Sensory neuron-specific receptor activation elicits central and peripheral nociceptive effects in rats

Abstract

The sensory neuron-specific G protein coupled receptors (SNSRs) have been described as a family of receptors whose expression in small diameter sensory neurons in the trigeminal and dorsal root ganglia suggests an implication in nociception. To date, the physiological function(s) of SNSRs remain unknown. Hence, the aim of the present study was to determine the effects of rat SNSR1 activation on nociception in rats. The pharmacological characterization of rat SNSR1 was initially performed in vitro to identify a specific ligand, which could be used subsequently in the rat for physiological testing. Among all ligands tested, γ2-MSH was the most potent at activating rat SNSR1. Structure–activity relationship studies revealed that the active moiety recognized by rat SNSR1 was the C-terminal part of γ2-MSH. The radiolabeled C-terminal part of γ2-MSH, γ2-MSH-6–12, bound with high affinity to membranes derived from rat skin and spinal cord, demonstrating the presence of receptor protein at both the proximal and distal terminals of dorsal root ganglia. To investigate the physiological role of SNSR, specific ligands to rat SNSR1 were tested in behavioral assays of pain sensitivity in rats. Selective rat SNSR1 agonists produced spontaneous pain behavior, enhanced heat and mechanical sensitivity when injected intradermally, and heat hypersensitivity when injected centrally, consistent with the localization of rat SNSR1 protein at central and peripheral sites. Together, these results clearly indicate that the SNSR1 plays a role in nociception and may provide novel therapeutic opportunities for analgesia.

Gprotein-coupled receptors constitute one of the largest gene family of proteins that have been exploited successfully as drug targets (1). With the near completion of the human genome project, bioinformatic analyses have revealed the existence of ≈145 orphan receptors (1, 2). In recent years, the “reverse pharmacology approach” has generated >40 ligand–receptor pairings (1, 3), and the subsequent investigation of these newly discovered ligand–receptor pairings should help in understanding and elucidating their potential physiological and pathophysiological roles.

We cloned a previously undescribed family of G protein-coupled receptors that we named the sensory neuron-specific receptors (SNSRs) because of their unique mRNA distribution in small nociceptive sensory neurons in dorsal root (DRG) and trigeminal ganglia (4). The SNSRs are phylogenetically related to the Mas oncogene receptor and belong to the Mas-related genes or Mrg family described for mouse and human by Dong et al. (5). Based on several analyses, this subfamily of receptors is comprised of four to six members in human (MrgX1–4 or SNSR1–6) and 32 receptors in mouse classified into three major subfamilies Mrg A, B, and C (4–7). Initially, only one SNSR gene was identified in rat (4); recently, Zylka et al. (8) have demonstrated that more than one rat SNSR/Mrg subtype exists. These receptors have been subclassified in a similar scheme as described for human and mouse, rMrg A (one gene), B (10 genes, four of which are predicted to be pseudogenes), C (one gene), and D (one gene). For the sake of simplicity, we refer to rSNSR/rMrgC as rSNSR1 in this study, which corresponds to the first gene described in small DRG neurons (4).

Several groups have reported the identity of many distinct and selective ligands capable of activating some members of this family. Dong et al. (5) demonstrated that Arg–Phe-amide (RF-amide) containing neuropeptides, such as FLRF-amide, were selective in eliciting calcium responses in cells expressing mouse MrgA1 and MrgA4. Adenine was recently identified from natural extracts derived from porcine brain cortex and proposed as a potent endogenous ligand for the rat ortholog of mouse MrgA10 (6) (rMrgA). However, adenine was reported not to activate human SNSRs/MrgX1–4 receptors nor the mouse MrgA1 and C11 receptor subtypes (6, 7). Fragments from the proenkephalin A gene, including BAM 22 and BAM 8–22, were identified as ligands capable of potently activating human SNSR3 and 4 (MrgX1) (4). More recently, Robas et al. (9) described cortistatin14 as a ligand with nanomolar affinity for human MrgX2.

Despite their pharmacological differences, many of these receptor subtypes have been shown to be located in small sensory nociceptive neurons by in situ hybridization studies (4–7). Moreover, several proposed ligands are neuropeptides known to be involved in the transmission of pain (4, 7, 10), suggesting a possible role for these receptors in modulating nociception.

