, Busan, Korea). Sham surgery was conducted by exposing the sciatic nerve in the same manner but without tying the nerve. Hargreaves and von Frey assays were performed with the injured mice 14 days after the surgery.
For a postoperative pain model, an incision pain model was modified according to a previous study [18] (Supplementary Figure S8D). Briefly, mice were anesthetized with 2% isoflurane and hind paws was sterilized with 10% povidone-iodine solution. With a no. 10 scalpel blade, a 5 mm longitudinal incision was made in the glabrous skin and fascia of a hind paw plantar. The underlying muscle was elevated with a sterile curved forcep, with the muscle origin and insertion intact. The incised skin was apposed with two nylon sutures (8-0; Ailee, Korea). Sham mice were not incised or sutured but anesthetized for the same duration.

Open Field Behavioral Tests
The open field was a square opaque white box (40 × 40 × 40 cm) that was divided into a central field (center, 20 × 20 cm) and an outer field (periphery). After drug injection, individual mice were exposed for 30 min to the open field box under dim light, and the total distance traveled, the time spent in the central field, and rearing numbers were monitored with TSE ActiMot/MoTil system (TSE systems, Bad Homburg, Germany) as described previously [19] (Supplementary Figure S8E).

Sticky-Tape Removal Tests
A circular piece of tape, 9 mm in diameter, was placed on the bottom of a hind paw of the animals. The latency of biting or licking to remove the tape was measured as described previously [20] (Supplementary Figure S8B).

Measurements of Food Intake, Water Intake, and Body Weight
Experiments were conducted as described previously [21]. Briefly, mice fasted for 12 h were treated with drugs and then returned to their individual cages. Cumulative food and water consumptions were recorded at 1, 2, 4, and 8 h after drug treatment. For intraperitoneal vehicle, 1% DMSO in sterile saline was used. For long-term measurement, drugs were administered on every third day for forty-eight days and food and water con-sumptions were recorded at 8 h after every drug treatment. Body weights were measured on every fifth day for fifty days from the first treatment.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and
Quantitative RT-PCR GPR171 mRNA expression was validated using RT-PCR or qRT-PCR as described previously [22]. We reverse-transcribed 1 µg RNA with the amfiRivert Platinum cDNA synthesis Master Mix (GenDEPOT, Baker, TX, USA). cDNA was subjected to qRT-PCR with the SYBR Green PCR Master Mix (Applied Biosystems, Woolston, UK) or RT-PCR with 5 × PCR Master Mix (Elpisbio, Daejeon, Korea). β-actin mRNA was the internal control. Primer sequences are listed below.

Cell Cultures and Transfections
Cultures of DRG neurons and HEK293 cells were prepared and transfected as described previously [11,23,24]. All cells were grown at 37 • C and 5% CO 2 and neurons were used at 48-72 h after culture. For in vivo transfection of small-interfering RNA (siRNA) or scrambled RNA (scRNA), 3 µg of RNA and 5% d-glucose in 7 µg lipofectamine 2000 (Invitrogen) were injected into the spinal cord or peri-sciatic region 24 or 48 h before experiments. Mouse GPR171-siRNA (target sequence: sense-CUGUACUAUCAUCUGUCAA, antisense-UUGACAGAUGAUAGUACAG) and a scrambled RNA were purchased from Bioneer Corporation (Daejeon, Korea). cDNA for human GPR171 was generously gifted by Dr. Jae Young Seong of Korea University.

Fluorescence Intracellular Ca 2+ Imaging Experiments
The Ca 2+ imaging experiments were conducted as described previously [25]. Briefly, cells were loaded with 5 µM Fura-2AM dye and 0.02% pluronic F127 for 30 min. The cells were resuspended in 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES (titrated to pH 7.4 with NaOH). Images of dye-loaded cells were obtained with a cooled charge-coupled device (CCD) camera (Retiga-SRV, Q-imaging Corp., Burnaby, BC, Canada) at a perfusion rate of approximately 4 mL/min of test solutions and at room temperatures. The ratio of fluorescence intensity at 340 nm/380 nm wavelengths in each experiment was analyzed using MetaFluor (Molecular Devices, Sunnyvale, CA, USA).

