钙释放通道放射配基分析法的建立
心肌|肌浆网|钙释放通道|放射配位体测定,关键词：
关键词：心肌；肌浆网；钙释放通道；放射配位体测定
【摘要】 目的 用心肌匀浆建立心肌肌浆网钙释放通道检测方法,并观察其在高血压中的变化。方法 用3 H标记兰尼定(ryanodine),探索在大鼠心肌匀浆中建立钙释放通道放射配基分析的最佳条件,并对自发性高血压大鼠(SHR)左室心肌钙释放通道进行饱和实验。 结果 ①保温时间在0～60分钟内,3 H-ryanodine与心肌钙释放通道结合量迅速增加,90分钟达平衡;匀浆蛋白在0～400μg,3 H-ryanodine结合量与蛋白质浓度呈直线关系;反应介质中游离钙浓度直接影响3 H-ryanodine的结合量。②SHR及正常对照组(WKY)标准饱和曲线及Scatchard作图显示,3 H-ryanodine的特异性结合高,呈单位点结合等特点,SHR组最大结合量(Bmax )值明显高于WKY组(P<0.01) 。结论 用3 H-ryanodine可在心肌匀浆中进行钙释放通道放射配基分析,且发现3 H-ryanodine Bmax 升高与高血压左室肥厚密切相关。
Radioligand assayof cardiac calcium release channel and its application in SHR
Zhu Baohua, ZhangJinan, Ma Wenzhu, et al. Department of Cardiology, The First Affiliated Hospital ofNanjing Medical University, Nanjing, 210029
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Abstract】 Purpose To establish the best conditionin assaying the calcium release channel (ryanodine receptor) in cardiac sarcoplasmicreticulum (CSR), and analyse the CSR ryanodine receptor in spantanous hypertensive rat(SHR). Methods 3 H-ryanodine was used as a radioligand to anylyse the binding in Sprague Dawleyrat cardiac homogenate in following conditions: varied protein concentrations, differentfree calcium concentrations, different incubation time. The effect of sarcoplasmicreticulum purifying process and ryanodine competitive binding were also studied. Usingthese best conditions, SHR and contral group (WKY) CSR ryanodine receptor were studied. Results ①There was a positive linear correlation between 3 H-ryanodinebinding and the homogenate protein concentraton. ②When the free calcium concentration was 30μ mol/L～ 1mmol/L, the 3 H-ryanodine binding reached themaximum. While the free calcium concentration was lower than lμ mol/L, there was no 3 H-ryanodine binding. ③The 3 H-ryanodinebinding kept increasing during incubation, from 0 to 60 min, and equilibrium reached by 90min. ④The ryanodinespecifically inhibited 3 H-ryanodine binding in cardiac homogenate. ⑤During the sarcoplasmic reticulumpurifying process, the 3 H-ryanodine binding in a unit amount of cardiachomogenate decreased with the centrifugal force and times applied in centrifugation. ⑥SHR and WKY CSR ryanodinereceptor saturation curve and Scatchard analysis showed this method produced a very highlevel of specific binding, up to 45nmol/L ryanodine, which infered a single class ofbinding sites. The Bmax value of CSR ryanodine receptor in SHR left ventriclewas siginificantly higher than that in WKY(P<0.01), but there is no difference betweentwo Kd values. Conclusions 3 H-ryanodine can be used as a radioligandto analyse the calcium release channel in cardiac homogenate, and ryanodine receptor mayplay an important role in hypertensive left ventricular remodeling process.......
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