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

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 32 P from [γ 32 P] ATP to myelin basic protein(MBP).Total PKC activity refers to PKC activity in “cytosol” fraction plus the one in “membrane” fraction.\; The results show that Alarelin could induce activation of PKC,which acted in time course manner,and the peak time of PKC activity was 3 minutes after treatment.When Alarelin was 10 -9 ,10 -8 ,10 -7 ,10 -6 ,10 -5 mol/L respectively,the PKC activity was increased gradually,and acted in dose dependent way.The peak value reached 781 40±14 74 pmol/min/mg when Alarelin was 10 -5 mol/L.The smallest value was 103 12±11 04 pmol/min/mg.After cells were preincubated with 10 mmol/L PMA 10 min,then the 10 -5 mol/L Alarelin was administered(Group B),we found that PKC activity was not different from Group A or Group B( P >0 05).After PKC activity was depleted by 12 hr treatment of PMA in Group C,then incubated with 10 -5 mol/L Alarelin,we found PKC activity were significantly lower than that of Group A and Group B( P <0 05).It was also observed that the alarelin stimulated PKC activity was dependent on the presence of extracellular calcium.These results indicated that PKC is an important signal molecule in the regulation of GnRH to cultured stomach smooth muscle cells and GnRH analogue could increase the PKC activity.