GPR171 Activation Modulates Nociceptor Functions, Alleviating Pathologic Pain

Abstract

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.

Keywords: GPR171; TRP channel; analgesia; nociceptor; pain.

Figure 1

PR171 expression in a subset of dorsal root ganglia (DRG) neurons. (A) Double immunostaining of GPR171 with other DRG markers in the lumbar DRG (scale bar, 50 μm). Arrowheads indicate the marker-expressing somas for example. (B) Co-expression ratios of GPR171 and other DRG markers. The ratios were calculated as mean percent values by counting the number of GPR171-positive neurons among that of marker-positive neurons from three different mice and vice versa. Data are presented as means ± S.E.M. (C) Size distribution of collected DRG neurons and GPR171-positive DRG neurons according to soma diameters. (D) Time course of the 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.

Figure 2

GPR171 expressions in Transient receptor potential (TRP)-expresser neurons and the effect on TRP-mediated nociceptions of GPR171 activation. (A) Double immunostaining of GPR171 with TRP channels in the lumbar DRGs (scale bar, 50 μm). (B) Co-expression ratios of GPR171 and TRP channels of the neurons from three different mice. Data are presented as means ± S.E.M. (C) Time course of the duration of 10 mM cinnamaldehyde (CA)-induced nociceptive behaviors in mice. Animals were intraplantarily pretreated with vehicle (red), or GPR171-specific agonist, MS15203 (2.5 μg, green), 30 min prior to the CA injection. From (C) to (E), black circles represent the mean data from the animals pretreated with MS150203, 30 min prior to the vehicle injection instead of TRP activators. Total durations of nociceptive responses for 5 min are quantified in the right histogram. (D) Time course of the duration of 0.1% capsaicin (CAP)-induced nociceptive behaviors and the effect on it of MS15203 were monitored in the same way as in (C). (E) Time course of the number of hypotonicity-induced flinches in prostaglandin E2 (PGE2)-primed mice. Animals were intraplantarily pretreated with vehicle (red) or MS15203 (2.5 μg, green), 30 min prior to the hypotonic stimulation. Total number of flinches for 10 min are quantified in the right histogram. (F) Time course of the duration of 140 mM KCl-induced nociceptive behaviors in mice. Animals were intraplantarily pretreated with vehicle (red) or MS15203 (2.5 μg, green), 30 min prior to the KCl injection. Total durations of nociceptive responses for 10 min are quantified in the right histogram. In (C–F), five animals were used for each data. Statistical differences are indicated by * for the effects of MS15203 compared to the responses upon chemical stimulations. *** p < 0.001.

Figure 3

GPR171 modulates TRP channel functions in DRG neurons. (A) MS15203 attenuated intracellular Ca²⁺ increases by 0.3 μM capsaicin-induced TRPV1 activation in Fura-2 Ca²⁺ imaging experiments using cultured murine DRG neurons (n = 30). Averaged Ca²⁺ peaks are quantified and normalized in the right histogram in (A) to (F). (B) Preincubation of pertussis toxin (PTX) for 18 h prevented the effect of GPR171 agonists shown in (A) (n = 10). (C) Co-application of 100 µM gallein did not prevent the effect of GPR171 agonists shown in (A) (n = 10). (D) MS15203 attenuated intracellular Ca²⁺ increases by 300 μM cinnamaldehyde-induced TRPA1 activation in Fura-2 Ca²⁺ imaging using cultured DRG neurons (n = 23). (E) MS15203 attenuated intracellular Ca²⁺ increases by 0.1 μM GSK1016790A (abbreviated to GSK)-induced TRPV4 activation in Fura-2 Ca²⁺ imaging using cultured DRG neurons (n = 12). (F) The µ-opioid receptor agonist [D-Ala², NMe-Phe⁴, Gly-ol⁵]-enkephalin (DAMGO), but not MS15203, attenuated intracellular Ca²⁺ increases by 60 mM KCl-induced voltage-gated channel activation in Fura-2 Ca²⁺ imaging using cultured DRG neurons (n = 36). Experiments shown in (A–F) were performed in triplicate. KCl stimulation was used for monitoring cell survival at the end of the fluorescence monitoring. (G–I) Representative traces of capsaicin-induced miniature excitatory postsynaptic currents (mEPSCs) in lamina 2 dorsal horn neurons (G) and its cumulative probability plot of the inter-event intervals (H) and the peak amplitudes (I) of recordings with or without 10 μM MS15203 treatment (n = 3). (J) Representative traces of capsaicin-induced inward currents in whole-cell voltage clamp recordings of cultured DRG neurons (left) and its quantification of peak amplitudes (right, n = 5) with or without 10 μM MS15203 treatment (inset: a current–voltage curve of capsaicin-induced current without MS15203 treatment subtracted by that with MS15203 treatment). The break indicates a 5 min omission on the trace. Data are presented as means ± S.E.M.