To further examine the physiological role(s) of Mrg/SNSR receptors, we have performed a pharmacological characterization of rSNSR1 (or rMrgC) (8). We have identified selective neuropeptide agonists for rSNSR1 derived from two distinct opioid precursors, proopiomelanocortin (POMC) and proenkephalin A, as well as peptides containing the RF-amide motif. The γ2-MSH peptide and its corresponding C-terminal fragment were found to be the most potent ligands at activating rSNSR1. Because the mRNA distribution for rSNSR1 is restricted to the DRGs, the rat receptor protein could be located in central or peripheral tissues. In the present study, we report the expression of rSNSR1 protein in the rat spinal cord and skin, and we have assessed the physiological role of rSNSR1 by testing the effect of these classes of ligands on pain behavior in rats.

Materials and Methods

Ligand Identification Studies. Fluorometric imaging plate reader (FLIPR) measurement of the mobilization of intracellular calcium in response to ligands was performed as described (4). Peptides were purchased from American Peptide (Sunnyvale, CA), Bachem, or Phoenix Pharmaceuticals (St. Joseph, MO), except for γ2-MSH-6–12 and its derivative (Tyr⁶)-γ2-MSH-6–12, which were synthesized in house. EC₅₀ values were obtained by using the prism 3.02 (GraphPad, San Diego) nonlinear regression method.

Binding experiments were performed by using membranes (P2 pellet, 30 μg of protein per well in 96-well format) prepared from rSNSR1/HEK293s Gα_qi5 or HEK293s Gα_qi5. Briefly, membrane preparations were incubated for 1.5 h at room temperature with 200 μl of binding buffer containing 50 mM Tris·HCl (pH 7.4), 5 mM MgCl₂, 2.5 mM EDTA, 0.1 mg/ml BSA fatty acid free, protease inhibitor mixture (Sigma), and various concentrations of Tyr(¹²⁵I)⁶-γ2-MSH-6–12 (1,800 Ci/mmol radiolabeled in house; 1 Ci = 37 GBq) in the presence or absence of 2 μM unlabeled (Tyr⁶)-γ2-MSH-6–12. In competition binding studies, the concentration of the tracer Tyr(¹²⁵I)⁶-γ2-MSH-6–12 used was 1 nM. After incubation, bound tracer was separated by filtration over GF/B Unitfilter plates (Packard) presoaked for 1 h in polyethylene imine 0.1%, then washed three times with 1 ml of ice-cold wash buffer containing 50 mM Tris·HCl (pH 7.0), 5 mM MgCl₂, and 1% BSA fatty acid free. Dried filters were counted by γ scintillation counting after the addition of 65 μl of MS-20 scintillation fluid per well. K_i, K_d, and B_max values were calculated with prism 3.02 software using the nonlinear regression analysis method.

Characterization of Rat SNSR Binding in Rat Tissues. Seventeen-day-old rat pups were decapitated, and tissues were collected on ice. The spinal cords, brains, and midbrains (including superior and inferior colliculus, tegmentum, substantia nigra, and cerebral peduncle) were separated by dissection and processed through a polytron homogenizer for 2 min at 4°C in a buffer solution containing 50 mM Tris·HCl (pH 7.4), 5 mM MgCl₂, 2.5 mM EDTA, and 0.5 mM PMSF. Tissues were then processed to obtain a P2 pellet. The rat hind paw skin tissues were collected and frozen at -80°C until membrane preparation and binding experiment were performed. A total of 20–40 rat hind paw skins were thawed at room temperature in Falcon plastic tube and treated with collagenase (Sigma), 2 mg/ml at 37°C in 20 ml of DMEM during 1 h. The rat skin preparation was homogenized by using polytron homogenizer for 5 min at 4°C. The homogenates were centrifuged for 20 min at 12,000 rpm in a Sorvall RC26 Plus, and the pellet was resuspended in a buffer solution containing 50 mM Tris·HCl (pH 7.4), 5 mM MgCl₂, 2.5 mM EDTA, and protease inhibitor mixture (Sigma) and homogenized by using a glass/glass homogenizer. Homogenate (50 μg of protein per well) was used to perform binding assays (in 96-well plate format) using the same conditions described for rSNSR1/HEK293s Gα_qi5 membranes.

In Vivo Studies. Central administration. Compounds or vehicle were administrated by intrathecal (i.t.) puncture in a volume of 20–30 μl. The injection was performed at the site between the dorsal aspect of L5 and L6. Thermal sensitivity of the tail was assessed by using a hot water tail immersion test. The rat was held horizontally over a water bath maintained at 49°C. When the animal's tail was motionless and pointing downwards, the tail was introduced vertically into the water to a 5-cm depth. The withdrawal latency of tail flick was recorded. The tail immersion test was performed before and 10, 20, and 40 min after i.t. administration of test compounds or vehicle for the time course and 10 min after i.t. administration for the dose–response experiment.

