钙释放通道放射配基分析法的建立

Radioligand assay of cardiac calcium release channel and its application in SHR

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３Ｈ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，３Ｈ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，３Ｈ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３Ｈ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，３Ｈ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３Ｈｒｙａｎｏｄｉｎｅ可在心肌匀浆中进行钙释放通道放射配基分析，且发现３ＨｒｙａｎｏｄｉｎｅＢｍａｘ升高与高血压左室肥厚密切相关。

Purpose To establish the best condition in assaying the calcium release channel (ryanodine receptor) in cardiac sarcoplasmic reticulum (CSR), and analyse the CSR ryanodine receptor in spantanous hypertensive rat (SHR) Methods 3H ryanodine was used as a radioligand to anylyse the binding in Sprague Dawley rat cardiac homogenate in following conditions: varied protein concentrations, different free calcium concentrations, different incubation time The effect of sarcoplasmic reticulum purifying process and ryanodine competitive binding were also studied Using these best conditions, SHR and contral group (WKY) CSR ryanodine receptor were studied Results ①There was a positive linear correlation between 3H ryanodine binding and the homogenate protein concentraton ②When the free calcium concentration was 30μmol/L～1mmol/L, the 3H ryanodine binding reached the maximum While the free calcium concentration was lower than lμmol/L, there was no 3H ryanodine binding ③The 3H ryanodine binding kept increasing during incubation, from 0 to 60 min, and equilibrium reached by 90 min ④The ryanodine specifically inhibited 3H ryanodine binding in cardiac homogenate ⑤During the sarcoplasmic reticulum purifying process, the 3H ryanodine binding in a unit amount of cardiac homogenate decreased with the centrifugal force and times applied in centrifugation ⑥SHR and WKY CSR ryanodine receptor saturation curve and Scatchard analysis showed this method produced a very high level of specific binding, up to 45nmol/L ryanodine, which infered a single class of binding sites The B max value of CSR ryanodine receptor in SHR left ventricle was siginificantly higher than that in WKY(P<0 01), but there is no difference between two Kd values Conclusions 3H ryanodine can be used as a radioligand to analyse the calcium release channel in cardiac homogenate, and ryanodine receptor may play an important role in hypertensive left ventricular remodeling process