大鼠心肌缺血再灌注时心肌细胞核1,3,4,5-四磷酸肌醇受体的结合特性改变

Change of cardiac myocyte nuclear inositol 1,3,4,5-tetrakisphosphate receptor binding proterties in rat with myocardial ischemic reperfusion

目的 观察大鼠心肌缺血再灌注时心肌细胞核 1, 3, 4, 5 四磷酸肌醇受体 (IP4R)的结合特性改变,进一步探索心肌缺血再灌注损伤时细胞凋亡的病理机制。方法 TUNEL技术检测心肌细胞核凋亡率。蔗糖密度梯度离心法提纯心肌细胞核,放射配体分析法检测心肌细胞核膜上IP4R的结合特性变化。结果 心肌缺血再灌注组大鼠心肌细胞凋亡率显著高于对照组 (P<0 01)。大鼠心肌细胞核膜上存在两个与IP4 配体结合容量不同、亲和力不同的单位点结合受体。心肌缺血再灌注组心肌细胞核膜上IP4R两结合位点解离常数值较对照组分别降低 63%及 55% (P<0 05),且高亲和位点IP4R受体密度(Bmax)显著高于对照组 (P<0 05 ),而低亲和位点IP4R的Bmax无显著改变。两组心肌细胞核经蛋白激酶C磷酸化后,IP4R的结合特性显著增高 (P<0 05),以心肌缺血再灌注组效应更明显。对照组大鼠心肌细胞核IP4R的结合特性对核外钙离子是浓度依赖性的,心肌缺血再灌注组大鼠在钙超载情况下,结合力也显著性增强。结论 大鼠心肌缺血再灌注病理情况下心肌细胞核IP4R结合特性显著增强,致核内钙浓度增高,这可能是心肌缺血再灌注损伤中心肌细胞凋亡的主要病理机制之一。

Objective To observe the alteration of cardiac myocyte nuclear inositol 1,3,4,5 tetrakisphosphate receptor (IP 4R)binding proterties in rat subjected to myocardial ischemic reperfusion in order to further make it clear whether this change is involved in the molecule mechanism of cell apoptosis of rat with myocardial ischemic reperfusion Methods Extracting of cardiac myocyte nucleus was accomplished by saccharose density gradient centrifugation method, the binding proterties of nuclear IP 4R in different conditions were detected by radioligand binding assay Apoptosis index of myocardial cell was determined by using TUNEL assay Results (1)Myocardial cell apoptosis index in rat heart underwent 30 min regional ischemia and 3 h reperfusion increased distinctly compared with that in control group( P <0 01) (2)There were two IP 4 binding sites located to the nuclear envelope (3)In ischemic reperfusion injury (IRI) group, Bmax from high affinity binding site of nuclear IP 4R significantly increased compared with that in sham operated group, whereas Bmax from low affinity binding site didn′t change Kd values of both sites were all significantly decreased by 63% and 55%, respectively (4)Phosphorylation of nuclear IP 4R by PKC increased markedly its binding ability both in IRI and control group( P <0 05), which was more apparent in IRI group (5)In sham operated group, the binding ability of nuclear IP 4R increased with increasing free calcium concentrations in cytoplasm, and the binding proterties of IP 4R in IRI group were also increased in the condition of calcium overloading Conclusion The increasing of binding proterties of nuclear IP 4R from ischemic reperfusion heart may be one of important mechanism involved in myocardial cell apoptosis, furthermore resulting in myocardial IRI