摘要
目的:探讨钙-钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)在收缩促进骨骼肌细胞葡萄糖转运子4(GLUT4)转位机制中的作用。方法:将接种在培养板中的C2C12GLUT4myc小鼠肌管随机分为对照组和氨乙酰胆碱(Cch)组,各组分为抑制剂亚组和对照亚组。分别用10μmol/LCaMKⅡ的特异性抑制剂KN93或KN62预孵育,用酶联免疫吸附法(ELISA)测定细胞膜上GLUT4myc的含量(转位);用KN93预孵育后,免疫印迹法测定蛋白激酶CaMKⅡ的磷酸化。结果:氨乙酰胆碱使细胞膜上GLUT4myc的水平显著增加。KN93和KN62抑制氨乙酰胆碱刺激的GLUT4myc转位。氨乙酰胆碱可增加CaMKⅡ的磷酸化,KN93不影响对照组CaMKⅡ的磷酸化水平,但可以抑制Cch刺激的CaMKⅡ磷酸化。结论:CaMKⅡ位于Ca2+下游并有介导收缩促进骨骼肌细胞GLUT4myc转位的作用。
Objective： To study the role of calcium-/calmodulin- dependent protein kinase Ⅱ （CaMK Ⅱ ） in the mechanism of contraction-stimulated translocation of glucose transporter 4（GLUT4） in skeletal muscle cells. Methods： C2C12GLUT4myc myotubes were divided into two groups treated with or without carbachol （Ceh）. CaMK Ⅱ inhibitor KN93 or KN62 was added to the medium prior to treatment, respectively. Then cell surface levels of GLUT4myc were measured by enzyme linked immunosorbent assay（ELISA）. The phosphorylation of CaMK Ⅱ was detected by immunoblotting after pre-incubation with KN93. Results： Carbaehol increased phosphorylation of CaMK Ⅱ which was reduced by KN93. KN93 and KN62 inhibited the gain of cell surface GLUT4myc induced by Cch. KN93 did not affect the phosphorylation of CaMK Ⅱ but it inhibited the CaMK Ⅱ phosphorylation stimulated by Cch. Conclusion： CaMK Ⅱ is the downstream signal of Ca^2＋ and me- diates contraction-stimulated GLUT4myc traffic in skeletal muscle cells. Key words ealeium-calmodulin-dependent protein kinase type 2 muscle, skeletal glucose transporter type 4 muscle contraction cells, cultured enzyme-linked immunosorbent assay blotting, Western carbaehol
出处
《天津医药》
CAS
北大核心
2011年第6期536-538,共3页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(项目编号:30570912)
国家自然科学基金委员会-加拿大卫生研究院健康研究合作计划项目(项目编号:30611120532)
天津市科委科技支撑计划项目(项目编号:09ZCZDSF04500)
关键词
钙-钙调素依赖性蛋白激酶型
肌
骨骼
葡萄糖转运体型
肌收缩
细胞
培养的
酶联免疫吸附测定
印迹法
蛋白质
氨乙酰胆碱
calcium-calmodulin-dependent protein kinase type 2 muscle, skeletal glucose transporter type 4muscle contraction cells, cultured enzyme-linked immunosorbent assay blotting, Western carbaehol
