Effects And Mechanisms Of Acid-sensing Ion Channels On Inflammation Response Of Cultured Rat Microglia

Part I Functional expression of ASICs in cultured rat microglia cellsAims:Acid-sensitive ion channels (acid-sensing ion channels, ASICs) is a ligand-gated ion channels, a subfamily of the ENaC/Deg superfamily of ion channels. Since ASICs were first found on the membrane of neurons in1980, it has been recieved more and more attention. ASICs are widely distributed in the forebrain, midbrain, brainstem, pons, cerebellum, on the hypothalamus, spinal cord and other parts in central nervous system, With further research, it was found that ASICs expression in other types of cells, including glial cells and immune cells. The ASIC subunits play an important role in a variety of physiological and pathological processes, including ischemic brain injury, pain, learning and memory, epilepsy, multiple sclerosis, migraine headaches, irritable bowel syndrome. Studies have shown that, there might be close relationship between ASICs and immune inflammation. However, whether ASICs are expressed in microglia is still unknown. This study was designed to identify the expression of ASICs from the gene and protein levels, and investigate the characteristics of the channels in the microglia.Methods:We identify the expression of ASICs in microglia from mRNA level by RT-PCR. Quantitative real-time PCR experiments were performed to quantitative analysis of mRNA encoding ASIC1, ASIC2a and ASIC3. western blotting was used to detected protein expression of ASICs. We determine the distribution of ASICs in microglia by The cellular immunofluorescence method. Whole-cell patch clamp was adopted to detected the current properties of microglia. calcium imaging was performed to investigate the ion permeability of ASICs in microglia.Results:(1) In this study, we confirmed that ASIC1,2a,3were expressed in microglia, real-time quantitative PCR analysis showed that, ASIC1was the most abundant subunit followed by ASIC3, ASIC2a least. ASIC1and ASIC3were distributed in the cytoplasm and nucleus, ASIC2a was mainly located in the nucleus.(2) ASIC-like current could be elicited by rapid decrease of extracellular pH in LPS-stimulated microglial cells, these currents were pH-dependent and the pH of half-maximal activation (pH0.5) was6.00±0.04. addition, the current could be suppressed by100μM amiloride, inhibition rate was63.15%±6.9%. Extracellular acidification caused increase of intracellular calcium in LPS-stimulated microglia, which was inhibited by100μM amiloride and10nM PcTxl, the inhibition rate reached61.7%±4.8%and47.3%±6.2%.Conclusion:ASICs are functional expressed in microlgia and ASICla is the most abundant, the membrane protein expression of ASIC la was increased in LPS-stimulated microglia, thus,, it is easy to induce inward current and intracellular calcium concentration by rapid decrease of extracellular pH. Part Ⅱ Effects and mechanisms of ASICs on inflammatory responses from cultured rat microgliaAim:Inflammation is often associated with the organization of acidification, the pH value is lower than normal, the low pH environment is considered to be a sign of inflammation. By feeling such change can play a role in the inflammatory response. Acid sensitive ion channels can feel the extracellular pH decline, which is activated by extracellular H+, formate inward currents, cause cell membrane depolarization, excitatory effect is produced. Some primary studies prompted that, the ASICs indeed play an important role in the inflammatory reaction, however, the exact role of ASICs in microglia during immune inflammation remains elusive. This study is designed to investigate the role of ASICs in the expression of inflammatory cytokines and scratch migration using microglia.Methods:The non-specific ASICs antagonist amiloride and specific homomeric ASIC1a blocker PcTx1were used to study the role of ASICs in the expression of iNOS and COX-2in LPS-induced microglia by westtern blotting and migration in scratch-induced microglial migration.Results:(1) the protein expression of ASICs were increased in LPS-stimulated microglia, membrane protein research results show that, the membrane proteins expression of ASIC1were significantly increased.(2) LPS induced iNOS and COX-2expression of microglia, this effect could be inhibited by ASICs channel blocker amiloride and PcTx1.(3) The non-specific ASICs antagonist amiloride and specific homomeric ASIC1a blocker PcTx1inhibited migration of microglia through inhibition of ERK phosphorylation.Conclusion:ASICs involved in the expression of inflammatory factors and migration of microglia.