Whole-Cell Patch Clamp Recordings
The slices after incubation as described were placed in a recording chamber and perfused with oxygenated recording solution at a rate of 5 mL/min at room temperatures. Whole-cell recording experiments were then performed on lamina 2 dorsal horn neurons. Borosilicate glass pipettes (Sutter Instrument, Novato, CA, USA) with resistance of 3-6 MΩ were filled with internal solution containing 130 mM potassium gluconate, 5 mM KCl, 4 mM Na 2 ATP, 0.5 mM NaGTP, 20 mM HEPES, and 0.5 mM EGTA with pH 7.28 adjusted with KOH and osmolality at 310-320 mOsm/kg. Data were acquired by pClamp 10.0 software (Molecular Devices) with MultiClamp 700B patch-clamp amplifier and Digidata 1550B (Molecular Devices). Data were low-pass filtered at 2 kHz and digitized at 5 kHz.

Ex Vivo Nociceptor Recordings
Ex vivo single-fiber recordings were carried out as previously described [26]. The two-chambered organ bath system (perfusion and recording chambers) was used. The perfusion chamber was continuously superfused with a synthetic interstitial fluid (SIF) composed of 3.5 mmol/L KCl, 107.8 mmol/L NaCl, 0.69 mmol/L MgSO 4 ·7H 2 O, 1.53 mmol/Ll CaCl 2 ·2H 2 O, 1.67 mmol/L NaH 2 PO 4 ·2H 2 O, 26.2 mmol/L NaHCO 3 , 9.64 mmol/L C 6 H 11 NaO 7 , 7.6 mmol/L sucrose, and 5.55 mmol/L glucose, which was saturated with a mixture of 95% O 2 and 5% CO 2 and maintained at 31 ± 1 • C. The hairy skin of the hind paw innervated by the saphenous nerve was dissected from mice sacrificed with CO 2 inhalation. The preparation was put in the perfusion chamber with the epidermal side down. The nerve connected to the skin flap was drawn to the recording chamber filled with paraffin oil. The nerve filaments teased from the nerve were used for single-fiber recordings using gold electrodes. Spikes from single C-fiber nociceptors were recorded with a differential amplifier (DP 311; Warner Instruments, Hamden, CT, USA), and the data were transferred to a personal computer via a data acquisition system (DAP5200a; Microstar Laboratory, Redmond, WA, USA). Data were analyzed using the window discrimination feature of the software (Dapsys 8; Bethel University, http://dapsys.net/ accessed on 5 March 2021) [27].

Compounds
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise described. BigLEN and MS15203 were purchased from Tocris Bioscience (Ellisville, MO, USA). Cinnamaldehyde was purchased from MP Biomedicals (Solon, OH, USA). Stock solutions were prepared using water or dimethyl sulfoxide and diluted with test solutions before use.

Data Analysis
Statistical significance of data was assessed using the two-tailed unpaired Student's t-test, one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test, or two-way ANOVA followed by Tukey's post hoc test (*** p < 0.001, ** p < 0.01, * p < 0.05). Data are shown as means ± S.E.M.

Data Availability
All relevant data are available from the authors upon request.

