Adrenomedullin Mediates TNF-?-induced Alterations In Dorsal Root Ganglion(DRG) Cells In Rats

Tumor necrosis factor alpha(TNF-a)is a pro-inflammatory cytokine and a key mediator in multiple signaling pathways,which has a variety of biological effects.TNF-a can induce plasticity in dorsal root ganglia(DRG)neurons.As a result,the excitability of DRG neurons,the cell bodies of nociceptors,is increased leading to peripheral sensitization.However,the underlying mechanisms are unclear.Adrenomedullin(AM),a member of the calcitonin gene-related peptide(CGRP)family,is a pain peptide.AM possess a character that recruit other pain mediators.It has been shown that AM is involved in the pathology of pathological pain.AM and TNF-a are distributed in the DRGThe techniques of real-time quantitative PCR,Western blotting and double-label immunofluorescence were used in the present study.This study investigated the possible involvement of AM in_TNF-?-induced responses contributing to neuronal plasticity in the DRG.We assessed the involvement of AM in TNF-a-induced activation of satellite cells and intracellular signaling pathways.The possible colocalization of AM with TNF-a in DRG neurons was examined.The results showed:1.Exposure of the DRG explant cultures to TNF-a(5 nM)for 48 hours upregulated the expression of AM mRNA.The treatment with TNF-a also increased the level of CGRP,substance P(SP),CC chemokine ligand 2(CCL2)and matrix metalloproteinase-9(MMP-9)mRNA in the cultured DRG.This increase was attenuated by the co-treatment with the selective AM receptor antagonist AM22-52(2 ?M);2.The blockade of AM receptors inhibited TNF-a-induced increase of the glial fibrillary acidic protein(GFAP),interleukin-1?(IL-1?),phosphorylated cAMP response element binding protein(pCREB)and nuclear factor kappa B(pNF-?B)proteins.The inhibition of AM activity did not change TNF-a-induced phosphorylation of extracellular signal-related kinasel/2(pERKl/2);3.The treatment with the AM receptor agonist AM1-50(10 nM)for 96 hours induced an increase in the level of GFAP,IL-1?,pCREB,pNF-?B,pERK1 and pERK2 proteins while this increase was abolished by the addition of AM22-52 in the cultures.AM22-52 treatment alone did not change the level of these proteins compared to the saline group;4.Immunofluorescence assay showed colocalization of AM-like immunoreactivity(IR)with TNF-a-IR or CCL2-IR in the soma of DRG neurons.In summary,our results indicate that the pronociceptive mediator AM not only was the effector of TNF-a,but also mediated the TNF-?-induced responses in the DRG including CGRP;SP,CCL2,MMP-9 and IL-1? productions,activation of satellite cells as well as the phosphorylation of CREB and NK-kB.These observations were further supported by the finding that AM is extensively colocalized with TNF-? or CCL2 in DRG neurons.The present study suggests that the increased AM receptor signaling mediated the many,but not all,TNF-a-induced activities.The results in this study showed that AM mediated TNF-a-induced responses in DRG.Our results suggest a novel cellular mechanism of biological effects of TNF-?.