GnRH类似物对培养的大鼠胃平滑肌细胞蛋白激酶C活性的影响  

EFFECT OF GnRH ANALOGUE ON PKC ACTIVITY IN CULTURED RAT STOMACH SMOOTH MUSCLE CELLS

作　　者：陈蕾[1] 李东红[2] 姚元庆[2] 刘丽宏[3] 黄威权[1] 

机构地区：[1]第四军医大学组织胚胎学教研室,西安710032 [2]第四军医大学唐都医院妇产科,西安710038 [3]第四军医大学药理学教研室,西安710032

出　　处：《动物学报》2002年第5期654-659,共6页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金资助项目 (No .3 9770 3 88)~~

摘　　要：研究GnRH类似物阿拉瑞林 (Alarelin)对大鼠胃平滑肌细胞蛋白酶C (PKC)活性的影响 ,采用放射性同位素法测定PKC的活性 ,发现 :(1)阿拉瑞林可使培养的大鼠胃平滑肌细胞中PKC活性明显升高 ,3min时达高峰 ,10min后作用明显降低 ,且成剂量依赖性。当阿拉瑞林为 10 -5mol/L时 ,PKC活性最高 ,10 -9mol/L时其对PCK的作用基本消失 ;(2 )当用佛波醇酯 (phorbol 12myristate 13 acetate ,PMA)短期作用激活PKC后 ,再加入阿拉瑞林 ,仍可使PKC活性略有升高 ,但无显著性差异 ;当用PMA长期持续作用耗竭PKC后 ,再加入阿拉瑞林作用 3min ,它不能激活PKC ,与单纯的阿拉瑞林作用 3min组相比差异显著 ;(3)在无外Ca2 +时 ,阿拉瑞林也可使PKC活性升高 ,但不如单独用阿拉瑞林作用 3min组的高。因此PKC活性的增高 ,并不完全依赖细胞外Ca2 + ,它可以动员胞内Ca2 + 的释放激活PKC ,说明GnRH类似物可使胃平滑肌细胞中PKC活性增加 ,PKC参与阿拉瑞林对胃平滑肌细胞调控的信号转导过程。Recently,Gonadotropin releasing hormone(GnRH)and its receptor have been found in rat digestive system.Our previous studies demonstrated that GnRH receptor and its mRNA both existed in cultured rat stomach smooth muscle cells(SSMCs),and also,we found GnRH analogue could increase the intracellular [Ca 2+ ] of SSMCs.In order to investigate the effect of GnRH analogue on protein kinase C(PKC)activity in cultured rat stomach smooth muscle cells,we used Radioimmuoassay to determine the PKC activity.SSMCs which we used in this experiment were passage 3～5.After several days in culture medium,SSMCs were washed and treated with different agents in the following manners:(1)SSMCs were treated with Alarelin at 10 -5 mol/L for different time(1,2,3,6,8 and 10 min)to find the time dependence.(2)The SSMCs were treated for 3 min with Alarelin at 10 -9 ,10 -8 ,10 -7 ,10 -6 ,10 -5 mol/L respectively to identify the optimal dosage of Alarelin(3)SSMCs were preincubated with phorbol 12 myristate 13 acetate(PMA)for 10 min in Group A and Group B,and 12 hr in Group C respectively.Then Group B and Group C were treated with 10 -5 mol/L Alarelin to confirm the effects of PMA on PKC activity of SSMCs.(4)In order to find the calcium dependence of GnRH analogue mediated PKC activity,the SSMCs from which extracellular calcium ion were removed the by the use of EGTA were treated with 10 -5 mol/L Alarelin.Then,total cells were scraped with a rubber policeman and transferred to plastic tubes containing the extraction PKC.Cell suspensions were centrifuged and cell pellets were resuspended and sonicated on ice.The homogenate was centrifuged at 100 000 ×g for 30 min at 4℃.The supernatant was used as the “cytosol” fraction,and the pellets were re homogenized in 1% Triton X 100.The second homogenate was centrifuged at 100 000 ×g for 30 min at 4℃,and the supernatant was used as the “membrane” fraction.PKC activity in both fractions were assayed by determining the transfer rate of

关 键 词：GNRH类似物 大鼠 胃平滑肌细胞 蛋白激酶C活性 PKC 放射性化学分析测定 

分 类 号：Q55[生物学—生物化学]