GPR171 Is Expressed in Nociceptors
We first examined GPR171 immunostaining in DRG, which initiate nociceptive signals. We found GPR171 expression in a subpopulation of DRG neurons (502 of 1498; 33.5%). We further examined whether other subpopulation markers were co-expressed with GPR171 ( Figure 1A,B). Peptidergic and non-peptidergic nociceptor markers including calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding components were co-localized with GPR171 expression as well as the marker for myelinated DRG neurons, neurofilament 200 (NF200). Over fifty percent of the GPR171-positive subpopulation co-expressed CGRP and at least a third of IB4-positive non-peptidergic nociceptors were also GPR171positive ( Figure 1B). Neuronal diameter analysis was also performed on the DRG neurons. Small-diameter neurons, which are mostly comprised of C-and or Aδ-fiber nociceptors, constituted the majority of GPR171-expressing neurons, particularly around neurons with 15-20 µm in diameter ( Figure 1C). Approximately half of these small-diameter neurons expressed GPR171, and these proportions were two times greater than the subpopulation with diameters 35 µm and larger. These data suggest that considerable numbers of nociceptor DRG neurons express GPR171. Immunostaining of the glabrous skin layers, spinal cord, and cultured DRG neurons of mice also revealed GPR171 expression (Supplementary Figure S1). DRG nerve termini in the skin epidermis partly showed its co-expression with CGRP but not with IB4 (Supplementary Figure S1A) [28,29]. Thus, these data indicate a potential role of GPR171 in nociceptor DRG neurons.
We then speculated whether GPR171 functions in nociception and examined the effect of its specific activation using the conventional formalin test. Intraplantar and intrathecal administration of endogenous or synthetic GPR171 agonists (bigLEN and MS15203, respectively) both significantly mitigated phase 2 nociceptive behaviors induced by 5% formalin injection ( Figure 1D,E). Interestingly, when we performed the Transient receptor potential ankyrin subtype 1 (TRPA1) activation-specific formalin test using a lower dose of formalin (0.5%) [30,31], both phase 1 and 2 nociceptive behaviors were reduced by GPR171 activation ( Figure 1F,G). The data suggest that GPR171 expressed in peripheral nociceptors may play a role in modulating their excitability, implicating that modality-specific components such as Transient receptor potential (TRP) ion channels may be involved in this mechanism.

Peripheral GPR171 Activation Attenuates TRP-Specific Acute Pain
Based on the data shown above, we hypothesize possible co-expressions of nociceptive TRP ion channels (TRPV1, TRPA1, and TRPV4). When immunostained, over a half of TRP-positive neurons co-expressed GPR171 and approximately a quarter of the DRG neuronal population expressing GPR171 also expressed at least one of those three TRP channels (Figure 2A,B). Therefore, we then focused on these TRP-specific pain modalities. Licking and flicking induced by intraplantar injection of cinnamaldehyde, which is a TRPA1 activator, and induced by the TRPV1 activator capsaicin as well as flinches in response to TRPV4 activation using hypotonic insult were all blunted when the GPR171 activator, MS15203, was peripherally pre-treated ( Figure 2C,E). In contrast, nociceptive behaviors by the activation of voltage-sensing components were not affected ( Figure 2F). Thus, rather than general excitability of the nociceptors, TRP-channel-specific mechanisms may be modulated by GPR171 action in these neurons, which may also explain the attenuation of phase 1 nociception because, at least partly, TRPA1 activation is known to be involved. Experiments were conducted as in (F) but animals were intrathecally pretreated with vehicle or drugs. Five animals were used for each data point of (D-G). Effects on the durations of the nociceptive responses of drugs in (D-G) at every 5 min (left panels) were compared with those with vehicle injection by two-way ANOVA and Tukey's test (* for MS15203 treatment and # for bigLEN treatment), and the phase comparisons (right panels) were conducted by one-way ANOVA and Bonferroni's test. Statistical differences are indicated by * for the effects of MS15203 and # for those of bigLEN, compared to those of the vehicles. *** p < 0.001, ** p < 0.01, * p < 0.05; ### p < 0.001, ## p < 0.01, # p < 0.05.
We then speculated whether GPR171 functions in nociception and examined the effect of its specific activation using the conventional formalin test. Intraplantar and intrathecal administration of endogenous or synthetic GPR171 agonists (bigLEN and MS15203, respectively) both significantly mitigated phase 2 nociceptive behaviors in- duration of 5% formalin-induced nociceptive behaviors in mice. Animals were intraplantarily (i.pl.) pretreated with vehicle (red), or GPR171-specific agonists, MS15203 (2.5 µg, green) or bigLEN (2.5 µg, blue), 30 min prior to the formalin injection. The formalin responses were quantified into the two phases of the right histogram. (E) Experiments were conducted as in (D) but animals were intrathecally (i.t.) pretreated with vehicle or drugs. (F) Experiments were conducted as in (D) but animals were treated with 0.5% formalin. (G) Experiments were conducted as in (F) but animals were intrathecally pretreated with vehicle or drugs. Five animals were used for each data point of (D-G). Effects on the durations of the nociceptive responses of drugs in (D-G) at every 5 min (left panels) were compared with those with vehicle injection by two-way ANOVA and Tukey's test (* for MS15203 treatment and # for bigLEN treatment), and the phase comparisons (right panels) were conducted by one-way ANOVA and Bonferroni's test. Statistical differences are indicated by * for the effects of MS15203 and # for those of bigLEN, compared to those of the vehicles. *** p < 0.001, ** p < 0.01, * p < 0.05; ### p < 0.001, ## p < 0.01, # p < 0.05. TRPV4 activation using hypotonic insult were all blunted when the GPR171 activator, MS15203, was peripherally pre-treated ( Figure 2C,E). In contrast, nociceptive behaviors by the activation of voltage-sensing components were not affected ( Figure 2F). Thus, rather than general excitability of the nociceptors, TRP-channel-specific mechanisms may be modulated by GPR171 action in these neurons, which may also explain the attenuation of phase 1 nociception because, at least partly, TRPA1 activation is known to be involved.

