Contribution of G Protein-Coupled Receptor 55 to Periaqueductal Gray-Mediated Antinociception in the Inflammatory Pain

Abstract

The brain mechanism of inflammatory pain is an understudied area of research, particularly concerning the descending pain modulatory system. The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol-sensitive receptor that has also been involved in cannabinoid signaling. It is widely expressed throughout the central nervous system, including the periaqueductal gray (PAG), a brainstem area and key element of the descending pain modulatory system. In this study, we used behavioral, stereotaxic injections, pharmacological tools, and two inflammatory pain models (formalin and carrageenan) to determine if GPR55 in the PAG plays a role in the pain associated with inflammation in rats. It was found that the blockade of GPR55 action in PAG can drive the descending pain modulatory system to mitigate inflammatory pain. These data show that GPR55 plays a role in the descending pain modulatory system in inflammatory pain.

Introduction

The periaqueductal gray (PAG) is a brainstem area and a key element of the descending pain modulatory system that regulates nociceptive neurotransmission at the level of the spinal cord dorsal horn through the rostral ventromedial medulla.^1,2 This system can exert both faciliatory and inhibitory effects on pain. For example, intense stress and fear are associated with descending inhibition and hypoalgesia, whereas inflammation and nerve injury have been linked to descending facilitation, hyperalgesia, and pain.^3,4

Current analgesics, including opioids, are far from satisfactory in providing pain relief. Safe and effective nonaddictive therapeutics remain a critical need. The G protein-coupled receptor 55 (GPR55) is widely expressed throughout the central nervous system,⁵ including the PAG.⁶ GPR55 has been proposed to be a potential candidate as an endocannabinoid receptor.⁷ Indeed, several cannabinoid receptor ligands are found to interact with GPR55, including the phytocannabinoid Δ⁹-tetrahydrocannabinol,⁸ and the endocannabinoid 2-AG.⁷ Although GPR55 activity can be modulated by certain phyto- and endocannabinoids, studies have suggested that L-α-lysophosphatidylinositol (LPI), which activates the GPR55, but not the cannabinoid 1 receptor (CB1) or cannabinoid 2 receptor (CB2), could be its endogenous ligand.⁹

GPR55 may play a role in pain modulation. GPR55 knockout mice did not develop mechanical hyperalgesia post-intraplantar injection of Freund's complete adjuvant or after partial nerve ligation.¹⁰ Gene deletion of GPR55 showed an antinociceptive effect in the hot plate test.¹⁰ However, recently, inflammatory and neuropathic nociception have been shown to be preserved in GPR55 knockout mice.¹¹ Gene deletion has been primarily used to determine the role of GPR55 in pain. During a collaborative project between our laboratory and the Sanford-Burnham screening center of the Molecular Libraries Probe Production Centers Network, we identified a series of GPR55 agonists and antagonists that belong to novel, unreported GPR55 chemotypes with EC₅₀s in the 0.26 to 2.72 μM range.¹² ML193 found to be potent (221 nM potency for GPR55) and selective for GPR55.¹²

In this study, we made use of this antagonist (for pharmacological manipulation of the GPR55), behavioral assays, stereotaxic injections, and two inflammatory pain models to determine if GPR55 in the PAG plays a role in the pain associated with inflammation in rats.

Materials and Methods

Animals

Sprague-Dawley male rats (Envigo), weighing 250–300 g, were housed in groups of three for at least 1 week in an animal room maintained at 22±1°C and ∼50±5% relative humidity. All experimental procedures were approved by the Institutional Animal Care and Use Committees at Texas Tech University Health Sciences Center.

Cannula implantation

Rats were anesthetized with 4% isoflurane for induction and 2% for maintenance. A sterilized stainless steel C313G guide cannula (26-gauge; Plastics One, Inc., Roanoke, VA) was implanted bilaterally into the vlPAG.¹³ For cannula implantation we used a digital stereotaxic alignment system (David Kopf Instruments, Model 942). The stereotaxic coordinates were as follows: 7.8 mm posterior to bregma, 0.5 mm from midline, and 4.5 mm ventral to the dura mater.¹⁴ A C313DC cannula dummy (Plastics One, Inc.) of identical length was inserted into the guide tube to prevent its occlusion. After a 7-day recovery period, rats were allowed to habituate to the test chambers for 1 hour before testing. The vehicle and drugs were microinjected bilaterally into the vlPAG through an internal cannula (26-gauge; Plastics One, Inc.) in awake animals. At the end of the behavioral experiments, standard histological procedures were used to verify the site of injection.^13,15,16