Peripheral administration. Test compounds or vehicle were administrated by intradermal (i.d.) injection in a volume of 10 μl. The injection was performed in the center of the plantar surface of the hind paw. Thermal sensitivity of the plantar surface of the hind paw was assessed according to the method of Hargreaves et al. (11). A paw thermal stimulator system (University of California at San Diego, La Jolla) was used to measure the paw withdrawal latency. Two trials were conducted with 5 min between each trial. The cutoff time was 20 s. Baseline latencies were obtained for each rat before any drug administration. Mechanical sensitivity was tested according to the up-and-down method (12) by touching the plantar surface of the animal hindpaw with a series of eight von Frey filaments (bending force ranging from 0.4 to 15 g). Licking or withdrawing of the stimulated paw was considered a pain-like behavior. Duration of nocifensive behaviors (paw licking, biting, and lifting) was recorded during 3 min after intradermal administration by using observer software.

Results

The C-Terminal Part of γ2-MSH Specifically Activates rSNSR1. We initiated a ligand identification campaign for rSNSR1 using a comprehensive collection of compounds commercially available and an assay of intracellular calcium mobilization (fluorometric imaging plate reader assay, FLIPR). Among all ligands tested, γ2-MSH was the most potent at activating rSNSR1 with an EC₅₀ value of 37 ± 10 nM as shown in (Table 1). Many peptides derived from preproenkephalin A, POMC, and RF-amide-related peptides dose-dependently stimulated the release of intracellular calcium in cells expressing rSNSR1, but these exhibited a lower affinity compared to γ2-MSH (Table 1). Other peptides derived from POMC, including αMSH, βMSH or ACTH were inactive on rSNSR1 (Table 1). Structure–activity relationship studies revealed that the active moiety was restricted to the C-terminal part of γ2-MSH, which has been shown to be inactive at melanocortin receptors (13). The C-terminal fragment of γ2-MSH, namely γ2-MSH-6–12, demonstrated full agonism and potency comparable to the full-length γ2-MSH, with an EC₅₀ value of 44 ± 25 nM. To radiolabel γ2-MSH-6–12, a tyrosine (Y) residue was added to replace the N-terminal phenylalanine (F) (Table 1). The resulting peptide, (Tyr⁶)-γ2-MSH-6–12, displayed a high affinity and potency for rSNSR1, EC₅₀ = 15 ± 2 nM, when tested in a functional FLIPR calcium assay (Table 1).

Table 1. The EC₅₀ values of various peptides as determined by fluorometric imaging plate reader (FLIPR) assay using HEK293s Gαqi5 cells expressing rat SNSR1.

Peptides	Sequences	EC50, nM
Met enkephalin	YGGFM-amide	Inactive
Met enkephalin RF-amide	YGGFMRF-amide	391 ± 120
BAM 22	YGGFMRRVGRPEWWMDYQKRYG	155 ± 25
BAM 8—22	VGRPEWWMDYQKRYG	120 ± 15
β neo-endorphin	YGGFLRKYP	Inactive
ACTH 1—39	SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF	Inactive
α MSH	SYSMEHFRWGKPV	Inactive
β MSH	AEKKDEGPYRMEHFRWGSPPKD	Inactive
γ1-MSH-amide	YVMGHFRWDRF-amide	63 ± 22
γ2-MSH	YVMGHFRWDRFG	37 ± 10
γ3-MSH	YVMGHFRWDRFGRRNGSSSSGVGGAAQ	1,192 ± 308
γ2-MSH-6—12	FRWDRFG	44 ± 25
(Tyr6)-γ2-MSH-6—12	YRWDRFG	15 ± 2
(Tyr6-Ala10)-γ2-MSH-6—12	YRWDAFG	Inactive
(Tyr6-Ala11)-γ2-MSH-6—12	YRWDRAG	Inactive
NPFF bovine	FLFQPQRF-amide	1,048 ± 367
NPAF bovine	AGEGLSSPFWSLAAPQRF-amide	594 ± 168
NPSF human	SQAFLFQPQRF-amide	2,092 ± 629
SHU 9119		Inactive
Adenine		Inactive

Inactive means that compounds were not active at the highest concentration tested, 10 μM. Data represent the mean of four independent experiments performed in duplicate.