GPR171 Modulates DRG Neuronal Functions
We further analyzed the mechanism of GPR171 action using in vitro and ex vivo preparations. In cultured DRG neurons, the TRPV1-mediated increase in intracellular Ca 2+ upon capsaicin perfusion was blunted by the GPR171-specific agonist, MS15203 or bigLEN, incubation ( Figure 3A and Supplementary Figure S3A). This result is reproducible in whole-cell voltage clamp experiments ( Figure 3J). The inhibitory effect disappeared when pretreated with pertussis toxin but not with gallein, which confirmed that Gi/o signaling is coupled and that Gα than Gβγ is critical in this GPR action ( Figure 3B Figure S4A-H) and suggests that TRP-channel-specific events were particularly modulable by GPR171 action. Ex vivo C-fiber recording in skin-nerve preparation also showed the same outcomes from peripheral perfusion of bigLEN for inhibition of capsaicin-induced firing (Supplementary Figure S4M,N). Therefore, TRP-mediated excitatory events can be regulated by GPR171 activation in the peripheral termini of nociceptors. We recorded the excitatory post-synaptic current (EPSC) of dorsal horn neurons to observe the effect of GPR171 in the spinal cord. The frequency of EPSC but not its amplitude evoked by capsaicin perfusion was significantly reduced with treatment with either bigLEN or MS15203, indicating that TRPV1-mediated presynaptic events in the DRG central termini were selectively attenuated by GPR171 activation (Figure 3G-I; Supplementary Figure  S4I-L). Altogether, these data suggest that TRP activation in nociceptor DRG neurons may be a crucial target of GPR171 action in peripheral nociceptive signaling.

