Development of an image-based system for measurement of membrane potential, intracellular Ca(2+) and contraction in arteriolar smooth muscle cells.
Development of an image-based system for measurement of membrane potential, intracellular Ca(2+) and contraction in arteriolar smooth muscle cells.
复制标题
开发基于图像的系统，用于测量膜电位、细胞内 Ca(2) 和小动脉平滑肌细胞的收缩。
DOI：
10.1111/j.1549-8719.2010.00059.x
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Hill,MichaelA
中科院分区：
文献类型：
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作者：
Ella,SrikanthR;Yang,Yan;Clifford,PhilipS;Gulia,Jyoti;Dora,KimA;Meininger,GeraldA;Davis,MichaelJ;Hill,MichaelA
请将本文引用为：Ella，Yang，Clifford，Gulia，Dora，Meininger，Davis and Hill（2010）。基于图像的系统的开发，用于测量小动脉平滑肌细胞的膜电位、细胞内Ca 2+和收缩。微循环17（8），629- 640。摘要目的：平滑肌细胞（SMC）膜电位（Em）的变化对于血管舒缩反应至关重要。由于荧光指示剂方法可以减少玻璃电极在收缩制剂中的限制，我们的目标是开发基于Forster（或荧光）共振能量转移（FRET）的Em.Methods测量：本研究中使用的FRET对（供体CC 2-DMPE [激发405 nm]和受体DisBAC 4（3））提供快速测量，其灵敏度是许多比率指示剂无法实现的。 该方法还结合了使用fluo-4和490 nm激发来测量Ca 2+的变化。结果：建立加载条件后，在电压钳下固定的FRET染料负载的HEK细胞中，Em与荧光信号之间呈线性关系。 在−70至+30 mV的电压范围内，斜率（FRET信号对电压，m）= 0.49 ± 0.07，r2= 0.96 ± 0.025。 在脑动脉SMC中获得了类似的数据，斜率（m）= 0.30 ± 0.02，r2= 0.98 ± 0.02。 在-70 mV至+30 mV的保持电位下，HEK细胞的FRET发射率变化为41.7 ± 4.9%，动脉SMC为30.0±2.3%。 FRET信号也显示出以浓度依赖性方式被KCl诱导的去极化调制。此外，在离体动脉SMC中，KCl诱导的去极化（60 mM）测量发生增加的fluo-4荧光发射（62 ± 9%）和收缩（-27 ± 4.2%）。结论：数据支持基于FRET的方法测量动脉SMC中Em的变化。 此外，Em的基于图像的测量可以与Ca 2 + i和收缩的时间变化的分析相结合。
Please cite this paper as:Ella, Yang, Clifford, Gulia, Dora, Meininger, Davis and Hill (2010). Development of an Image‐Based System for Measurement of Membrane Potential, Intracellular Ca2+and Contraction in Arteriolar Smooth Muscle Cells.Microcirculation17(8), 629–640.AbstractObjective:Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)‐based measurement for Em.Methods:The FRET pair used in this study (donor CC2‐DMPE [excitation 405 nm] and acceptor DisBAC4(3)) provide rapid measurements at a sensitivity not achievable with many ratiometric indicators. The method also combined measurement of changes in Ca2+iusing fluo‐4 and excitation at 490 nm.Results:After establishing loading conditions, a linear relationship was demonstrated between Em and fluorescence signal in FRET dye‐loaded HEK cells held under voltage clamp. Over the voltage range from −70 to +30 mV, slope (of FRET signal vs. voltage, m) = 0.49 ± 0.07, r2= 0.96 ± 0.025. Similar data were obtained in cerebral artery SMCs, slope (m) = 0.30 ± 0.02, r2= 0.98 ± 0.02. Change in FRET emission ratio over the holding potential of −70 to +30 mV was 41.7 ± 4.9% for HEK cells and 30.0 ± 2.3% for arterial SMCs. The FRET signal was also shown to be modulated by KCl‐induced depolarization in a concentration‐dependent manner. Further, in isolated arterial SMCs, KCl‐induced depolarization (60 mM) measurements occurred with increased fluo‐4 fluorescence emission (62 ± 9%) and contraction (−27 ± 4.2%).Conclusions:The data support the FRET‐based approach for measuring changes in Em in arterial SMCs. Further, image‐based measurements of Em can be combined with analysis of temporal changes in Ca2+iand contraction.