CAJ | 학술논문

目的研究足细胞是否表达有功能的代谢型谷氨酸受体(mGluR).方法以RT-PCR法检测墓因表达,Western印迹、免疫荧光双染色和免疫电镜检测蛋白质表达.酶免疫实验(EIA)、电泳迁移位移实验(EMSA)和Western印迹法检测细胞环腺苷酸(cAMP)的产生和转录因子cAMP反应元件结合蛋白(CREB)的活化.激光共聚焦显微镜观察细胞内游离钙的变化.结果足细胞表达mGluR1和5的基因和蛋白质,肾小球中mGluR1的表达和足细胞标志蛋白synaptopodin表达共定位.免疫电镜显示mGluR1位于足细胞体和足突细胞膜下层.mGluR1和5的激动剂3,5-二羟基苯甘氨酸(DHPG)诱导足细胞快速地产生cAMP,转录因子CREB被激活,磷酸化CREB表达增高.这些改变能被mGluR1和5的选择性抑制剂1-氨基茚-1,5-二羧酸(AIDA)以及腺苷酸环化酶抑制剂(SQ22536)抑制,而2-氨基乙氧化联苯-甲硼烷(2APB)则不具有上述抑制作用.DHPG诱导足细胞内游离钙缓慢持续地增加,细胞孵育于不含钙的培养液中、AIDA预处理以及2APB预处理抑制了DHPG诱导的细胞内游离钙离子增加.结论足细胞表达有功能的mGluR1和5.
목적 연구족세포시부표체유공능적대사형곡안산수체(mGluR).방법 이RT-PCR법검측묘인표체,Western인적、면역형광쌍염색화면역전경검측단백질표체.매면역실험(EIA)、전영천이위이실험(EMSA)화Western인적법검측세포배선감산(cAMP)적산생화전록인자cAMP반응원건결합단백(CREB)적활화.격광공취초현미경관찰세포내유리개적변화.결과 족세포표체mGluR1화5적기인화단백질,신소구중mGluR1적표체화족세포표지단백synaptopodin표체공정위.면역전경현시mGluR1위우족세포체화족돌세포막하층.mGluR1화5적격동제3,5-이간기분감안산(DHPG)유도족세포쾌속지산생cAMP,전록인자CREB피격활,린산화CREB표체증고.저사개변능피mGluR1화5적선택성억제제1-안기인-1,5-이최산(AIDA)이급선감산배화매억제제(SQ22536)억제,이2-안기을양화련분-갑붕완(2APB)칙불구유상술억제작용.DHPG유도족세포내유리개완만지속지증가,세포부육우불함개적배양액중、AIDA예처리이급2APB예처리억제료DHPG유도적세포내유리개리자증가.결론 족세포표체유공능적mGluR1화5.
Objective To investigate the expression of metabotropic glutamate receptor (mGluR) in murine podocytes.Methods Conditional immortalized podocytes were used in the research.RT-PCR was used to estimate the mRNA expression.Western blotting,immunofluorescence staining and immunoelectron microscopy were employed to determine the protein production.EIA,EMSA and Western blotting were used to examine the cAMP generation and cAMP response element-binding protein (CREB) activation.Intracellular calcium was investigated using confocal microscopy.Results mGluR1 and 5 mRNA and protein were expressed in murine brain and podocytes.In glomeruli,most of mGluR1 expression located in podocytes and was expressed in the submembrane space of the podocytes.Podocytes treated with (S)-3,5-dihydroxyphenylglycine (DHPG,an agonist for mGluR1/5) rapidly generated cAMP and activated CREB.(RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA,a selective antagonist of mGluR1/5) and SQ22536 (an adenylate cyclase inhibitor),but not 2-aminoethoxydiphenyl borate (2-APB an antagonist of canonical transient receptor potential) blocked DHPG-induced cAMP generation and CREB activation.Following DHPG treatment,intracellular calcium level rose and was prevented by pre-treatment with AIDA and 2-APB.DHPG-induced calcium influx was also prevented by incubation with calcium-free medium.Conclusion Podocytes express functional mGluR1 and mGluR5.