Peripheral GPR171 Activation Attenuates Pathologic Pain
To determine whether the anti-nociceptive mechanisms of GPR171 work in pathologic pain states in vivo, we first tested peripheral injection of GPR171-specific agonists in complete Freund's adjuvant (CFA)-induced hind paw inflammatory pain. Two injection routes were selected to examine whether the targeting of peripheral and central termini of nociceptors works. Allodynic mechanical hypersensitivity and hyperalgesic heat hypersensitivity were both improved by intrathecal agonist injection ( Figure 4A,B) or by its ipsilateral intraplantar injection ( Figure 4C,D). These analgesic effects increased in a dosedependent manner, which indicates that the analgesia is receptor-dependent and that doses of hundreds nanomolar to micromolar concentrations are relatively effective ( Figure 4E,F and Supplementary Figure S5). We also tested the effect of the specific agonists on chronic constriction injury (CCI)-induced pain. The analgesic effects were largely similar but more prominent in mechanical allodynia compared with those observed in CFA-induced pain ( Figure 5A-D). These effects also showed dose dependence ( Figure 5E,F; Supplementary Figure S6). In an incisional pain model of mice, GRP171 activation was also effective at allodynic and hyperalgesic pain alleviation ( Figure 6).

Peripheral GPR171 Activation Attenuates Pathologic Pain
To determine whether the anti-nociceptive mechanisms of GPR171 work in pathologic pain states in vivo, we first tested peripheral injection of GPR171-specific agonists in complete Freund's adjuvant (CFA)-induced hind paw inflammatory pain. Two injection routes were selected to examine whether the targeting of peripheral and central termini of nociceptors works. Allodynic mechanical hypersensitivity and hyperalgesic heat hypersensitivity were both improved by intrathecal agonist injection ( Figure 4A,B) or by its ipsilateral intraplantar injection ( Figure 4C,D). These analgesic effects increased in a dosedependent manner, which indicates that the analgesia is receptor-dependent and that doses of hundreds nanomolar to micromolar concentrations are relatively effective (Figure 4E,F and Supplementary Figure S5). We also tested the effect of the specific agonists on chronic constriction injury (CCI)-induced pain. The analgesic effects were largely similar but more prominent in mechanical allodynia compared with those observed in CFAinduced pain ( Figure 5A-D). These effects also showed dose dependence ( Figure 5E,F; Supplementary Figure S6). In an incisional pain model of mice, GRP171 activation was also effective at allodynic and hyperalgesic pain alleviation ( Figure 6).   single intrathecal treatment of bigLEN 24 h after CFA inflammation; inverted triangle, double intrathecal treatments of bigLEN 24 and 48 h after CFA inflammation). Statistical differences are indicated by * for the effects of MS15203 and # for those of bigLEN, compared to those of the vehicles. *** p < 0.001, ** p < 0.01, * p < 0.05; ### p < 0.001, ## p < 0.01, # p < 0.05.   Figure S6). Symbols indicate different drug treatments in mice with CCI (circle, 1 h after intraplantar treatment of MS15203; square, 2 h after intraplantar treatment of MS15203; diamond, 1 h after intraplantar treatment of bigLEN; inverted triangle, 2 h after intraplantar treatment of bigLEN). *** p < 0.001, ** p < 0.01; ### p < 0.001, ## p < 0.01.
