Molecular Mechanisms For Noradrenergic α2Receptor To Inhibit ERK1/2in Spinal Dorsal Horn Of Animals With Inflammatory Pain

Activation of noradrenergic α2receptor in spinal dorsal horn effectively alleviates the pathological pain hypersensitivity, however, the precise molecular mechanisms are not fully understood as yet. There have been some studies implicating that Extracellular signal-regulated kinases1and2(ERK1/2), which is closely related to nociceptive response, is a key player in spinal sensitization. In the present study, inflammatory pain model was induced by intraplantar injection of Complete Freund’s Adjuvant (CFA) in mice, and western blot, immunohistochemistry, co-immunoprecipitation and behavioral tests were used to investigate the possible regulation by noradrenergic α2receptor of ERK1/2after peripheral tissue lesions. Our data showed that:(1) intrathecal application of noradrenergic a2receptor agonist, clonidine (0.05u.g), could suppress the ERK1/2activity in spinal dorsal horn of mice with inflammatory pain, while it could not alter the ERK1/2activity in intact animals;(2) pretreatment with pertussis toxin (PTX), a Gi protein inhibitor, blocked the regulatory effect of clonidine on ERK1/2activity in spinal dorsal horn of mice with inflammatory pain;(3) in intact mice, intrathecal application of forskolin (6μg), an agonist of cAMP-dependent protein kinase (PKA), mimicked the effects of peripheral tissue injury by stimulating the activation of ERK1/2in spinal dorsal horn, which was reversed by clonidine. These data suggested that Gai/PKA pathway was involved in the regulation of ERK1/2activity by noradrenergic a2receptor;(4) intrathecal injection of forskolin elevated the phosphorylation level of61kD isoform of Striatal-enriched protein phosphatase (striatal-enriched protein tyrosine phosphatase61; STEP61) at Ser221when it stimulated the activation of ERK1/2, and clonidine revesed the phosphorylation of STEP61at Ser221induced by forskolin. These data implicated that noradrenergic a2receptor might resume the inhibition conferred by STEP61on ERK1/2;(5) additionally, intrathecal application of clonidine after periphery tissue lesions also reversed the phosphorylation of STEP61at Ser221;(6) co-immunoprecipitation results showed that, with the dephosphorylation of STEP61at Ser221, clonidine resumed the interaction between STEP61and ERK1/2in spinal dorsal horn of mice with inflammatory pain;(7) direct overexpression of STEP61mutation---STEP61(S221A) in spinal dorsal horn to block the phosphorylation of STEP61at Ser221could effectively repress CFA-induced inflammatory pain hypersensitivity and ERK1/2hyperactivity in spinal dorsal horn. These results suggested that Gi protein-coupled noradrenergic a2receptor might suppress ERKl/2-dependent pain symptom through Gai/PKA/STEP61signaling pathway.