The synthetic peptides (Tyr⁶-Ala¹⁰)-γ2-MSH-6–12 and (Try⁶-Ala¹¹)-γ2-MSH-6–12, were devoid of activity at rSNSR1 (Table 1). The rank order of potency obtained for some of the most potent peptides tested was as follows: (Tyr⁶)-γ2-MSH-6–12, γ2-MSH-6–12 = γ2-MSH > BAM 8–22 > Met-Enkephalin-RF-amide > NPAF. BAM 22 and its C-terminal fragment, BAM 8–22, were full agonists at rSNSR1 (Table 1); however, their potencies were relatively poor when compared to the values obtained for human SNSRs (4) and the value of γ2-MSH for rSNSR1. As with the human SNSRs, the N-terminal opioid Met-enkephalin motif YGGFM is not able to activate rSNSR1. Treatment with the nonselective opioid antagonist naloxone or more selective compounds including naltrindole, morphine, or SNC80 did not abolish the calcium-mediated response induced by BAM fragments nor γ2-MSH (data not shown). Moreover, the calcium response elicited by rSNSR1 agonists was not affected by pretreatment with pertussis toxin, suggesting that rSNSR1 is a Gαq coupled-receptor which is in agreement with Han et al. (7). In a recent study, Bender et al. (6) cloned an orphan rat G protein-coupled receptor sharing 48% identity with rSNSR1 and identified adenine as the endogenous ligand, hence the receptor was named the adenine receptor (6). Even though the adenine receptor or rMrgA, is the closest homolog of rSNSR1/rMrgC (8), none of the ligands active at rSNSR1, were able to activate the adenine receptor, except for adenine itself (data not shown). Similarly, adenine did not bind nor activate rSNSR1, indicating that rSNSR1 and the adenine receptor are not only phylogenetically distinct but also exhibit different ligand specificities (Fig. 1 C and D and Tables 1 and 2). The recently discovered rMrg B and D subtypes (8) were not tested for potential selectivity issues of (Tyr⁶)-γ2-MSH-6–12 because of their lower identity to rSNSR1 compared to that of rMrgA.

Fig. 1.

Tyr(¹²⁵I)⁶-γ2-MSH-6–12 specifically binds to rat SNSR1. (A) Saturation binding isotherm of Tyr(¹²⁵I)⁶-γ2-MSH-6–12. Filled triangles indicate specific binding using HEK293s-rSNSR1 membranes. (B) Saturation binding isotherm of Tyr(¹²⁵I)⁶-γ2-MSH-6–12; specific binding obtained using rat spinal cord (blue circles), skin (filled squares), midbrain (red triangles), and brain (green diamonds) membranes. Competition of Tyr(¹²⁵I)⁶-γ2-MSH-6–12's binding to HEK293s-rSNSR1 (C) or rat spinal cord membranes (D) using BAM 8–22 (green triangles), (Tyr⁶)-γ2-MSH-6–12 (blue squares), γ2-MSH-6–12 (blue inverted triangles), γ2-MSH (red diamonds), neuropeptide FF (NPFF) (open circles), adenine (open squares), (Tyr⁶–Ala¹¹)-γ2-MSH-6–12 (open triangles), or αMSH (filled circles). Data represent a single experiment representative of four independent experiments performed in quadruplicate.

Table 2. The K_i values of various peptides as determined by binding assays using HEK293s-rSNSR1, rat spinal cord, and skin membrane preparations.

		Ki, nM
Peptides	Sequences	HEK 293s-rSNSR1	Spinal cord	Skin
Met-Enkephalin	YGGFM	>10,000	>1,000
Met-Enkephalin-RF-amide	YGGFMRF-amide	60 ± 10	654 ± 44
BAM 22	YGGFMRRVGRPEWWMDYQKRYG	153 ± 33	59 ± 17
BAM 8—22	VGRPEWWMDYQKRYG	94 ± 11	250 ± 29	242 ± 40
ACTH 1—39	SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF	Inactive	Inactive
β neo-endorphin	YGGFLRKYP	>10,000	>1,000
α MSH	SYSMEHFRWGKPV	Inactive	Inactive	Inactive
β MSH	AEKKDEGPYRMEHFRWGSPPKD	Inactive	Inactive
γ1 MSH-amide	YVMGHFRWDRF-amide	36 ± 5	140 ± 14
γ2 MSH	YVMGHFRWDRFG	25 ± 3	25 ± 3	21 ± 7
γ3 MSH	YVMGHFRWDRFGRRNGSSSSGVGGAAQ	50 ± 6	>1,000
γ2 MSH-6—12	FRWDRFG	6 ± 1	36 ± 7	15 ± 4
(Tyr6)-γ2-MSH-6—12	YRWDRFG	1.64 ± 0.3	8 ± 0.9	29 ± 7
(Tyr6-Ala10)-γ2-MSH-6—12	YRWDAFG	980 ± 59	Inactive	>1,000
(Tyr6-Ala11)-γ2-MSH-6—12	YRWDRAG	785 ± 74	Inactive	Inactive
NPFF bovine	FLFQPQRF-amide	>1,000 nM	>10,000
NPAF bovine	AGEGLSSPFWSLAAPQRF-amide	377 ± 68	830 ± 95
NPSF human	SQAFLFQPQRF-amide	643 ± 50	>1,000
SHU9119		Inactive	Inactive
Adenine		Inactive	Inactive	Inactive

