Molecular Mechanisms Underlying The Modulation By Spinophilin Of Chronic Inflammatory Pain

F-Actin and Protein phosphatase-1(PP1)-binding protein Spinophilin (SP) plays a critical role in the regulation of excitatory glutamatergic synaptic transmission and plasticity. However, the distribution and function of SP in spinal cord remain largely unknown as yet. The present study performed behavioral tests, immunohistochemistry, immunoblotting and co-immunoprecipitation to investigate the role of SP in inflammatory pain and explore the molecular mechanisms for SP to modulate the nociceptive processing. Our data showed that:(1) SP was specifically present at lamina Ⅰ-Ⅱ of rat spinal dorsal horn, which co-localized with PP1at postsynaptic density, suggesting that SP, as the targeting protein of PP1, might direct PP1into spinal synapses to modulate the nociceptive transmission and integration;(2) Intrathecal injection of pEGFP vector encoding wild-type SP [SP(WT)] produced little effects on the nociceptive behavioral responses of intact rats, whereas intrathecal application of pEGFP vector encoding SP mutant, SP(F451A), to specifically interfere with SP-mediated PP1synaptic targeting dose-dependently evoked mechanical allodynia. Moreover, non-specific PP1inhibitor okadaic acid (0.16ng), when intrathecally given, completely ruled out the pronociceptive action of SP(F451A). These data suggested that SP-targeted PP1operated to inhibit the conveyance of painful signals; Disturbance of SP/PP1interaction eliminated the inhibition conferred by SP-targeted synaptic PP1on nociceptive transmission, resulting in the formation of allodynia;(3) Spinal administration of Ca/Calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93(6μg), cAMP-dependent protein kinase (PKA) inhibitor H-89(5μg), Protein kinase C (PKC) inhibitor chelerythrine (0.4μg), phosphoinositide-3-kinase (PI3K) inhibitor wortmannin (0.5μg) or Src family protein tyrosine kinases (SFKs) inhibitor PP2had no ability to attenuate the allodynia induced by SP(F451A). Only Extracellular Signal-Regulated Kinases1and2(ERK1/2) inhibitor U-0126(5μg) ameliorated SP(F451A)-evoked pain sensitization, suggesting that ERK1/2acted downstream of SP/PP1signaling.(4) Overexpression of SP(F451A) in spinal dorsal horn of intact rats significantly enhanced the synaptic concentration of NMDA receptor NR1and NR2B subunits, with that of NR2A subunit unaltered; Behavioral tests verified that NR2B receptor-selective antagonist ifenprodil (10μg) potently alleviated SP(F451A)-induced allodynia, implicating that disruption of SP-mediated PP1synaptic targeting specifically elicited NR2B receptor hyperfunction;(5) Intraplantar injection of Complete Freund’s Adjuvant (CFA) to induce inflammatory pain dramatically reduced the synaptic localization of endogenous SP in spinal dorsal horn, which, however, could be blocked by intradermal application of lidocaine (2%;50μg), a chemical reagent inhibiting noxious stimuli input, or by intrathecal application of PKA inhibitor H-89(5μg), implying that activation of spinal PKA by ongoing input of noxious stimuli from the injuried sites dispersed endogenous SP out of synapses, thus abolishing the inhibitory effects of SP-targeted PP1on nociceptive conveyance;(6) Overexpression of exogenous SP(WT) to resume SP-mediated PP1synaptic targeting selectively repressed the synaptic concentration of NR2B receptors in spinal dorsal horn of inflamed rats, leading to the alleviation of inflammatory pain. These data suggested that SP-mediated PP1synaptic targeting exerted a tonic inhibition on nociceptive transduction in spinal dorsal horn; Peripheral tissue lesion-evoked SP/PP1mistargeting at synapses was involved in the initiation and maintenance of inflammatory pain; Expression of exogenous SP generated analgesia against inflammatory pain.