Role Of Gβγ Subunit In Nordrenergic α2A Receptor-mediated Suppression Of Chronic Inflammatory Pain

Objective:The inhibitory G protein (Gi)-coupled nordrenergic a2A receptor triggers multiple signaling pathways through a and Py subunits of Gi protein.α2A adrenergic receptor (a2A-R), a subtype of a2 adrenoceptor, is expressed in the spinal cord and plays a critical role in modulation of nociceptive transmission. To date, most studies have investigated the relevance of cAMP signaling pathways trigged by Gia subunit of G protein to pain modulation. However, the functions of Gβγ subunit are still unknown. The present study aimed to investigate the role of signaling pathways trigged by Gβγ subunit in nordrenergic α2A receptor-mediated analgesia against chronic inflammatory pain.Method:Complete Freund’s Adjuvant (CFA) was subcutaneously injected into the plantar surface of mouse hindpaws to establish the animal model of chronic inflammatory pain. Inhibition of Gβγ activity was achieved by intrathcally injecting recombinant adenovirus that encoded βARKct, a specific inhibitor of GPy (the carboxyl-terminal 195 amino acids of β-adrenergic receptor kinase-1). Nociceptive behavioral tests, immunoblotting, patch clamp whole-cell recordings and immunoprecipitation were used to investigate the effects of a2A adrenergic receptor-specific agonist Guanfacine on inflammatory pain.Results:(1) Intrathecal injection of a2A receptor agonist Guanfacine effectively ameliorated the thermal hyperalgesia and mechanical allodynia induced by CFA, which could be abolished by βARKct; (2) Through Gβγ subunit, a2A receptor might regulate the synaptic expression of N-Methyl-D-Aspartate (NMDA)-subtype glutamate receptors, because βARKct prevented guanfacine from reducing the protein contents of NMDA receptor GluN1 and GluN2B subunits at synaptosomal membrane fraction; (3) Immunoblotting analysis showed that a2A receptor, acting via Gpy subunit, decreased the phosphorylation of GluN2B subunit at Tyr1472 (GluN2B-Tyrl472), which led to the enhanced endocytosis of GluN2B subunit-containing NMDA receptors; (4) Immunoprecipitation experiments illustrated that Src, a protein tyrosine kinase responsible for GluN2B-Tyr1472 phoshporylation, was a key downstream target of a2A receptor/Gβγ signaling. Guanfacine decreased the phoshoryaltion level of Src at Tyr418 and increased the phoshoryaltion of Src at Tyr529. Spinal expression of PARKct, however, blocked the effect of guanfacine on Src activity; (5) C-Terminal Src Kinase (CSK) might be involved in the regulation of Src activity by a2A receptor/Gβγ subunit signaling. We found that βARKct abolished CSK redistribution at synapse induced by spinal application of Guanfacine in inflamed mice; (6) In order to explore the role of CSK in nociceptive modulation, the recombinant adenovirus encoding catalytically inactive CSK mutant [CSK(K222R)] was intrathecally injected in intact mice. We found that spinal expression of CSK(K222R) increased the phosphorylation of GluN2B-Tyrl472. Meanwhile, CSK(K222R) blocked the inhibitory effect of guanfacine on NMDA receptors in inflamed mice; (7) More importantly, spinal expression of wild-type CSK [CSK(WT)] through intrathecal injection of recombinant adenovirus mimicking the effects of Guanfacine in inflamed mice by decreasing the synaptic expression of GluNl/GluN2B subunits, reducing the phosphorylation level of GluN2B-Tyr1472 and repressing NMDA receptor-mediated nociceptive transmission. Consequently, spinal CSK(WT) expression resulted in a significant alleviation of chornic inflammatory pain.Conclusion:α2A adrenoceptor inhibited Src-mediated phosphorylation and synaptic expression of GluN2B subunit through the Gβγ/CSK signaling pathways, which might be an important mechanism for a2A adrenoceptors to regulate the chronic pain.