K_i values were determined by using Tyr(¹²⁵I)⁶-γ2-MSH-6—12 binding displacement by nonlabeled ligands using HEK-SNSR1, rat spinal cord, and skin membrane preparations. Inactive means that compounds were not active at the highest concentration tested, 10 μM. Data represent the mean of three independent experiments performed in quadruplicate. NP, neuropeptide.

rSNSR1 Binding Profiles Are Similar in HEK293s Cells, Spinal Cord, and Skin Membrane Preparations. To validate the selectivity of (Tyr⁶)-γ2-MSH-6–12, competition binding experiments were performed on a panel of >30 G protein-coupled receptors and channels including the melanocortin MC1, MC4, and neuropeptide FF (NPFF) receptors (in addition to histamine opioids, muscarinic, dopamine, adrenergic, γ-aminobutyric acid receptors). No specific displacement was observed at 10 μM (data not shown), thus demonstrating the selectivity of this ligand for rSNSR1. We evaluated the binding properties of rSNSR1 on membrane preparations derived from HEK 293s Gαqi5 cells stably expressing rSNSR1 (HEK-rSNSR1) and rat membranes derived from the spinal cord, midbrain, brain, and hind paw skin tissues. Using Tyr(¹²⁵I)⁶-γ2-MSH-6–12 as a radioligand and HEK-rSNSR1 membranes, a single population of binding sites was identified (K_d = 1.7 ± 0.4 nM; B_max = 350 ± 100 fmol/mg protein; Fig. 1A). No specific binding was observed when membrane derived from nontransfected cells was used (data not shown). The same approach showed that, of the CNS tissues tested, the spinal cord was the only one displaying specific binding that was concentration dependent and saturable with the following parameters: K_d = 6.5 ± 0.7 nM and B_max of 105 ± 14 fmol/mg protein (Fig. 1B). Similar to the observation in HEK-rSNSR1, a single population of binding sites was detected by using spinal cord membranes. Various neuropeptides were used to displace Tyr(¹²⁵I)⁶-γ2-MSH-6–12 binding using HEK-rSNSR1 (Fig. 1C) and rat spinal cord (Fig. 1D) membranes. For all of the peptides tested, a similar rank order of potency was obtained in spinal cord and HEK293s-rSNSR1 (Fig. 1 and Table 2), confirming that a single population of binding sites exists in the spinal cord that is specific for rSNSR1. The rank order of potency observed for all these peptides was similar to the results obtained with the intracellular calcium mobilization assay (Table 1 and 2). The melanocortin receptor ligands, αMSH, ACTH and SHU9119, were inactive at competitively displacing Tyr(¹²⁵I)⁶-γ2-MSH-6–12 binding in the spinal cord membranes, suggesting that the receptor labeled by Tyr(¹²⁵I)⁶-γ2-MSH-6–12 is not a melanocortin receptor subtype but indeed rSNSR1 (Table 2). Because rSNSR1 mRNA was detected in small diameter DRG neurons, we surmised that peripheral primary afferent terminals may express rSNSR1 protein. In this regard, rat paw skin membranes were prepared from naïve animals. Rat skin membranes derived from the paw displayed specific Tyr(¹²⁵I)⁶-γ2-MSH-6–12 binding but to a lower extent as compared to rat spinal cord, K_d = 2.7 ± 0.9 nM and B_max = 66 ± 20 fmol/mg protein (Fig. 1B). Only a limited pharmacological characterization was performed by using rat skin membranes (Table 2) because of the difficulty in obtaining a significant amount of quality material. The pharmacological profile obtained by using Tyr(¹²⁵I)⁶-γ2-MSH-6–12 (Fig. 1 and Table 2) clearly indicates that rSNSR1 is expressed in peripheral primary afferent nociceptive neurons.

SNSR Agonists Evoke Pronociceptive Effects in Vivo After Spinal Administration. Because two distinct ligand families activate rSNSR1, the metabolic stability of the two peptides [(Tyr⁶)-γ2-MSH-6–12 and BAM 8–22] was examined before conducting in vivo studies. In control incubations carried out without any tissue homogenates, (Tyr⁶)-γ2-MSH-6–12 and BAM 8–22 did not show any significant protein-independent degradation and were chemically stable in pH 7.4 phosphate buffer (see supporting information, which is published on the PNAS web site). Both peptides showed significant degradation in the rat brain, spinal cord homogenates, and rat cerebrospinal fluid (Fig. 2A and supporting information). (Tyr⁶)-γ2-MSH-6–12 was undetectable in brain or spinal cord homogenates after only 10 min of incubation. BAM 8–22 was more stable, as 40% remained in spinal cord homogenate after 30 min of incubation and >45% remained in rat cerebrospinal fluid after 60 min of incubation (Fig. 2A and supporting information).

