再灌注损伤大鼠心肌细胞核3H-Ryanodine结合特性的变化

目的观察缺血-再灌注损伤(IRI)心肌细胞核兰尼定受体(RyR)结合特性改变.方法雄性Wistar大鼠随机分为IRI组和假手术(Sham)组,结扎冠状动脉左主干建立IRI模型,3H-Ryanodine饱和结合法分析分离的心肌细胞核上RyR最大结合容量(Bmax)和解离常数(Kd).结果大鼠心肌细胞核存在高亲和力的RyR,IRI组较Sham组Bmax降低29%(P<0.01),Kd差异无显著性(P>0.05).IRI组和Sham组心肌细胞核经佛波酯(PMA)+磷脂酰丝氨酸(PS)处理后,Bmax均显著增加(P均<0.01),但IRI组的增加幅度较小(P<0.01);IRI组和Sham组心肌细胞核经Ca2+-钙调素(CaM)处理后,Bmax均降低(P均<0.05),但IRI组降低幅度较小(P<0.01);上述处理对两组Kd均无显著影响(P均>0.05).结论 IRI心肌细胞核RyR的Bmax呈降低趋势;经PMA+PS和Ca2+CaM处理后,Bmax的改变削弱,而Kd无改变.

Changes in the feature of 3H-Ryanodine binding to cardiomyocyte nuclei in reperfusion injury in rat

Objective To investigate the changes in binding features of 3HRyanodine cardiomyocytic nuclei in reperfusion injury in the rat, and the effects of phosphorylation regulation on the binding characteristics. Methods Healthy male Wistar rats were randomly divided into ischemia/reperfusion injury(IRI) group and shamoperation group. IRI model was reproduced by ligating the left main coronary artery for 30 minutes followed by reperfusion of the heart for 3 hours, while in the shamoperation group the animals underwent a thoracotomy only for 3 5 hours. Cardiomyocytic nuclei were isolated by sucrose density gradient centrifugation. The maximum binding capacity (Bmax) and the dissociating ratio ( K d) of Ryanodine receptors(RyRs) were measured by radioligand binding analysis as well as Scatchard plot. Results There was a high affinity of RyRs to bind with 3HRyanodine on the nuclear wall of rat cardiomyocytic nucleus. Compared with that of the shamoperation group, Bmax of RyRs of IRI cardiomyocytic nuclei was decreased by 29%( P <0 01), but there was no difference in K d between two groups( P >0 05). With phosphorylation by activating the endogenous protein kinase C(PKC) with phorbol myristate acetate (PMA)+phosphatidyl serine(PS), Bmax of both sham and IRI groups were increased markedly ( P <0 01), but in the latter group it was less increased( P <0 01). With phosphorylation by Ca 2+ calmodulin (CaM), Bmax was decreased in both shamoperation and IRI groups(both P <0 05), but was less decreased in the latter( P <0 01). However, both PMA+PS and Ca 2+ CaM did not change the K d of nuclear RyR in either group(both P >0 05). Conclusion After IRI, Bmax of 3HRyanodine of cardiomyocytic nuclei is decreased and the impact of PMA+PS and Ca 2+ CaM on the Bmax is impaired, but the affinity of 3 HRyanodine to cardiomyocytic nuclei is not altered under above circumstances.