square, 2 h after intrathecal treatment of MS15203; diamond, 1 h after intrathecal treatment of bigLEN; inverted triangle, 2 h after intrathecal treatment of bigLEN). (F) Percent M.P.E.s were produced from treatments with different doses of GPR171 activators (details are listed in Supplementary Figure S6). Symbols indicate different drug treatments in mice with CCI (circle, 1 h after intraplantar treatment of MS15203; square, 2 h after intraplantar treatment of MS15203; diamond, 1 h after intraplantar treatment of bigLEN; inverted triangle, 2 h after intraplantar treatment of bigLEN). *** p < 0.001, ** p < 0.01; ### p < 0.001, ## p < 0.01.  To determine whether the agonist-induced analgesic effect is GPR171 specific, we conducted RNA interference to downregulate GPR171 expression in the DRG by pre-sciatic or intrathecal injection of its small-interfering RNA and observed the in vivo parameters from the CFA model. Despite bigLEN or MS15203 treatment, pain deteriorated in a similar fashion to those treated with vehicle (Figure 7), indicating that these agonists may specifically act by GPR171 activation. We further determined whether any contribution of psychiatric or motor mechanisms may occur by this GPR activation in the central nervous system, particularly when intrathecally injected, but no such sign was detected in the open field test (Figure 8). We also wondered whether our local intraplantar and intrathecal administrations of GPR modulators may affect eating behaviors, which was previous shown by hypothalamic modulation [21]. We reproduced the mild increase in food intake when intraperitoneally injected, but our local injections did not significantly affect food and water intake and weight gain (Supplementary Figure S2I-M). This tolerance was repeated in mice with GRP171 knockdown (Supplementary Figure S7). We further checked whether innocuous somatosensory functions are affected and showed that those are largely ignorable (Supplementary Figure S2A-H). Overall, our results suggest that the inhibitory actions of GPR171 activation on the nociceptor activity can be extrapolated to the in vivo level with pathologic pain, and thus, GPR171 may be a novel peripheral target for pain modulation.
intrathecal injection of its small-interfering RNA and observed the in vivo parameters f the CFA model. Despite bigLEN or MS15203 treatment, pain deteriorated in a similar f ion to those treated with vehicle (Figure 7), indicating that these agonists may specific act by GPR171 activation. We further determined whether any contribution of psychia or motor mechanisms may occur by this GPR activation in the central nervous system, ticularly when intrathecally injected, but no such sign was detected in the open field ( Figure 8). We also wondered whether our local intraplantar and intrathecal administrat of GPR modulators may affect eating behaviors, which was previous shown by hypo lamic modulation [21]. We reproduced the mild increase in food intake when intraperito ally injected, but our local injections did not significantly affect food and water intake weight gain (Supplementary Figure S2I-M). This tolerance was repeated in mice w GRP171 knockdown (Supplementary Figure S7). We further checked whether innocu somatosensory functions are affected and showed that those are largely ignorable (Sup mentary Figure S2A-H). Overall, our results suggest that the inhibitory actions of GPR activation on the nociceptor activity can be extrapolated to the in vivo level with pathol pain, and thus, GPR171 may be a novel peripheral target for pain modulation.  on Frey thresholds in complete Freund's adjuvant (CFA)-inflamed mice. Animals were intraplantarily treated with drugs isted in the inset immediately after 24 and 48 h threshold monitoring. The left dot plot shows individual threshold values. G) Time course of Hargreaves latencies in CFA-inflamed mice. Animals were treated with drugs in the same manner as n (F). The left dot plot shows individual latency values. (H) Expression levels of GPR171 observed by quantitative RT-CR with lumbar DRGs of the animals peri-sciatically treated with siRNA compared to those with scrambled RNA. *** p 0.001, ** p < 0.01; ### p < 0.001.