Fig. 2.

Stability and pronociceptive effects produced by rSNSR1 agonists after i.t. administration. (A) Stability of rSNSR1 agonists in rat spinal cord homogenates. (B) Time course of tail withdrawal latency (TWL) changes after rSNSR1 agonists i.t. administration. (C) Decrease of TWL induced by rSNSR1 agonists and N-methyl-d-aspartate (NMDA) by i.t. administration. i.t. administration of (Tyr⁶–Ala¹⁰)-γ2-MSH-6–12 [at the highest dose used for both (Tyr⁶)-γ2-MSH-6–12 and BAM 8–22] did not change TWL. All data are given as mean ± SEM with n ≥ 7. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. vehicle group.

Application of the rSNSR1-specific agonists, (Tyr⁶)-γ2-MSH-6–12 and BAM 8–22, to the surface of the spinal cord by i.t. injection evoked a dose-dependent and statistically significant thermal hyperalgesia (decrease in the tail withdrawal latency, TWL) 10 min after administration (Fig. 2 B and C). The pronocieptive effect produced by SNSR ligands was equivalent to that evoked by application of the excitatory amino acid, N-methyl-d-aspartate, an established pronociceptive stimulus (Fig. 2C). The thermal hyperalgesia produced by BAM 8–22 was longer lasting than that of (Tyr⁶)-γ2-MSH-6–12 (Fig. 2B). The control peptide, (Tyr⁶–Ala¹⁰)-γ2-MSH-6–12, had no effect on TWL after i.t. injection (Fig. 2 B and C). Considering the relative affinities observed for these two peptides at rSNSR1, the higher metabolic stability of BAM 8–22 is likely responsible for its greater efficacy and long duration of action compared to (Tyr⁶)-γ2-MSH-6–12 in vivo.

rSNSR1 Agonists Evoke Pain Behaviors, Heat Hyperalgesia, and Mechanical Allodynia in Vivo After Peripheral Administration. The heat hyperalgesia we saw after spinal administration of rSNSR1 agonists supported an excitatory role for SNSR in the pain pathway. We investigated this further by examining the spontaneous behavioral response of rats after intradermal injection of rSNSR1 agonists. Agents that excite the peripheral terminals primary “damagesensing” neurons (nociceptors) like capsaicin, formalin, and ATP produce licking, biting, and lifting of the treated paw after local administration (10, 14, 15). We found that intradermal injection of (Tyr⁶)-γ2-MSH-6–12 evoked dose-dependent spontaneous nocifensive (pain-related) behaviors very similar to those observed after capsaicin administration (Fig. 3A), further supporting a role for rSNSR1 in nociception. Activation of peripheral nociceptors by painful stimuli like capsaicin also produces a long-lasting hypersensitivity to thermal and mechanical stimuli (16). We next tested whether the rSNSR1 agonists also produced the behavioral manifestations of sensitization after intradermal injection in rats. Intradermal injection of (Tyr⁶)-γ2-MSH-6–12 and BAM 8–22 evoked a dose-dependent and statistically significant decrease in the paw withdrawal latency for the injected paw 20 min after inoculation (Fig. 3B). Moreover, the heat hyperalgesia was similar to that produced by the same concentration of capsaicin (Fig. 3B). The heat hyperalgesia produced by rSNSR1 agonists was likely caused by a specific action on rSNSR1 because the groups treated with the control peptides, (Tyr⁶–Ala¹⁰)-γ2-MSH-6–12 and (Tyr⁶–Ala¹¹)-γ2-MSH-6–12, did not show any evidence of heat hyperalgesia (Fig. 3 B and C). Intradermal injection of SNSR agonists also produced a long-lasting hypersensitivity to light touch (allodynia), again equivalent to that evoked by intradermal capsaicin (Fig. 3D).

Fig. 3.

Intradermal injection of rSNSR1 ligands evokes spontaneous pain behaviors. (Tyr⁶)-γ2-MSH-6–12, as capsaicin, produces spontaneous pain behaviors (A). At 90 nmol, (Tyr⁶)-γ2-MSH-6–12 alters similarly to capsaicin both thermal and mechanical sensitivity (B–D). BAM8–22 evokes a dose-dependent heat hyperalgesia (B). Control peptides [(Tyr⁶–Ala¹⁰)-γ2-MSH-6–12 and (Tyr⁶–Ala¹¹)-γ2-MSH-6–12] have no effect on paw thermal sensitivity (B and C). All data are given as mean ± SEM with n ≥ 7. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (for vehicle versus treated groups with the one-way ANOVA or repeated-measures two-way ANOVA).

