活性氧簇介导血管紧张素Ⅱ对延髓神经元胞内游离Ca^(2+)水平的调节作用

ROS mediates regulation of intracellular Ca^(2+) induced by angiotensin Ⅱ in primarily cultured medullary neurons

目的:探讨活性氧簇(reactive oxygen species,ROS)介导血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对延髓神经元胞内游离Ca^(2+)的调节作用及其机制。方法:原代培养延髓神经元;免疫荧光双标法鉴定培养神经元的特征;给予Ang Ⅱ处理后,用二氢乙啶荧光探针法测定神经元ROS水平;同时或单独给予Ang Ⅱ和NADPH氧化酶抑制剂apocynin或自由基清除剂TEMPOL后,Fura-2/AM钙瞬变法记录神经元胞内游离Ca^(2+)的水平;CCK-8法检测神经元活性。结果:原代培养的延髓神经元多数为谷氨酸阳性的神经元;Ang Ⅱ(5μmol/L)可在10 min内显著升高神经元ROS水平(P<0.01);给予Ang Ⅱ处理后延髓神经元胞内Ca^(2+)水平显著升高(P<0.01);给予apocynin/TEMPOL预处理后,Ang Ⅱ引起的延髓神经元胞内Ca^(2+)的升高则被抑制(P<0.05)。实验浓度的Ang Ⅱ对神经元无毒性作用。结论:ROS介导Ang Ⅱ诱导的延髓神经元胞内Ca^(2+)的升高作用,可能是Ang Ⅱ在中枢诱导氧化应激作用的潜在细胞内信号机制。

AIM：To investigate the role of reactive oxygen species（ ROS） in the regulation of intracellular Ca^2＋induced by angiotensin Ⅱ（ Ang Ⅱ） in the primarily cultured medullary neurons.METHODS：Primarily cultured medullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study.The identification of medullary neurons was assessed by double-labeling immunofluorescence.To explore the role of ROS,mainly the superoxide（ O_2--·）,the O_2--·generation was measured using the fluorogenic probe dihydroethidium（ DHE）.To determine intracellular free calcium concentration（ [Ca^2＋]i）,the neurons were loaded with the Ca^2＋-specific dye Fura-2/AM.The cell viability after adding Ang Ⅱ was also examined using CCK-8 assay.RESULTS：Most of the cultured cells were medullary neurons,more than 80% of which were glutamate positive neurons.Ang Ⅱ（ 5 μmol/L） increased the level of ROS within 10 min in the medullary neurons.Ang Ⅱ at 5 μmol/L induced a significant [Ca^2＋]iincrease in the medullary neurons,and the effect of Ang Ⅱ occurred rapidly and reached a peak within 20 min after administration.The level of [Ca^2＋]istarted to decline after washout.The Ca^2＋elevation induced by Ang Ⅱ was significantly decreased by apocynin or TEMPOL.No significant difference in the cell viability between control group and 5 μmol/L Ang Ⅱ treatment group was observed.CONCLUSION：ROS is involved in the regulation of [Ca^2＋]iinduced by Ang Ⅱ in the primarily cultured medullary neurons,suggesting a potential intracellular signaling mechanism involved in the Ang Ⅱ-mediated oxidant regulation of central neural control of blood pressure.