Discussion
Our study proposes a new GPR-coupled regulatory mechanism of pain. Use of the neuropeptide ligand, bigLEN, and synthetic ligand, MS15203, has confirmed GPR171 action, as shown in previous studies [7,21]. In peripheral nociceptors, the similar Gi/omediated effect of GPR171 activation operates with these two ligands. Either intraplantar injection or intrathecal application of these ligands avoided brain-related behaviors observed in the studies above, suggesting that pain-specific phenotypes can be produced when peripherally modulated.
We did not detect any GPR171 in tissues near the DRG termini, which indicates that GPR171 action in nociceptors rather than non-neuronal actions may be predominant for neuronal modulation. However, the secretion of endogenous ligands for GPR171 including bigLEN and other unknown ones remains as an open question because of the lack of available antibodies. Since neuron-specific expression of Pcsk1n protein, which is the precursor protein for bigLEN cleavage, has been shown, including in embryonic DRG, but not neighboring non-neuronal tissues, paracrine and/or autocrine actions on the nociceptor-secreted bigLEN in some inflamed conditions can be hypothesized [32,33].
In the spinal cord superficial laminae, some neuronal components, but no glial component, expressed GPR171 as shown by immunohistochemical assays. Nonetheless, firing of dorsal horn neurons was only presynaptically modulated by perfusion of GPR171 ligands. Further studies will clarify whether and/or how these GPR171-positive dorsal horn neurons contribute to the modulation of the ascending pain signals. This may also help to fully understand why phase 2 nociception in the formalin test was more consistently affected than that of phase 1 because many other components as well as presynaptic TRPs are known to contribute to phase 2-inducing synaptic sensitization.
DRG is an important painkilling target because DRG-specific modulation may avoid central adverse effects. Neuropeptide-activated and Gi/o-coupled GPRs such as somatostatin and neuropeptide Y receptors, subtypes of which are expressed in a subset of DRG neurons, are currently being revisited in exploring peripheral analgesic efficacy [34][35][36]. Of note, the studies proposed TRP channel inhibition as the downstream effector mechanism. Although Gi/o-coupled signaling may affect the general excitability of DRG neurons, which mainly involves the altered functions of voltage-gated channels or cyclic nucleotidegated channels, only TRP channel-mediated effects were detected in our study. Thus, the analgesic outcomes observed here may share a common molecular paradigm with previous studies in which peripheral GPR modulation prompts TRP inhibition. Nonetheless, with the current lack of approaches using voltage-dependent subunits, the possibilities need to be carefully monitored to determine whether specific voltage-dependent effectors are influenced in some particular pathological states and locations in the future.
TRP ion channels expressed in nociceptors play a central role in monitoring harmful events in tissues and transforming those into pain signals. In addition to the direct and specific inhibition of each TRP, triggering an inhibitory upstream signal that may commonly attenuate different TRP actions can be an efficient approach for controlling many aspects of pain events. In this context, our results indicate a novel GPR-mediated mechanism that inhibits multiple TRP actions. Our study demonstrated TRPA1, TRPV1, TRPV2, TRPV4, TRPM3, and TRPC4/C5 inhibitions by GPR171 activation. Many of these TRPs have been shown to be engaged in mechanical and thermal nociceptions to some extent, which seems to explain our analgesic phenotypes from the treatment of GRP171 agonists [37]. Such effector-specific outcomes may not be limited to these TRP-mediated ones in this GPR action and other possible important pathologies should be explored in the future.
Very recently, GRP171 is proposed to be present in the periaqueductal gray (PAG) and to regulate opioid signaling there in µ-opioid receptor-dependent fashions [38]. Its activation alone in PAG was not significantly effective, but reinforced µ-receptor-mediated signals, finally enhanced the opioid-induced analgesia. Thus, the outcomes observed here regarding the peripheral mechanisms and those potentially involving the descending inhibitory pathways appear to commonly contribute to pain relief. Further attempts on these analgesic concepts operating in different circuits in more practical ways such as quantitative comparisons in pharmacology and target accessibility may extend the applicable options for promoting the utilities of GPR171 modulators. In conclusion, our results suggest a novel GPR171-mediated pain-relieving mechanism. Such findings may lead to future opportunities to devise novel analgesic strategies.
Supplementary Materials: The following are available online at https://www.mdpi.com/2227-905 9/9/3/256/s1. Figure S1. GPR171 expression in tissues. Figure S2. Effects of treatments of GPR171 activators on acute somatosensations, food intake, water intake, and weight gain. Figure S3. BigLEN modulates DRG neuronal TRP channel functions. Figure S4. BigLEN modulates dorsal horn and DRG neuronal current response upon capsaicin treatment but not voltage-dependent currents in DRG neurons. Figure S5. Administration of GPR171 agonists at various doses alleviates inflammatory pain. Figure S6. Administration of GPR171 agonists at various doses alleviates neuropathic pain. Figure S7. Effects of treatments of GPR171 activators on food intake, water intake, and weight gain in mice with knockdown. Figure S8. Summarized illustrations for animal experiments conducted in this study.