Figure 4

GPR171 activation alleviates inflammatory pain. (A) Time course of von Frey paw-withdrawal thresholds (PWTs) in complete Freund’s adjuvant (CFA)-inflamed mice. Animals were intrathecally treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) immediately after 24 and 48 h threshold monitoring. The right line plot shows the mean thresholds. BLs indicate baseline thresholds or latencies under normal condition before inflammation. Red arrows indicate the time for treatments. (B) Time course of Hargreaves paw-withdrawal latencies (PWLs) in CFA-inflamed mice. Animals were treated with drugs in the same manner as in (A). The right line plot shows the mean latencies. (C) Time course of von Frey thresholds in CFA-inflamed mice. Animals were intraplantarily treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) in the ipsilateral hind paws immediately after 24 and 48 h threshold monitoring. The right line plot shows the mean thresholds. (D) Time course of Hargreaves latencies in CFA-inflamed mice. Animals were treated with drugs in the same manner as in (C). The right line plot shows the mean latencies. Five animals were used for each data point. Statistical significance is represented using different symbols (* for MS15203 treatment and # for bigLEN treatment). (E) Maximum possible analgesic effects (percent M.P.E.) were produced from treatments with different doses of GPR171 activators (details are listed in Supplementary Figure S5). Percent M.P.E.s were calculated from comparisons of the subtractions of withdrawal thresholds (or latencies) of the ipsilateral side before and after inflammation with vehicle treatment, to those with drug treatment. Symbols indicate different drug treatments in CFA-inflamed mice (circle, single intrathecal treatment of MS15203 24 h after CFA inflammation; square, double intrathecal treatments of MS15203 24 and 48 h after CFA inflammation; diamond, single intrathecal treatment of bigLEN 24 h after CFA inflammation; inverted triangle, double intrathecal treatments of bigLEN 24 and 48 h after CFA inflammation). (F) Percent M.P.E.s were produced from treatments with different doses of GPR171 activators (details are listed in Supplementary Figure S5). Symbols indicate different drug treatments (circle, single intrathecal treatment of MS15203 24 h after CFA inflammation; square, double intrathecal treatments of MS15203 24 and 48 h after CFA inflammation; diamond, 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 5

GPR171 activation alleviates neuropathic pain. (A) Time course of von Frey paw-withdrawal thresholds in mice with chronic constriction injury (CCI). Animals were intrathecally treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) immediately after 14 days of threshold monitoring. The right line plot shows the mean thresholds. (B) Time course of Hargreaves paw-withdrawal latencies in mice with CCI. Animals were treated with drugs in the same manner as in (A). The right line plot shows the mean latencies. (C) Time course of von Frey thresholds in mice with chronic constriction injury (CCI). Animals were intraplantarily treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) in the ipsilateral hind paws immediately after 14 days of threshold monitoring. The right line plot shows the mean thresholds. (D) Time course of Hargreaves latencies in mice with CCI. Animals were treated with drugs in the same manner as in (C). The right line plot shows the mean latencies. Five animals were used for each data point. Statistical significance is represented using different symbols (* for MS15203 treatment and # for bigLEN treatment). (E) 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 intrathecal treatment of MS15203; 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.

