The Expression And Function Of Acid-sensing Ion Channels In Trigeminal Ganglion And RAW264.7Cells

Partâ… . The expression and function of acid-sensing ion channels(ASICs) in trigeminal ganglion (TG)Objective: ASICs, belong to the degenerin/epithelial Na+channel superfamily(DEG/ENaC), can be activated by acute drop in the extracellular pH. Six subunits havebeen verified, they are ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3and ASIC4. Previousstudies demonstrate that ASICs are widely expressed in peripheral nervous system andASICs participate the pathophysiological process of pain, such as migrain, The orofacialpain is a common clinical symptom and trigeminal ganglion (TG) is the only sensory nerveinnervating orofacial region. However the exact expression of ASICs in TG and the exactfunction of ASICs in orofacial inflammatory pain remain elusive. In this part, weinvestigate the expression of different ASIC subtypes in TG neurons and the role of ASICsin orofacial inflammatory pain.Methods: We testified the expression of ASIC1, ASIC2a, and ASIC3in the mouse TGneurons by using RT-PCR, quantatitive real-time PCR, western blotting and whole-cellpatch clamp technology. We used immunofluroscence assay to examine the distribution ofASIC1, ASIC2a, and ASIC3in the TG neurons innervation orofacial region by injection ofretrograde labeling fluorescent dye DiI. The functional ASICs on acute dissected TGneurons were examined by whole-cell patch clamp. The change of ASICs proteinexpression was examined by western blot and immunofluroscence assay. The change offunction of ASICs was examined by whole-cell patch clamp. By formalin model, weinvestigated the effect of amiloride, PcTx1and genetic deletion of ASIC1on pain behivor.Results: We detected the protein and mRNA expression of ASIC1, ASIC2a, and ASIC3inTG neurons, and the ASIC3was the most aboundant transcript, following by ASIC1.ASIC1, ASIC2a, and ASIC3were high expressed in the plasma membrane and cytoplasmof TG neurons innervating orofacial vibrissa pad. The amplitude of ASICs current in TG neurons was increased in the formalin-induced orofacial inflammatory model. In addition,western blot and immunofluoresecnce assay also demonstrated a significantly higherexpression of ASICs in TG neurons during orofacial inflammation compared with thecontrol group. Furthermore, both pharmacological blockade of ASICs and genetic deletionof ASIC1attenuated the inflammatory pain behivor.Conclusion: ASIC1, ASIC2a, and ASIC3are high expressed in TG neurons, and ASIC3isthe most abundant transcript. Both the protein expression and the function of ASICs areincreased in the orofacial inflammatory pain. Pharmacological blockade of ASICs andgenetic deletion of ASIC1attenuate the symptom in orofacial formalin model. In brief,ASICs, especially ASIC1, participate in the procession of orofacial inflammatory pain. Part â…¡. The expression and function of acid-sensing ionchannels (ASICs) in RAW264.7cellsBackground: RAW264.7cells are mouse macrophage-like cell line. Macrophagesparticipate in the inflammation by triggering the inflammatory response, engulfing cells andantigen. In the inflammatory process, macrophage migrate and aggregate in inflammationregion. Acidosis is a common feature of inflammation, and our previous study demonstratethat acidosis promote the maturation of Dentritic cells. Thus, in this part, we investigate theexpression and function of ASICs in RAW264.7cells.Methods: We first testified the expression and distribution of ASIC1, ASIC2a, and ASIC3in RAW264.7cells by using RT-PCR, western blotting and immunofluroscence assay.The functional ASICs were examined by calcium image. The effect of ASICs on thesecretion of pro-inflammatory cytokines was examined by western b lotting. The effect ofASICs on the migration of RAW264.7cells was examined by scaring test. By flowcytometry analysis, we investigated the effect of ASICs on the apoptosis of RAW264.7.Results: We detected the mRNA and protein expression of ASIC1, ASIC2a, and ASIC3inRAW264.7cells. ASIC1are mainly located in the nucleus of RAW264.7cells, whileASIC2a and ASIC3are mainly expressed in the membrane and cytoplasm. Acidextracellular solution induces the elevation of intracellular [Ca2+]iin the LPS-stimulatedRAW264.7cells. The non-specific ASICs antagonist amiloride and specific homomericASIC1a blocker PcTx1reduced production of iNOS and COX-2in LPS-stimulatedRAW264.7cells. Pretreatment with amiloride and PcTx1attenuated the migration ofRAW264.7cells in scaring test. Amilorode and PcTx1protected the RAW264.7cells fromLPS induced apoptosis.Conclusion: ASIC1, ASIC2a, and ASIC3are expressed in the RAW264.7cells. Theinhibitors of ASICs reduce the production of pro-inflammatory cytokines and apoptosis inLPS-stimulated cells. The inhibitors of ASICs also attenuated the migration of RAW264.7cells in injury sites.