At the highest concentration tested, the rSNSR1 agonists produced a moderate but significant increase in paw volume (≈20–25% of increase) (supporting information). One of the control peptides, (Tyr⁶–Ala¹¹)-γ2-MSH-6–12, produced similar edema without evidence of heat hyperalgesia (supporting information), suggesting that the heat hyperalgesia evoked by rSNSR1 agonist peptides was not secondary to the inflammatory effects. The presence of an Arg at position 10 in one of the control peptides [(Tyr⁶–Ala¹¹)-γ2-MSH-6–12] might explain why it produces edema without hyperalgesia, because it was clearly shown that the potency for histamine release from rat mast cells increases in parallel with the number of positively charged amino acids (Lys, Arg). This effect is not receptor specific, but rather ligand specific (14, 15). Therefore, intradermal injection of rSNSR1 agonists produced a behavioral profile of spontaneous nocifensive behaviors, heat hyperalgesia, and mechanical allodynia very similar to well established algogens.

Discussion

The present study was designed to investigate the physiological role(s) of rSNSR1, particularly its potential involvement in nociception. When injected locally, centrally, or peripherally, selective rSNSR1 agonists evoked a behavioral profile similar to that of established pain-provoking agents like capsaicin, including spontaneous nocifensive behaviors, thermal hyperalgesia, and mechanical allodynia. These data suggest that rSNSR1 has a role in activating the pain pathway.

The POMC-Derived Peptide γ2-MSH Is a Potent Ligand for rSNSR1. We have identified distinct families of neuropeptides capable of activating rSNSR1 including peptides derived from POMC and proenkephalin A. Fragments derived from proenkephalin A, including bovine adrenal medulla peptides BAM 22 and BAM 8–22, activate rSNSR1 with affinities of 100–150 nM. We previously identified these peptides as potent ligands for human SNSR3 and 4 (hMrgX1) (4), revealing BAM 22 as an opioid ligand capable of targeting both opioid and SNSR receptors. There are some common features shared between human and rSNSRs. First, both receptors are activated by opioid peptides (e.g., BAM 22) via a nonopioid mechanism, as demonstrated by their insensitivity to classical opioid agonists and antagonists. Second, both share a similar structure–activity relationship of BAM 22.

In addition, we have demonstrated that the most potent agonist capable of activating and binding to rSNSR1 is γ2-MSH, and structure–activity relationship studies revealed that the active moiety is restricted to the C terminus, which ends in the amino acids “RFG.” Replacing the arginine residue with an alanine in the RF motif contained within γ2-MSH-6–12 resulted in a significant decrease in rSNSR1 activation and binding. Recently, Han et al. (7) reported γ2-MSH as a potent ligand for mouse MrgC11, reinforcing the notion that the rat SNSR1 is indeed the ortholog of mMrgC11. The expression of POMC, the precursor of γ2-MSH, has been detected in rat spinal cord, dorsal root ganglia, and skin (17–19), three different tissues that are the major site of action for rSNSR1 as assessed by our in vivo studies. In the last decade, γ2-MSH has been proposed to be the endogenous ligand for MC3 melanocortin receptor based on the pharmacological profile obtained in vitro (17, 20). Immunohistochemistry analysis has demonstrated that the γ2-MSH peptide is distributed in various structures of the central nervous system where MC3 is absent, or at least poorly expressed, suggesting that there may be other unidentified orphan receptors activated by this neuropeptide (17). Interestingly, the expression of MC3 receptor has never been demonstrated in rat spinal cord and skin tissues (17, 21). Moreover, the colocalization of rSNSR1 and γ2-MSH precursor proteins indicates that γ2-MSH and its C-terminal fragment(s) could be potential endogenous ligands for rSNSR1. Whether additional proteolytic cleavage of γ2-MSH peptide gives rise to smaller peptides such as the rSNSR1-selective ligand, γ2-MSH-6–12 still remains to be determined. Until then, the purpose of γ2-MSH6–12 will be as a selective tool to study the physiological role of rSNSR1.

