不同因素对肺动脉平滑肌细胞线粒体游离钙的作用效应

The Changes on Mitochondrial [Ca^(2+)] of Pulmonary Artery Smooth Muscle Cells by Different Stimulation

目的:通过观察肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)在化学缺氧、细胞膜去极化和血管舒缩活性物质等不同因素作用下线粒体游离钙([Ca2+]mit)的动态变化特点,探讨:[Ca2+]mit与缺氧性肺动脉收缩(hypoxia pulmonary artery constriction,HPV)发生之间的关联。方法:分离成年Wistar大鼠PASMCs进行体外培养,负载线粒体钙荧光指示剂Rhod-5F(5μmol/L),分组施加2mmol/L连二亚硫酸钠(Na2S2O4)化学缺氧、18mmol/LKCl、10-6mol/L血管紧张素II(Ang II)和100μmol/L一氧化氮供体(SIN-1)等不同刺激因素,在激光共聚焦扫描显微镜下观察[Ca2+]mit的动态的变化。结果:用Na2S2O4对PASMCs施加缺氧刺激,[Ca2+]mit荧光强度在缺氧刺激后迅速增高,持续约30s后回降至低于起始值,随后开始回升,达到并逐渐高于起始值,继而于100s缓慢回升,逐渐高于起始时荧光强度;向PASMCs加入KCl后,[Ca2+]mit迅速升高,40s后缓慢下降,于400s后恢复并维持在初始值;向PASMCs加入AngII后,[Ca2+]mit迅速小幅度升高,于20s时开始降低,于100s时至最低,形成低谷平台,持续至400s时逐渐回升,于600s恢复接近初始水平;向PASMCs加入SIN-1后,[Ca2+]mit迅速降低,于80s时降至最低,形成低谷平台持续至160s时开始回升,于200s时回升至平稳水平但仍低于初始值。结论:给予PASMCs缺氧、细胞膜去极化、舒缩血管活性物质等刺激,可以引起线粒体Ca2+发生相应变化,线粒体[Ca2+]mit可能在HPV发生中也起着重要作用。

Objective： To explore the mechanisms of hypoxia pulmonary artery constriction （HPV） by researching the fluorescence intensity change of mitochondrial [Ca^2＋] of pulmonary artery smooth muscle cell （PASMC）. Methods. PASMCs from adult Wister rats were isolated, cultured and identified. The different stimulation was obtained by addition of 2mmol/L Na2S2O4, 18mmol/L KCI, 10-6 mol/LAngⅡ, 100μmol/L SIN--1 to the incubating media. The fluorescent intensity of cell membrane potential, cytoplasmic and mitochondrial [Ca^2＋] was measured respectively by confocal microscopy after PASMCs loaded with the fluorescent indicators Rhod--F（5μmol/L）. Results： The fluorescent intensity of mitochondrial [Ca^2＋] increased quickly after adding Na2 S2 O4, and decreased quickly after 30s, then increased gradually at 100s and higher than the beginning at last; the fluorescent intensity of mitochondrial[Ca^2＋]increased quickly after adding KCI but decreased gradually after 40s and lasted to beginning level after 400s; the fluorescent intensity of mitochondrial[Ca^2＋]increased quickly after adding AnglI then decreased at 20s and had a lower level at 100s until to 400s. This level subsequently increased and reached to the beginning value at 600s; the fluorescent intensity of mitochondrial [Ca^2＋]decreased quickly after adding SIN--1 and had a lower at 80s until 160s, but it recovered to a lower level at 200s compared with initial level. Conclusions. There is different change of the fluorescent intensity of mitochondrial [Ca^2＋] due to stimulation of hypoxia, cell membrane depolarization and diastole --contraction vasoactive substance. The results suggest that the mitochondrial [Ca^2＋] maybe play a important role in HPV.