大鼠心肌细胞胞浆和核Ca^(2+)震荡现象及其机制探讨

Nuclear calcium oscillation in cultured neonatal rat myocytes

目的 :在培养的新生大鼠心肌细胞上 ,观察多种刺激因素对细胞胞浆Ca2 + ([Ca2 + ]c)和核Ca2 +([Ca2 + ]n)的影响 ,探讨其时间和空间关系及其机制。方法 :以Fluo - 4 /AM荧光指示剂负载培养的心肌细胞 ,应用激光共聚焦扫描显微镜检测胞浆和核Ca2 + 震荡的变化。结果 :正常心肌细胞内核Ca2 + 的荧光强度高于核周与细胞浆 ,三者均存在小幅度的钙震荡 ,分别加入去甲肾上腺素 (1 0 - 5mol/L)、异丙肾上腺素 (1 0 - 4mol/L)和三磷酸腺苷(ATP 3× 1 0 - 3mol/L) ,均使心肌细胞钙震荡幅度明显升高 (P <0 0 1 ) ,L -型Ca2 + 通道阻滞剂维拉帕米 (verapamil 5×1 0 - 4mol/L)、Ca2 + -ATPase抑制剂thapsigargin (1 0 - 6 mol/L)和KCl (2 0mmol/L)使钙的周期性震荡消失。用thapsigar gin 1 0 - 6 mol/L预先阻断心肌细胞钙库的Ca2 + 摄取 ,去甲肾上腺素 (1 0 - 5mol/L)则不能引起钙震荡和荧光强度增加。结论 :心肌细胞胞浆和核Ca2 + 均能产生钙震荡 ,而钙震荡的维持则依赖于细胞外Ca2 + 的内流、胞内钙库的Ca2 + 摄取和释放以及正常膜电位的维持。核与胞浆Ca2 + 变化不一致 ,提示细胞核上可能存在相对独立的Ca2 + 调节系统。

AIM: To determine whether nuclear Ca 2+ is independently regulated from the cytosolic Ca 2+ and nuclear Ca 2+ oscillation induced by many modulating factors in cultured rat neonatal myocytes and its mechanism. METHODS: Rat neonatal cardiac myocytes were cultured, and fluo-4/AM was loaded as calcium probe. The changes of cytosolic and nuclear Ca 2+ were observed by confocal laser microscopy. RESULTS: Calcium fluorescent intensity oscillated slightly in myocardiocytes and the average intensity was much higher in the nucleus than that in the cytosole. Ca 2+ oscillation in nucleus and cytosole induced by norepinephrine, isoproperenol, ATP were completely blocked by Ca 2+ -ATPase antagonist thapsigargin (10 -6 mol/L),L-type Ca 2+ channel blocker verapermil (500 μmol/L) and KCl (20 mmol/L). Ca 2+ -ATPase antagonist thapsigargin completely blocked the propagation of Ca 2+ waves and simutaneouly induced a temporary Ca 2+ increase followed by a magnificient drop and loss of response to norepinephrine. CONCLUSIONS: The results suggested that generation and maintenance of calcium oscillation both in cytosole and nucleus depended on extracellular Ca 2+ influx, membrane potential, Ca 2+ release and uptake of cytosolic and nuclear calcium stores. The difference between cytosolic calcium and nuclear calcium indicated that calcium regulating system relatively independent of cytosole may exist in nucleus.