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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 observe the effects of Ang Ⅱ and NO on the membrane potential, cytoplasmic calcium concentration ([Ca2 +] i) and mitochondrial calcium concentration ([Ca2 +] mit) in pulmonary artery smooth muscle cells. Methods: Pulmonary artery smooth muscle cells (PASMCs) were loaded with fluorescent probes: Fluo-4 / AM, Di-8-ANEPPS, (Rhod-5F), and then given a certain concentration of angiotensin Ⅱ (Ang Ⅱ) and nitric oxide (NO), measured by confocal laser scanning microscopy [Ca 2+] i fluorescence, cell membrane potential, Ca2 +] mit fluorescence. Results: AngⅡ basically did not affect the cell membrane potential, but caused a transient increase of [Ca2 +] i. The increase of [Ca2 +] i resulted in a slight increase of [Ca2 +] mit and then the release of mitochondrial Ca2 + Ca2 +] mit significantly reduced; NO can rapidly weaken the PASMCs membrane potential fluorescence intensity, the cells in a marked hyperpolarization state, while [Ca2] i decreased rapidly to reach the minimum 300s, and maintained at this level, Ca2 +] mit also rapidly decreased, forming a trough platform quickly rebounded, and basically stable at, slightly below the initial level. CONCLUSIONS: Ang II contraction may be related to mitochondrial calcium release and [Ca2 +] mit reduction. NO relaxation may be related to hyperpolarization, decrease of [Ca2 +] i and [Ca2 +] mit.