再灌注损伤大鼠心肌细胞核3H-Ryanodine结合特性的变化
再灌注损伤大鼠心肌细胞核3H-RyanoDInE结合特性的变化,ChaRaCTERIsTICsInThEFEaTuREoF3H-RyanoDInEBInDInGToCaRDIomyoCyTICnuClEIInREPERFusIonInjuRyInRaT
唐维平 司良毅 张乐之 周红 何华美【摘要】 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。
【关键词】 缺血-再灌注损伤，心肌； 细胞核； 兰尼定受体
Characteristics in the feature of 3H-Ryanodine binding to cardiomyocytic nuclei in reperfusion injury in rat TANG Wei-ping, SI Liang-yi, ZHANG Le-zhi, ZHOU Hong, HE Hua-mei,Department of Pharmacology, College of Fundamental Medical Sciences, Third Military Medical University, Chongqing 400038, China
OBJECTIVE: To investigate the changes in binding features of 3H -Ryanodine 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 sham-operation 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 sham-operation 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 (Kd) 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 3H-Ryanodine on the nuclear wall of rat cardiomyocytic nucleus. Compared with that of the sham-operation group, Bmax of RyRs of IRI cardiomyocytic nuclei was decreased by 29% (P<0.01), but there was no difference in Kd 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 Ca2+-calmodulin (CaM), Bmax was decreased in both sham-operation and IRI groups (both P<0.05), but was less decreased in the latter (P<0.01). However, both PMA+PS and Ca2+-CaM did not change the Kd of nuclear RyR in either group (both P>0.05). CONCULSION: After IRI, Bmax of 3H -Ryanodine of cardiomyocytic nuclei is decreased and the impact of PMA+PS and Ca2+-CaM on the Bmax is impaired, but the affinity of 3H -Ryanodine to cardiomyocytic nuclei is not altered under above circumstances. ......
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