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, gamma2-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 gamma2-MSH. The radiolabeled C-terminal part of gamma2-MSH, gamma2-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.

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.

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.

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).