Formalin test

Nociceptive behavior was induced by subcutaneous injection of 50 μL of 5% formalin into the hind paw using a 1-mL syringe and a 28-gauge needle. Nociceptive behavior of rats was recorded at 12 intervals of 5 min each for a total of 60 min. During each 5-min time bin, the duration spent performing pain–response behaviors was recorded. The nociceptive behaviors were separated into three categories: (0) the injected paw has little weight placed on it; (1) the injected paw is raised off of the ground; (2) the injected paw is licked, shaken, or bitten. The amount of time spent in each category was quantified and weighted with the composite pain score-weighted scores technique (CPS-WST_0,1,2), resulting in a CPS for each 5-min interval between 0 (no pain behaviors) to 2 (maximal pain behavior).¹⁷

Carrageenan test

Rats received an intraplantar injection of 100 μL of carrageenan (Sigma-Aldrich) into the hind paw. Mechanical allodynia¹⁸ was assessed using an electronic von Frey device (IITC Life Sciences, Woodland Hills, CA). Rats were placed in individual plastic cages on an elevated wire mesh platform and allowed to habituate to the testing apparatus until exploratory behavior was no longer observed. A rigid filament was applied with increasing force (g) until a paw withdrawal response was elicited. The force at which this response occurred was recorded automatically by the apparatus and is designated as the paw withdrawal threshold. Three thresholds were taken at each time point.

Heat hypersensitivity was assessed by measuring the limb withdrawal time after application of an infrared heat stimulus¹⁹ (Plantar test, Ugo Basile, Varese, Italy). The animals were placed in a clear plexiglass box with a dry glass floor and allowed to acclimatize. A focused beam of radiant light (active intensity of 40%) was used to heat the plantar surface of the hind paw, and the latency to flinch, lick, or withdraw the hind paw was recorded or until a cutoff time of 20 sec was reached (to prevent tissue damage). The paw withdrawal latency (sec) to this stimulus was recorded.

Drugs

ML-193 was purchased from Tocris Biosciences. Formalin, carrageenan, SR144528, and SR141716A were obtained from Sigma-Aldrich (St. Louis, MO). The vehicle for the cannabinoid antagonists was composed of cremaphor+DMSO+saline, in a 1:1:18 ratio.

Statistical and histological analysis

One-way analysis of variance (ANOVA) was employed. Significant differences were probed using Bonferroni or Fisher post hoc pair-wise comparisons. For comparisons between two group means in which the response was affected by a single variable, an unpaired t-test was performed. The p<0.05 was taken as the significant level of difference. At the end of the behavioral experiments, standard histological procedures were used to verify the site of injection.^13,15,16

Results

The design of the experiment is shown schematically in Figure 1A. Compared with the vehicle, bilateral vlPAG administration of ML-193 (1–50 ng/0.5 μL) 5 min before formalin injection significantly suppressed the second phase (20–40 min post-formalin) of formalin-induced nociception; this effect was dose dependent. The maximal effect was observed with 50 ng (Fig. 1C, one-way ANOVA, p<0.01, n=6). No effect was observed on the first phase (0–15 min post-formalin) (Fig. 1B, one-way ANOVA, p>0.05, n=6). Cannula placement was confirmed using histological verification (Fig. 1D). Intraplantar injection of ML-193 (0.1–1 μg) did not affect formalin-induced pain-like behaviors (Fig. 1E, F, t-test [t=0.05, df=10], p>0.05, n=6), which supports a central role of GPR55 in pain control.

FIG. 1.

The design of the experiment (A). PAG pretreatment with ML-193 reduced the second phase (C) of formalin-induced pain-like behaviors, but not the first phase (B). Illustration of cannula placement, site of action, and schematic representation of targeted (Bregma −7.32 mm) (D). Intraplantar injection of ML-193 (0.1–1 μg) did not affect formalin induced pain-like behaviors (E, F). SR141716A and SR 144528 failed to affect the analgesic effect of ML-193 (G, H). Data are presented as mean±SEM. **p<0.01, *p<0.05 (ML-193 vs. vehicle). PAG, periaqueductal gray; ns, not significant.

To determine if the endocannabinoid system contributes to the analgesic effect of ML-193, the animals were pretreated with SR141716A (CB1 cannabinoid receptor antagonist), SR144528 (CB2 cannabinoid receptor antagonist), or the vehicle (Fig. 1G, H). The CB1 and CB2 cannabinoid receptor antagonists (1 μg/0.5 μL) did not affect ML-193's analgesic effect (50 ng/0.5 μL), suggesting that ML-193's analgesic effect is independent of CB1 and CB2 receptors (Fig. 1G, H, one-way ANOVA p>0.05, n=6). The doses used for CB1 and CB2 cannabinoid receptor antagonists were based on their behavioral effects in pain models when administered in the PAG.²⁰ The antagonists were administered 15 min before ML-193.

