Background: The G-protein-coupled receptor 40 (GPR40) is suggested to function as a transmembrane receptor for medium- to long-chain free fatty acids and is implicated to play a role in free fatty acids-mediated enhancement of glucose-stimulated insulin secretion from pancreas. However, the functional role of GPR40 in nervous system including somatosensory pain signaling has not been fully examined yet. Results: Intrathecal injection of GPR40 agonist (MEDICA16 or GW9508) dose-dependently reduced ipsilateral mechanical allodynia in CFA and SNL models and thermal hyperalgesia in carrageenan model. These anti-allodynic and anti-hyperalgesic effects were almost completely reversed by a GPR40 antagonist, GW1100. Immunohistochemical analysis revealed that GPR40 is expressed in spinal dorsal horn and dorsal root ganglion neurons, and immunoblot analysis showed that carrageenan or CFA inflammation or spinal nerve injury resulted in increased expression of GPR40 in these areas. Patch-clamp recordings from spinal cord slices exhibited that bath-application of either MEDICA16 or GW9508 significantly decreased the frequency of spontaneous excitatory postsynaptic currents in the substantia gelatinosa neurons of the three pain models. Conclusions: Our results indicate that GPR40 signaling pathway plays an important suppressive role in spinal nociceptive processing after inflammation or nerve injury, and that GPR40 agonists might serve as a new class of analgesics for treating inflammatory and neuropathic pain.

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP–PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation.Results: We found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level.Conclusion: Our data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia.

1 . We investigated the role of nitric oxide (NO) in modulating spinal synaptic responses evoked by electrical and noxious sensory stimuli in the neonatal rat spinal cord in vitro. 2 . Potentials were recorded extracellularly from a ventral root (L3-L5) of the isolated spinal cord preparation or spinal cord-saphenous nerve-skin preparation of 0- to 2-day-old rats. Spinal reflexes were elicited by electrical stimulation of the ipsilateral dorsal root or by noxious skin stimulation. 3 . In the spinal cord preparation, single shock stimulation of a dorsal root at C-fibre strength induced mono-synaptic reflex followed by a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath-application of NO gas-containing medium (10⁻⁴-10⁻² dilution of saturated medium) and NO donors, 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC12, 3–300 μm), S-nitroso-N-acetyl-D, L-penicillamine (SNAP, 3–300 μm) and S-nitroso-L-glutathione (GSNO, 3–300 μm), produced an inhibition of the slow VRP and a depolarization of ventral roots. Another NO donor, 3-morpholinosydononimine (SIN-1, 30–300 μm), also depressed the slow VRP but did not depolarize ventral roots. These agents did not affect the mono-synaptic reflex. 4 . In the spinal cord-saphenous nerve-skin preparation, application of capsaicin (0.1-0.2 μm) to skin evoked a slow depolarizing response of the L3 ventral root. This slow VRP was depressed by NOC12 (10–300 μm) and SIN-1 (100–300 μm). When the concentration of NOC12 was increased to 1 mM, spontaneous synaptic activities were augmented and the depressant effect of NOC12 on the slow VRP became less pronounced. 5 . A NO-scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(carboxy-PTIO, 100–300 μm) prevented the depressant effect on the dorsal root-evoked slow VRP and ventral root depolarizing effects of NO donors. Carboxy-PTIO increased spontaneous synaptic activities and markedly potentiated the slow VRP. A NO synthase (NOS) inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 0.03-1 μm), but not D-NAME (0.03-1 μm), also markedly potentiated the slow VRP and this effect was reversed by L-arginine (300 μm). 6 . 8-Bromo-cyclic guanosine 3′: 5′-monophosphate (8-Br-cyclic GMP, 100–300 μm) produced both an inhibition of the slow VRP and a depolarization of ventral roots. A cyclic GMP-dependent protein kinase inhibitor, KT5823 (0.3 μm), partly inhibited the depressant effects of NO donors and 8-Br-cyclic GMP on the dorsal root-evoked slow VRP. In contrast, KT5823 did not inhibit the depolarizing effects of NO donors. 7 . Perfusion of the spinal cord with medium containing tetrodotoxin (0.3 μm) and/or low Ca²⁺ (0.1 mM)-high Mg²⁺ (10 mM) markedly potentiated the depolarizing effect of NO donors. The SNAP-evoked depolarization in the tetrodotoxin-containing low Ca²⁺-high Mg²⁺ medium was significantly inhibited by excitatory amino acid receptor antagonists D-(−)−2-amino-5-phosphonovaleric acid (30 μm) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 μm). 8 . The present study suggests that inhibitory and excitatory mechanisms meditated by the NO-cyclic GMP cascade are involved in the primary afferent fibre-evoked nociceptive transmission in the neonatal rat spinal cord. The inhibitory mechanism, but not the excitatory mechanism, appears to be partly mediated by cyclic GMP-dependent protein kinase. It is also suggested that Ca²⁺-independent release of excitatory amino acid neurotransmitters contributes to the depolarizing response to NO of ventral roots.

Background: The phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund's adjuvant (CFA) models. Results: We have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1–100 pmol) or IC261 (0.1–1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 μM) or IC261 (1 μM) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models. Conclusions: These results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.