摘要
目的:探讨牛磺胆酸钠对小鼠胰腺腺泡细胞氯通道的激活作用及途径。方法:胶原蛋白酶消化分离FVB/N小鼠胰腺腺泡细胞,采用膜片钳技术记录牛磺胆酸钠激活的全细胞电流;去除电极内液配方中的三磷酸腺苷(ATP),观察其胞内ATP依赖性;阴离子置换实验分析其阴离子选择性;利用氯通道阻断剂检测其药理学特性;采用47%高渗灌流液观察其容积敏感性;清除胞外等渗灌流液中的Ca^(2+)分析通道激活对胞外Ca^(2+)的依赖性;使用透膜荧光探针2',7'-二氯荧光黄双乙酸盐检测胞内活性氧(ROS)水平。结果:5 mmol/L牛磺胆酸钠可激活小鼠胰腺腺泡细胞产生电流,该电流呈外向整流优势,对细胞内液的ATP具有依赖性,其阴离子通透性顺序为:I->Br^(−)≥Cl^(−)>gluconate-(P<0.01);该电流能被氯通道阻断剂4,4'-二异硫氰酸十八烯-2,2'-二磺酸二钠盐水合物(DIDS)和tamoxifen抑制,且可被细胞外47%高渗刺激阻抑(P<0.01)。N-乙酰-L-半胱氨酸清除细胞内ROS不能阻断牛磺胆酸钠诱导胰腺腺泡细胞产生氯电流;氯通道阻断剂DIDS不影响牛磺胆酸钠诱导的ROS生成。清除细胞外Ca^(2+)后,牛磺胆酸钠未能诱导胰腺腺泡细胞产生氯电流。结论:牛磺胆酸钠可激活小鼠胰腺腺泡细胞氯通道,该通道的激活对胞外Ca^(2+)具有依赖性,但不依赖于ROS途径。
AIM:To investigate the activation of Cl-channels by sodium taurocholate(NaTC)in mouse pancreatic acinar cells.METHODS:The single isolated pancreatic acinar cells from FVB/N mice were prepared using collagenase digestion method.Whole-cell patch clamp technique was performed to record the currents.Intracellular adenosine triphosphate(ATP)dependence of the channels was examined via eliminating ATP from the pipette solution.Anion permeability of the channels was investigated with ion-exchange method.The pharmacological characteristics of the channels was confirmed by two Cl^(−)channel blockers.The volume sensitivity of the channels was detected using 47%hypertonic bathing solution.Extracellular Ca^(2+)dependence of activating the channels was examined through eliminating Ca^(2+)from the bathing solution.Intracellular reactive oxygen species(ROS)level was detected by an oxidation-sensitive fluorescent probe,2´,7´-dichlorofluorescin diacetate.The experiment was repeated 6 times in each group.RESULTS:Extracellular application of 5 mmol/L sodium taurocholate induced a Cl-current,exhibiting the properties of outward-rectification,a selectivity sequence of I->Br^(−)≥Cl->gluconate-and intracellularATP dependence(P<0.01).The currents were inhibited by chloride channel blocker 4,4´-diisothiocyanatostilbene-2,2´-disulfonic acid disodium salt hydrate(DIDS)and tamoxifen and by 47%hypertonicity stimulation(P<0.01).When ROS production was scavenged by N-acetyl-L-cysteine,the sodium taurocholate-induced Cl-currents were unaffected.The effect of sodium taurocholate on ROS production did not alter with the treatment with DIDS.Sodium taurocholate failed to induce Cl^(−)currents when Ca^(2+)was absent in extracellular bathing solution(P>0.05).CONCLUSION:Sodium taurocholate activates Cl^(−)channels in mouse pancreatic acinar cells,which is dependent on extracellular Ca^(2+)but not ROS pathway.
作者
阳小雅
林嘉伟
叶东
赵婵
王立伟
陈丽新
YANG Xiaoya;LIN Jiawei;YE Dong;ZHAO Chan;WANG Liwei;CHEN Lixin(Department of Physiology,Guangzhou Health Science College,Guangzhou 510450,China;Department of Breast Surgery,The First People's Hospital of Foshan,Foshan 528000,China;School of Basic Medical Sciences,Guangdong Pharmaceutical University,Guangzhou 510006,China;Department of Biology,Zhangjiajie Minzu Secondary School,Zhangjiajie 427000,China;School of Medicine,Jinan University,Guangzhou 510632,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2024年第10期1806-1814,共9页
Chinese Journal of Pathophysiology
基金
2022年广州市教育局高校科研项目(No.202235423)
广东省科技计划项目-对外科技合作项目(No.2017A050501021)
2022年度广州市高等教育教学质量与教学改革工程项目(No.2022JXMS029)。
关键词
牛磺胆酸钠
胰腺腺泡细胞
氯离子通道
钙离子
活性氧
sodium taurocholate
pancreatic acinar cells
chloride channel
calcium
rreactive oxygen species
