Tyrosine Phosphorylation Of Spinal NMDA Receptors In The Formation Of Allodynia

Objective:The hyperfunction of NMDA (N-methyl-D-aspartate)-subtype glutamate receptors plays an essential role in the initiation and development of chronic pathological pain. Previous studies have demonstrated that activation of NMDA receptor per se is able to trigger NMDA receptor hyperfunction and allodynia by inducing a specific and aberrant increase in the synaptic expression of NR2B subunit-containing NMDA receptors (NR2B receptors). Moreover, recent studies have implicated that the abnormal increase in NR2B synaptic concentration requires the involvement of NR2B tyrosine phosphorylation. However, the molecular mechanisms underlying the dynamic changes in NR2B tyrosine phosphorylation in spinal dorsal horn are largely unknown as yet. The present study aimed to explore the intracellular signaling cascades that regulated NR2B tyrosine phosphorylation.Methods:Healthy male mice were intrathecally injected with NMDA or intradermally injected with Complete Freund’s Adjuvant (CFA) to stimulate NMDA receptors in spinal dorsal horn. The relationship between the alteration in NR2B tyrospine phosphorylation and the aberrant expression of synaptic NR2B receptors was investigated, and the roles and significance of Src-family protein tyrosine kinases (SFKs) and protein tyrosine phosphatases (PTPs) in NMDA receptor-induced NR2B tyrosine phosphorylation were examined.Results:(1) Intrathecal application of NMDA (10-60ng) dose-dependently elicited allodynia in naive mice, which was associated with the specific enhancement of the synaptic contents of spinal NMDA receptor NR1/NR2B subunits without any changes in the total protein levels of NMDA receptor NR1/NR2A/NR2B subunits. Intrathecal administration of a NR2B receptor-selective antagonist ifenprodil completely blocked NMDA-induced allodynia.(2) A specific SFKs inhibitor PP2(1.5μg), when given intrathecally, not only alleviated NMDA-induced allodynia but also reversed the abnormal expression of NR2B/NR1subunit at synapses evoked by NMDA.(3) As a control, KN-93, the inhibitor of Ca2+/Calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) that worked as another regulator of NR2B receptor functions, produced little effects on NMDA-induced synaptic concentration of NR2B/NR1subunit, albeit its potent alleviation of NMDA-induced allodynia.(4) NMDA dose-dependently enhanced the tyrosine phosphorlyation of NR2B at residue1472(NR2B-Y1472) in spinal dorsal horn of mice, which could be eliminated by SFKs inhibitor PP2, suggesting that tyrosine phosphorylation of NR2B by the activated SFKs might represent one of important mechanisms underlying NMDA receptor-induced NR2B synaptic expression.(5) In addition to SFKs, protein tyrosine phosphatases (PTPs) might also be involved in the regulation by NMDA receptors of NR2B functions, because intrathecal injection of PTPs inhibitor Na3VO4simultaneously attenuated NMDA-induced allodynia and the aberrant synaptic expression of NR2B receptors.(6) Furthermore, intrathecal Na3VO4application also repressed NR2B synaptic concentration induced by peripheral tissue injury to alleviate inflammatory pain through depressing NR2B-Y1472phosphorylation.Conclusion:NMDA receptors in spinal dorsal horn catalyzed NR2B-Y1472phosphorylation to promote the synaptic delivery of NR2B receptors by stimulating the intracellular SFKs and PTPs signalings, leading to NR2B receptors hyperfunction and the formation of allodynia.