大鼠心肌细胞胞浆和核Ca2+震荡现象及其机制探讨

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(10^-5mol/L)、异丙肾上腺素(10^-4mol/L)和三磷酸腺苷(ATP3×10^-3mol/L),均使心肌细胞钙震荡幅度明显升高(P＜0.01),L-型Ca²⁺通道阻滞剂维拉帕米(verapamil5×10^-4mol/L)、Ca²⁺-ATPase抑制剂thapsigargin(10^-6mol/L)和KCl(20mmol/L)使钙的周期性震荡消失。用thapsigargin10^-6mol/L预先阻断心肌细胞钙库的Ca²⁺摄取,去甲肾上腺素(10^-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。

Nuclear calcium oscillation in cultured neonatal rat myocytes

AIM: To determine whether nuclear Ca²⁺ is independently regulated from the cytosolic Ca²⁺ and nuclear Ca²⁺ 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 Ca²⁺ 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. Ca²⁺ oscillation in nucleus and cytosole induced by norepinephrine, isoproperenol, ATP were completely blocked by Ca²⁺-ATPase antagonist thapsigargin (10^-6 mol/L),L-type Ca²⁺ channel blocker verapermil (500 μmol/L) and KCl (20 mmol/L). Ca²⁺-ATPase antagonist thapsigargin completely blocked the propagation of Ca²⁺ waves and simutaneouly induced a temporary Ca²⁺ 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 Ca²⁺ influx, membrane potential, Ca²⁺ 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.