rSNSR1 Protein Is Expressed at Central and Peripheral Terminals of the DRGs. The structure activity relationship of rSNSR1 ligands led us to establish a specific radioligand binding assay using Tyr(¹²⁵I)⁶-γ2-MSH-6–12. To determine the distribution of rSNSR1 protein, we performed radioligand binding studies with Tyr(¹²⁵I)⁶-γ2-MSH-6–12 on rat central and peripheral tissues. Tyr(¹²⁵I)⁶-γ2-MSH-6–12-specific binding sites were found at both levels in the spinal cord and in paw skin. In addition, a similar pharmacological profile for rSNSR1 was observed in cells recombinantly expressing this receptor as well as in rat spinal cord and skin membranes. These results demonstrate that rSNSR1 protein is synthesized in the DRGs with subsequent transport to peripheral nociceptive terminals and to central superficial layer of the spinal cord (lamina I and II most probably). These structures are integral component of the pain pathway. It is unlikely that Tyr(¹²⁵I)⁶-γ2-MSH-6–12 interacts with another receptor in the spinal cord or skin, because this peptide was inactive in a broad receptor screen that included melanocortin MC1–MC4 receptors, neuropeptide FF (NPFF) receptor, and opioid receptors. Because several rMrg mRNAs, namely rMrgA, rMrgB4, rMrgB5, rMrgC, and rMrgD were shown to be expressed in a subset of DRG neurons, the possibility exists that Tyr(¹²⁵I)⁶-γ2-MSH-6–12 may interact with some of these entities. We have tested several rSNSR1-selective ligands including γ2-MSH-6–12 and BAM 8–22 at rMrgA (rat adenine receptor; ref. 6), which shares the highest identity at the amino acid level to rSNSR1 (48%), and all of these peptides were completely inactive at concentrations up to 10 μM. Furthermore, the radioligand binding saturation and competition data from the spinal cord or skin preparation do not support the existence of multiple binding sites. Finally, the amino acid divergence analyses across the Mrg family performed by Choi et al. (22) clearly indicates that the probability for the Mrg subtypes sharing the same ligand is very low. All of the data presented support the notion that γ2-MSH-6–12 is a selective tool to study the function of rSNSR1.

rSNSR1 Activation Produces Spontaneous Pain Behavior. To study the role of rSNSR1 in modulating pain sensation, representative peptides from POMC [(Tyr⁶)-γ2-MSH-6–12] and proenkephalin A (BAM 8–22) families were injected centrally and peripherally. The peptides tested evoked pain-related spontaneous behaviors, an increase in sensitivity to noxious heat stimuli (heat hyperalgesia) and hypersensitivity to light touch (mechanical allodynia) in awake rats. Moreover, the control peptides with only a single amino acid difference were completely ineffective. There were, however, some differences in the in vivo effects of these peptides. After intradermal administration, the two peptides showed very similar effects, despite a 100-fold difference in affinity between (Tyr⁶)-γ2-MSH-6–12 and BAM 8–22 (see Tables 1 and 2). After spinal (i.t.) administration, the mismatch was even more pronounced. Here, BAM 8–22 was the most effective compound with the longest duration of action. This apparent discrepancy between the in vivo and in vitro potency of the peptides can be at least partially explained by the poor stability in cerebrospinal fluid and spinal tissue of (Tyr⁶)-γ2-MSH-6–12, which was undetectable after only 10 min of incubation. In contrast, BAM 8–22 was more stable, and therefore would be expected to have a much greater biological effect in vivo despite a relatively poor EC₅₀ value in our in vitro functional assays. Physical properties of the peptides such as the penetration into the tissue may also contribute to these differences. Nonetheless, one can never exclude the possibility of some of these peptides interacting at other targets, though we have performed a broad receptor screen to rule this out. Cao et al. (23), using an electrophysiological approach, reached a similar conclusion regarding rSNSR1's role in nociception. In their study, intrathecal administration of BAM 8–22 was shown to potentiate the rat nociceptive flexor–reflex responses to touch, pinch, and heat in a dose-dependent manner (23). Collectively, these results indicate that SNSR ligands that specifically bind to rSNSR1 are pronociceptive, suggesting that rSNSR1 plays a role in pain transmission or modulation.

Undoubtedly, more work is needed to understand how this receptor enhances nociceptive behaviors in rat. Furthermore, the evolutionary divergence of this family of receptors is truly intriguing and presently the phylogenicity is poorly understood; in this regard, Zylka et al. (8) have made a significant effort to further the knowledge.

Lastly, our results support the notion that the ligand divergence for this family of receptors is extensive beacuse we have identified another distinct peptide, γ2-MSH-6–12, as a selective agonist for rSNSR1. Our findings demonstrate that rSNSR1 protein is expressed at both the peripheral and central terminals of dorsal root ganglion neurons. Furthermore, we have shown that SNSR activation elicits pronociceptive actions in the central and peripheral nervous systems, consistent with the distribution of the rSNSR1 receptor. In conclusion, our data emphasize the importance of the SNSR/Mrg family as a promising pharmacological target for the management of pain.