In vivo pathway of thermal hyperalgesia by intrathecal administration of α,β-methylene ATP in mouse spinal cord: Involvement of the glutamate-NMDA receptor system

1. The aim of the present study is to characterize the role of the P2X receptor in spinal nociceptive processing in vivo. We investigated the mechanisms of the P2X receptor agonist α,β-methylene ATP (α,βmeATP)-induced modulation of acute nociceptive signalling in mouse spinal cord. 2. Intrathecal administration of α,βmeATP produced a significant and dose-dependent thermal hyperalgesic response. This response was completely blocked by intrathecal pretreatment with the non-selective P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and the selective P2X₁, P2X₃ and P2X₂₊₃ receptor antagonist, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). Pretreatment with α,βmeATP 15, 30 and 60 min prior to administration of a second dose of α,βmeATP diminished the α,βmeATP-induced thermal hyperalgesia. 3. A potent agonist for the P2X₁ receptor, β,γ-methylene-L-ATP, did not show the hyperalgesic response, indicating that the P2X₁ receptor is not involved in the spinal nociceptive pathway. 4. In fura-2 experiments using mouse dorsal root ganglion (DRG) neurons, α,βmeATP (100 μM) increased intracellular Ca²⁺ ([Ca²⁺](i)). This was not produced by a second application of α,βmeATP. The same DRG neurons also showed a marked [Ca²⁺](i) increase in response to capsaicin (3 μM). 5. Intrathecal pretreatment with the Ca²⁺-dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by α,βmeATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonopentanoate (APV), dizocilpine and ifenprodil. 6. These findings suggest that α,βmeATP-induced thermal hyperalgesia may be mediated by the spinal P2X₃ receptor subtype that causes unresponsiveness by repetitive agonist applications, and that α,βmeATP (perhaps through P2X₃ receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.