芥子碱对PC12细胞内游离钙升高的影响  

Effects of sinapine,a Chinese herb component,on the increase in cytosolic free calcium in PC12 cells

作　　者：何玲[1,2] 刘丽芳[1] 李海涛[2] 郭胜伟[2] 邱嘉斌[1] 李富[1] 蔡宝昌[2] 

机构地区：[1]中国药科大学药理教研室,南京210009 [2]南京中医药大学博士后科研流动站,南京210029

出　　处：《中国新药杂志》2008年第19期1679-1683,共5页Chinese Journal of New Drugs

基　　金：中国博士后科学基金面上资助项目(20070410343);江苏省博士后科研资助项目(0601050B);江苏省药效研究平台项目(BM2005103)

摘　　要：目的:考察芥子碱对高钾除极和谷氨酸引起细胞外钙内流以及咖啡因和环匹阿尼酸(cyclopia-zonic acid,CPA)引起细胞内钙释放所导致的PC12细胞内游离钙升高的影响。方法:PC12细胞用终浓度为5μmo.lL-1的钙离子荧光指示剂Fluo-3/AM于37℃避光负载孵育。细胞内游离钙([Ca2+]i)用VictorⅡ1420多功能标记分析仪检测,以荧光强度(FI)表示。结果:当有外钙存在时,PC12细胞静息[Ca2+]i的FI值为(1 188±163),75 mmol.L-1KCl和10 mmol.L-1谷氨酸引起细胞外钙内流,使[Ca2+]i增高,FI峰值分别增至(4 270±982)和(3 096±402)。芥子碱(0.01,0.1,1.0,10,100μmol.L-1)对静息[Ca2+]i没有明显的影响,但浓度相关性地抑制高钾和谷氨酸诱发的[Ca2+]i增高,抑制率分别为10.2%,39.5%,53.7%,71.8%,88.4%和30.3%,55.7%,68.5%,79.2%,94.1%。当无外钙存在时,PC12细胞静息[Ca2+]i的FI值为(813±112)。咖啡因(40 mmol.L-1)和CPA(30μmol.L-1)分别引起Caffeine-ryanodine和三磷酸肌醇(InsP3)敏感型-钙池释放而使[Ca2+]i升高,FI值分别增至(2 944±371)和(1 844±332)。芥子碱(0.1,1.0,10,100μmol.L-1)浓度相关性地抑制咖啡因和CPA诱发的[Ca2+]i增高,抑制率分别为23.7%,61.9%,77.5%,88.2%和10.9%,23.8%,44.6%,68.3%。结论:芥子碱能显著抑制电压依赖性和受体依赖性钙通道开放所引起的外钙内流,且对受体依赖性钙通道的抑制作用更强。此外,芥子碱对Caffeine-ryan-odine受体和InsP3敏感型钙池的内钙释放也有明显抑制作用,且对Caffeine-ryanodine受体敏感型钙池的抑制作用更强。这提示芥子碱有望成为很有研发前景的新型钙阻滞剂。Objective： To investigate the effects of sinapine, a Chinese Herb component, on high K^＋ or glutamate-induced extracellular calcium influx, and caffeine or cyclopiazonic acid （CPA）-induced calcium release from internal stores in PC12 cells. Methods： PC12 cells were loaded with calcium fluorescent indicator Fluo-3/AM at the final concentration of 5 μmol·L^-1 at 37 ℃. The intracellular cytosolic free Ca^2＋（ [ Ca^2＋] i ） was measured as fluorescent intensity （FI） by a Wallac 1420 VictorⅡ muhilabel counter （excitation： 488 nm; emission： 535 nm）. Results： When PC12 cells were exposed to extracellular Ca^2＋ 2.0 mmol·L^-1, the FI for resting [ Ca^2＋ ]i was （1 188 ± 163） ; high K^＋（75 mmol·L^-1） and glutamate （10 mmol·L^-1） induced an increase in [ Ca^2＋ ]i with the peak values of （4270±982） and （3096±402）, respectively. Sinapine （0.01, 0.1, 1.0, 10, 100 μmol·L^-1） had no apparent effect on resting [ Ca^2＋]i, but inhibited high K^＋ and glutamate-induced increase in [ Ca^2＋]i in a concen- tration-dependent manner with the inhibiting rates of 10.2% , 39.5% , 53.7% , 71.8% , 88.4% , and 30.3% , 55.7% , 68.5% , 79.2% , 94.1% , respectively. When PC12 cells were exposed to Ca^2＋ -free solution, the FI for resting[ Ca^2＋ ]i was （813 ± 112） ; caffeine（40 mmol·L^-1） and CPA（30 μmol·L^-1 ） stimulated Ca^2＋ release from caffeine-ryanodine and inositol 1,4,5-tris-phosphate （InsP3）-sensitive internal calcium stores, inducing an increase in [Ca^2＋]ito （2944 ±371） and （1844±332）, respectively. Sinapine （0.1, 1.0, 10, 100μmol· L^-1） inhibited caffeine and CPA-stimulated intracellular calcium release in a concentration-dependent manner with the inhibiting rates of 23.7% , 61.9% , 77.5% ,88.2% , and 10.9% , 23.8% , 44.6% and 68.3% , respectively. Conclusion ： Sinapine significantly inhibits Ca^2＋ influx induced by stimulation of voltage-gated and receptor-dependent calcium channel with more potent inhibition

关 键 词：芥子碱 细胞内游离钙 PCI2细胞 Fluo-3/AM 钙阻滞剂 

分 类 号：R965.1[医药卫生—药理学] R348.1[医药卫生—药学]