The analgesic effect of ML-193 was reproduced in another inflammatory pain model, carrageenan. The design of the experiment is shown schematically in Figure 2A. ML-193 (50 ng/0.5 μL) or vehicle was injected 2 hours post-carrageenan injection (at this point the pain response is maximum and stable). ML-193 suppressed mechanical allodynia (Fig. 2B, t-test [t=0.13, df=34], p<0.001, n=6), and thermal hypersensitivity produced by carrageenan (Fig. 2C, t-test [t=10.37, df=34], p<0.001, n=6) in the ipsilateral paw. No effect was observed in the contralateral paw in von Frey (Fig. 2B, t-test [t=7.5, df=34], p>0.05, n=6) and Hargreaves tests (Fig. 2C, t-test [t=7.5, df=34], p>0.05, n=6).

FIG. 2.

The design of the experiment (A). PAG pretreatment with ML-193 reduced the mechanical allodynia (B) and thermal hypersensitivity (C) induced by carrageenan. SR141716A and SR 144528 failed to affect the analgesic effect of ML-193 in carrageenan-induced mechanical allodynia (D) and thermal hypersensitivity (E). Data are presented as mean±SEM. ***p<0.001. ns, not significant.

We also determined the effect of SR141716A and SR144528 on the effect of ML-193 in the carrageenan test (Fig. 2). CB1 and CB2 cannabinoid antagonists (1 μg/0.5 μL) failed to affect ML-193's (50 ng/0.5 μL) analgesic effect (Fig. 2D, E, one-way ANOVA, p>0.05, n=6) in the ipsilateral paw in both tests. CB1 is expressed in PAG.²¹ Therefore, the lack effect of the CB1 antagonist is not due to the lack of CB1 expression in the PAG, rather ML-193 does not act through CB1.

Discussion

The present data show that GPR55 plays a role in the descending pain modulatory system in inflammatory pain.

Our data show that the blockade of GPR55 action in PAG can drive a descending pain modulatory system to mitigate inflammatory pain. This suggests failure to block the action of GPR55 in the PAG as an inflammatory pain brain mechanism. In support of the role of GPR55 in the descending pain modulatory system, this receptor in the PAG has been shown to be involved in neuropathic pain,²² and the peripheral blockage of GPR55 did not affect inflammatory pain, which supports a central role of GPR55 in pain control.

Previously, it has been shown that the levels of the endogenous GPR55 ligand, LPI, are increased in the caudal medulla oblongata of carrageenan-injected rats.²³ Furthermore, administration of LPI in the PAG showed a nociceptive effect.⁶ Therefore, one potential mechanism by which GPR55 in the PAG is involved in pain control is that during inflammatory pain there is an increased endogenous level of LPI in the PAG, leading to increased activation of GPR55, promoting descending pain facilitation, and the blockade of the action of LPI on this receptor results in pain inhibition.

Our data show that the pharmacological blockade of GPR55 by ML-193 produces an analgesic effect independently of the endocannabinoid system. This shows that the analgesic effect observed in inflammatory pain is due to ML-193–GPR55 interaction, and excludes any potential functional link between ML-193 and CB1/CB2. Given that our data show that the pharmacological blockade of GPR55 by ML-193 produces an analgesic effect independently of CB1, suggests that GPR55 is a potential target for analgesic therapeutic strategies that are devoted to CB1 side effects and abuse potential.

In summary, the present data show that GPR55 plays a key role in the descending pain modulatory system.

Abbreviations Used

ANOVA

analysis of variance

CB1

cannabinoid 1 receptor

CB2

cannabinoid 2 receptor

CPS

composite pain score

GPR55

G protein-coupled receptor 55

LPI

L-α-lysophosphatidylinositol

ns

not significant

PAG

periaqueductal gray

Authors' Contributions

K.B. and M.A. conceived and designed research. H.B., S.M., and S.A. conducted experiments. K.B. wrote the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by grant DA035926 (to K.B. and M.A.) from the National Institutes of Health.

Cite this article as: Blanton H, Armin S, Muenster S, Abood M, Benamar K (2022) Contribution of G protein-coupled receptor 55 to periaqueductal gray-mediated antinociception in the inflammatory pain, Cannabis and Cannabinoid Research 7:3, 274–278, DOI: 10.1089/can.2022.0006.