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首页> 外文期刊>American Journal of Physiology >Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2.
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Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2.

机译：用Rhod 2荧光测量兔心脏灌注钙的瞬变。

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摘要

Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators. Ultraviolet light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wave-length fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techniques to account for changes in cytosolic dye concentration. We have developed a method for in vivo quantitation of cytosolic rhod 2 concentration that in conjunction with calcium-dependent fluorescence measurements permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded into myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calcium concentration. Endothelial cell loading of rhod 2 was found to be minimal (< 5%), and dye leak rate out of the cytosol was slow, with approximately 5% loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, determined by manganese quenching, was also minimal (< 5%). The dissociation constant of rhod 2 for calcium was measured in vitro to be 500 nM, and this value increased to 710 nM in the presence of 0.5 mM myoglobin. On the basis of this value and in vivo fluorescence measurements, cytosolic calcium concentration in the rabbit heart was found to be 229 +/- 90 nM at end diastole and 930 +/- 130 nM at peak systole, with peak fluorescence preceding peak ventricular pressure by approximately 40 ms. This technique should facilitate detailed analysis of calcium transients from the whole heart.
机译：由于许多常用的钙指示剂都需要使用紫外线激发光，因此跳动心脏的表面荧光光谱法测量胞质钙受到了限制。心脏中的紫外线会产生高水平的背景荧光，并且会被高度吸收，从而限制了组织的渗透。可以使用可见光波长的荧光染料，例如rhod 2。然而，由于缺乏钙结合引起的光谱移动，因此无法使用比例技术来解释胞浆染料浓度的变化。我们已经开发出一种用于体内定量胞浆Rhod 2浓度的方法，该方法与钙依赖性荧光测量相结合，可以估算灌注兔心脏中胞浆钙的水平。负载在心肌上的rhod 2激发光的反射吸收率用作染料浓度的指标，荧光强度与吸收率的比值用作衡量胞质钙浓度的指标。发现rhod 2的内皮细胞负载极小（<5％），染料从胞质溶胶中泄漏的速度很慢，在染料加载后10到30分钟之间发生了约5％的染料荧光损失。通过锰淬灭法测定，Rhod 2进入亚细胞区室的装载量也很小（<5％）。在体外测量，rhod 2对钙的解离常数为500 nM，在存在0.5 mM肌红蛋白的情况下，该值增加至710 nM。根据该值和体内荧光测量结果，发现兔心脏舒张末期的胞质钙浓度为229 +/- 90 nM，收缩期峰值为930 +/- 130 nM，荧光峰值在心室压之前大约40毫秒此技术应有助于详细分析整个心脏的钙瞬变。

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