Figure 6

GPR171 activation alleviates incision-induced pain. (A) Time course of von Frey thresholds in mice of an incision pain model. Animals were intrathecally treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) immediately after the first post-incision threshold monitoring. Threshold monitoring began immediately after the completion of incision operation. The right line plot shows the mean thresholds. (B) Time course of Hargreaves latencies in mice before and after incision. Animals were treated with drugs in the same manner as in (A). The right line plot shows the mean latencies. (C) Time course of von Frey thresholds in mice before and after incision. Animals were intraplantarily treated with vehicle (red), MS15203 (2.5 μg, green), or bigLEN (2.5 μg, blue) in the ipsilateral hind paws immediately after 24 and 48 h threshold monitoring. The right line plot shows the mean thresholds. (D) Time course of Hargreaves latencies in mice before and after incision. Animals were treated with drugs in the same manner as in (C). The right line plot shows the mean latencies. In (A–D), five animals were used for each data point. Statistical significance is represented using different symbols (* for MS15203 treatment and # for bigLEN treatment). *** p < 0.001, ** p < 0.01; ^### p < 0.001, ^## p < 0.01, ^# p < 0.05.

Figure 7

Animals with GPR171 knockdown are tolerant to its pharmacological modulation. (A) A cartoon describing the intrathecal (i.t.) treatment of siRNA downregulating GPR171 expression for experiments from (B–D). Scrambled RNAs (SC) were injected in the same way. (B) Time course of von Frey thresholds in complete Freund’s adjuvant (CFA)-inflamed mice. Animals were intrathecally treated with drugs listed in the inset immediately after 24 and 48 h threshold monitoring. The left dot plot shows individual threshold values. (C) Time course of Hargreaves latencies in CFA-inflamed mice. Animals were treated with drugs in the same manner as in (B). The left dot plot shows individual latency values. (D) Expression levels of GPR171 observed by quantitative RT-PCR with lumbar DRGs of the animals intrathecally treated with siRNA compared to those with scrambled RNA. (E) A cartoon describing the peri-sciatic nerve treatment of siRNA downregulating GPR171 expression for experiments from (F–H). Scrambled RNAs were injected in the same way. (F) Time course of von Frey thresholds in complete Freund’s adjuvant (CFA)-inflamed mice. Animals were intraplantarily treated with drugs listed 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 in (F). The left dot plot shows individual latency values. (H) Expression levels of GPR171 observed by quantitative RT-PCR 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.

Figure 8

The effect of GPR171 agonist injection on open field behaviors. (A) Time spent in the central field of an open field box by mice was monitored for 30 min. Mean seconds ± S.E.M. are shown for five mice with or without intrathecal drug treatment. (B) Percent time spent in the central field by mice for 30 min as in (A) are shown in the histogram. Mean seconds ± S.E.M. are shown for five mice with or without intrathecal drug treatment. (C) Distance traveled with or without intrathecal drug treatment in five mice was averaged during every 5 min period. (D) Representative tracks for 30 min of a mouse under open field test are shown (left, vehicle treated; middle, MS15203 treated; right, bigLEN treated). The central field is indicated as a green square. (E) Numbers of rearing of mice inside open field box were monitored for 30 min. Mean numbers ± S.E.M. are shown for five mice with or without intrathecal drug treatment. (F) Time spent by mice for rearing inside an open field box was monitored for 30 min. Mean seconds ± S.E.M. are shown for five mice with or without intrathecal drug treatment. (G) Time spent by five mice for rearing inside an open field box with or without intrathecal drug treatment was averaged every 5 min period. No statistical difference was detected at any data point in one-way ANOVA and Tukey’s analysis.