Apparent PKA activity responds to intermittent hypoxia in bone cells: a redox pathway?
Apparent PKA activity responds to intermittent hypoxia in bone cells: a redox pathway?
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表观 PKA 活性对骨细胞间歇性缺氧作出反应：氧化还原途径？
DOI：
10.1152/ajpheart.01073.2009
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Chachisvilis,Mirianas
中科院分区：
文献类型：
--
作者：
Zhang,Yan-Liang;Tavakoli,Hesam;Chachisvilis,Mirianas
我们研究了缺氧诱导的动态变化之间的平衡PKA和PKA-抵消磷酸酶在微流体环境中的单细胞使用皮秒荧光光谱和分子内荧光共振能量转移（FRET）为基础的传感器PKA活性。首先，我们发现骨细胞（MC 3 T3-E1细胞）和内皮细胞（牛主动脉内皮细胞）中PKA的表观活性受到培养基中O2水平的快速而敏感的调节。当含葡萄糖培养基中的O2浓度由于微流控室中细胞的O2消耗而降低时，表观PKA活性增加;缺氧条件下细胞的再氧合导致表观PKA活性快速（102 min）降低。第二，培养基中缺乏葡萄糖导致PKA活性降低，并且PKA活性对缺氧和复氧的反应发生逆转。第三，缺氧条件下细胞中的表观PKA活性主要通过cAMP非依赖性途径调节，因为1）使用cAMP FRET传感器未检测到细胞中cAMP水平的变化，2）cAMP水平的衰减太慢而不能解释PKA活性水平响应于再氧合的快速降低，腺苷酸环化酶抑制剂（MDL-12，330 A）对缺氧/复氧引起的PKA表观活性反应无影响。第四，在MC 3 T3-E1细胞遭受缺氧和氧化还原水平的敏感性的表观PKA活性的ROS积累的立即发作表明，在本研究中，在缺氧和复氧过程中的表观PKA活性的变化可以连接到响应于间歇性缺氧的氧化还原电位的变化，通过调节PKA-抵消磷酸酶的活性，如蛋白磷酸酶1。最后，我们的研究结果表明，PKA活性的检测可以用于真实的实时监测细胞对缺氧的反应。
We studied hypoxia-induced dynamic changes in the balance between PKA and PKA-counteracting phosphatases in the microfluidic environment in single cells using picosecond fluorescence spectroscopy and intramolecular fluorescence resonance energy transfer (FRET)-based sensors of PKA activity. First, we found that the apparent PKA activity in bone cells (MC3T3-E1 cells) and endothelial cells (bovine aortic endothelial cells) is rapidly and sensitively modulated by the level of O2in the media. When the O2concentration in the glucose-containing media was lowered due to O2consumption by the cells in the microfluidic chamber, the apparent PKA activity increases; the reoxygenation of cells under hypoxia leads to a rapid (∼2 min) decrease of the apparent PKA activity. Second, lack of glucose in the media led to a lower apparent PKA activity and to a reversal of the response of the apparent PKA activity to hypoxia and reoxygenation. Third, the apparent PKA activity in cells under hypoxia was predominantly regulated via a cAMP-independent pathway since1) changes in the cAMP level in the cells were not detected using a cAMP FRET sensor,2) the decay of cAMP levels was too slow to account for the fast decrease in PKA activity levels in response to reoxygenation, and3) the response of the apparent PKA activity due to hypoxia/reoxygenation was not affected by an adenylate cyclase inhibitior (MDL-12,330A) at 1 mM concentration. Fourth, the immediate onset of ROS accumulation in MC3T3-E1 cells subjected to hypoxia and the sensitivity of the apparent PKA acitivity to redox levels suggest that the apparent PKA activity change during hypoxia and reoxygenation in this study can be linked to a redox potential change in response to intermittent hypoxia through the regulation of activities of PKA-counteracting phosphatases such as protein phosphatase 1. Finally, our results suggest that the detection of PKA activity could be used to monitor responses of cells to hypoxia in real time.