心肌细胞核Ca^(2+)库特点及其调节的离体研究

INVESTIGATION OF NUCLEAR Ca2+ REGULATION IN THE ISOLATED CARDIAC NUCLEI

研究核外Ca^(2+)浓度对核Ca^(2+)的影响,及细胞核Ca^(2+)摄取和释放的关系,以探讨核Ca^(2+)转运的调节机制。采用差速离心和密度梯度离心法分离纯化心肌细胞核,以Fluo-4/AM荧光指示剂负载心肌细胞核,应用激光共聚焦扫描显微镜和荧光分光光度计进行观察和测定。结果显示,分离纯化的成年大鼠心肌细胞核内自由[Ca^(2+)]随着核外[Ca^(2+)]的增加而逐渐增加,孵育液[Ca^(2+)]为1000 nmol/L达高峰,但二者增加的程度并不一致,之后随核外[Ca^(2+)]浓度的增加而呈降低趋势。ATP和100—600nmol/L的核外游离Ca^(2+),使心肌细胞核显示核被膜腔Ca^(2+)荧光,ATP和1000nmol/L的核外游离Ca^(2+)则进一步引起核浆内的Ca^(2+)荧光强度升高。荧光染色观察可见IP_3受体染色主要位于核内膜,而钙泵和ryanodine受体染色主要位于核外膜。IP_3和Ryancodine使核Ca^(2+)短暂升高1.68倍和1.93倍(P<0.001),而钙泵抑制剂Thapsigargin和IP_3受体抑制剂Heparin则分别使核Ca^(2+)降低64%和35.6%(p<0.05)。ryanodine使IP_3升高的核Ca^(2+)显著回落至正常水平以下(p<0.001)。Thapsigargin不能阻断IP_3和Ryanodine所致的核Ca^(2+)释放增加(p<0.05),但事先采用钙泵抑制剂Thapsigargin预处理心肌细胞核,则能显著的阻断IP_3和Ryanodine所致的核Ca^(2+)升高作用(Ca^(2+)释放作用)(p<0.05)。结果提示大鼠心肌细胞核可能也是细胞内的钙库之一,心肌细胞核上存在Ca^(2+)-ATPase、ryanodine受体和IP_3受体等Ca^(2+)转运系统,可能参与核Ca^(2+)摄取和释放的调节。

To investigate the regulation of Ca2+ in the isolated cardiac nuclei from rats which may illuminated the mechanism of nuclear calcium transport system. Elocity and isopyknic gradient centrifugation were employed to fractionate rat cardiac nuclei. Then fluo-4 confocal microscopy techniques was used to verify the changes of nuclear Ca2+ . There are calcium-dependent Ca2+ uptake in the cardiac nuclear obtained from normal rats, The accumulation Ca2 + of cardiac nuclei in vitro from the incubating medium were not consistent with free [Ca2 + ] in incubating medium. The nuclear envelope was initially loaded with Ca + (1 mmol/L ATP and approximately 100 nmol/L Ca2+ ), Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope and nucleoplasm. Exposure of Ca2 +-loaded nuclei to IP3, ryanodine or ryanodine+ thapsigargin, respectively, resulted in a rapid and transient elevation of nucleoplasmic Ca 2+ free concentration, this effects were abolished by pretreatment of cardiac nuclei with Ca2+ -ATPase inhibitor thapsigargin. Thapsigargin and IP3 receptor antagonist heparin induced nucleoplasmic Ca 2+ free concentration decrease. Fluorescence experiments indicated that both ryanodine receptors and Ca2+ -ATPase were distributed in the outer layer of nuclear envelope, and in-ositol 1,4,5-trisphosphate receptors mainly dispersively localized at inner layer of nuclear envelope. The present study demonstrates that nuclear calcium were regulated by free Ca2+ , IP3 and ryanodine, The results suggested calcium transport system might be present in the myocardial nuclei, the myocardial nuclei might served as one of calcium pools in myocardial cell.