舒缩血管物质对肺动脉平滑肌细胞膜电位、胞浆及线粒体Ca^(2+)的作用效应

The Effects ofsystolic and Diastolic Vasoactivesubstances on Pulmonary Arterialsmooth Muscle Cell Membrane Potential,Cytosolic and Mitochondrial Ca^(2+) Concentration

目的:观察血管紧张素II(AngⅡ)和一氧化氮(NO)对肺动脉平滑肌细胞膜电位、胞浆钙离子浓度([Ca2+]i)和线粒体钙离子浓度([Ca2+]mit)的影响。方法:肺动脉平滑肌细胞(pulmona-ry arterysmooth muscle cells,PASMCs)分别负载相应的荧光探针:胞浆钙荧光指示剂(Fluo-4/AM)、细胞膜荧光指示剂(Di-8-ANEPPS)、细胞线粒体钙荧光指示剂(Rhod-5F),然后给予一定浓度的血管紧张素Ⅱ(AngⅡ)和一氧化氮(NO),在激光共聚焦扫描显微镜下,测定[Ca2+]i荧光、细胞膜电位、[Ca2+]mit荧光。结果:AngⅡ基本不影响细胞膜电位,但可一过性引起[Ca2+]i升高,这种[Ca2+]i的升高可导致[Ca2+]mit小幅升高,进而启动线粒体钙离子的释放和[Ca2+]mit的大幅度降低;NO可迅速减弱PASMCs膜电位荧光强度,使细胞处于明显的超极化状态,同时,[Ca2+]i迅速下降,300s时达到最低,并维持在这一水平,[Ca2+]mit也迅速降低,形成低谷平台后迅速回升,并基本稳定于、稍低于初始值的水平。结论:AngⅡ收缩血管可能与线粒体钙释放和[Ca2+]mit的大幅降低有关;NO舒张血管可能与细胞超极化、[Ca2+]i、[Ca2+]mit降低有关。

Objective:To explore the affection of Angiotensin Ⅱ and Nitric oxide on calcium level of mitochondria by observing the change of pulmonary arterialsmooth muscle cells(PASMCs)membrane potential,cytosolic free calcium and mitochondrial free calcium.Methods:Loading the cytosolic calcium fluorescent indicator Fluo-4/AM,membrane fluorescent indicator Di-8-ANEPPS and mitochondrial calcium fluorescent indicator Rhod-5F on the different cells group.After incubating in the Carbon dioxide incubator about 30 minutes,the pulmonary arterysmooth muscle cells membrane potential,cytosolic free calcium and mitochondrial free calcium were tested under AngiotensinⅡ and Nitric oxide.Results:The cells membrane potential were increased generally after 80s with Angiotensin and reached a peak at 160s,then decreased lower than the beginning;the cytosolic free calcium increased quickly and reached a peak at 50s then decreased andstained at the beginning;the mitochondrial calciumshowed exaltation,reduced and rebound.The cells membrane potential decreasedsharply at 20s with NO reached and maintained the lowest at 200s;the cytosolic free calcium decreasedsharply at 60s,reached and maintained the lowest at 300s;the mitochondrial calcium decreasedsharply,reached and maintained the lowest at 80s,then rebounded at 160s untilstabled at 200s,the fluorescent were lower than the beginning.Conclusions:AngⅡ did not affect the membrane potential,but it can rise cytoplasmic level,and increased the mitslightly then the mitochondrial calcium release and mit decreased through the calcium-induced calcium release process(CICR) mechanisms.