IP_(3)R与RyR对新生大鼠心肌细胞钙振荡调控的研究

Regulation of calcium oscillations by IP_(3)R and RyR in neonatal rat cardiomyocytes

目的拟探讨新生期心肌细胞中肌浆网上两种主要的钙离子释放通道1,4,5-三磷酸肌醇受体(1,4,5-trisphosphate receptors,IP_(3)Rs)及雷诺丁受体(ryanodine receptors,RyRs)对细胞内钙振荡的调节作用。方法以体外培养原代新生SD大鼠心肌细胞为实验材料,通过钙离子指示剂fura-2荧光成像,分别利用IP_(3)Rs及RyRs通道的激动剂phenylephrine(PE)、caffeine,以及抑制剂2-APB和tetracaine作用于细胞,研究这两类通道对细胞内钙振荡信号的影响。结果培养的单层新生大鼠心肌细胞有自发的同步的钙振荡;PE处理使得钙振荡频率增加,而2-APB则抑制胞内钙振荡的频率,但它们对钙振荡幅度无明显影响;caffeine增加钙振荡频率但扰乱细胞跳动节律,而tetracaine则完全抑制心肌细胞的钙振荡信号,使细胞停止跳动。此外,应用PE分别作用于培养3 d、7 d及10 d的新生大鼠心肌细胞后,发现其对钙振荡频率的调增作用随培养时间增加而明显下降。结论新生大鼠心肌细胞内钙振荡频率主要受IP_(3)Rs调节,而RyRs是大量钙离子释放的主要通道,决定钙振荡的幅度。

Objective To explore the roles of 1,4,5-trisphosphate receptor(IP_(3)Rs)and ryanodine receptors(RyRs),the two main Ca^(2+)release channels in sarcoplasmic reticulum(SR),in the regulation of intracellular Ca^(2+)oscillations in neonatal rat cardiomyocytes(NRCMs).Methods We isolated and cultured NRCMs for different days,then loaded them with Ca^(2+)indicator fura-2 and performed real-time fluorescent imaging.To distinguish the effects of IP_(3)Rs and RyRs,NRCMs were pre-treated with phenylephrine(PE,IP_(3)Rs agonist),caffeine(RyRs agonist),2-APB(IP_(3)Rs antagonist),and tetracaine(RyRs antagonists),respectively.Results The cultured monolayer NRCMs showed spontaneous synchronized Ca^(2+)oscillations.PE activation or 2-APB blockade of IP_(3)Rs increased or reduced the frequency of Ca^(2+)oscillations in NRCMs,accordingly,with no significant effect on the amplitude of Ca^(2+)oscillations.Activation of RyRs with caffeine increased the frequency of Ca^(2+)oscillations,but unsynchronized the intercellular rhythm of calcium release and beating pace,while blocking RyRs with tetracaine completely abolished the Ca^(2+)oscillations and beats in NRCMs.In addition,the effect of PE stimulation on Ca^(2+)oscillation frequency gradually decreased along with cultured days.Conclusion IP_(3)Rs regulate the rhythm of calcium oscillations,whereas RyRs are the main channel for bulky store Ca^(2+)release.