Hancox J C, Evans S J, Levi A J
Department of Physiology, School of Medical Sciences, University Walk, University of Bristol, Bristol, BS8 1TD, UK.
Pflugers Arch. 1996 Jun;432(2):215-24. doi: 10.1007/s004240050127.
We used the whole-cell patch-clamp method to investigate the voltage dependence of the L-type Ca current (ICa,L) and intracellular Ca (Cai) transient in ventricular myocytes isolated from the rat heart. Intracellular Ca was monitored using Fura-2 and the experiments were carried out at 36 degrees C. We measured ICa,L by using a caesium-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak ICa,L amplitude was bell-shaped: ICa,L was maximal at +10 mV and declined at more positive potentials. When ICa,L was integrated over the first 25 ms to estimate the magnitude of Ca entry, this had a very similar voltage dependence to peak ICa,L. In all cells, phasic Fura-2 transients were abolished by 5 microM ryanodine (a blocker of the sarcoplasmic reticulum, SR) showing that the Fura-2 transient provided an index of the magnitude of SR Ca release. For experiments measuring the Cai transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. In 30-40% of cells, we found that the Fura-2 transient had a bell-shaped voltage dependence. This suggests that, in these cells, the primary trigger mechanism for Ca-induced Ca-release might have been Ca entry via ICa,L. In the remaining 60-70% of cells, the voltage dependence of the Fura-2 transient was not bell-shaped. The Fura-2 transient reached a maximum with a pulse to +10 mV, and the amplitude of the transient did not decline significantly at more positive potentials to this. In cells with a non-bell-shaped voltage dependence of the Fura-2 transient, pulses to potentials as far positive as +140 mV elicited phasic Fura-2 transients. Since this potential exceeded the Nernst potential for Ca, it was unlikely there was any trigger Ca entry via ICa,L at this potential. This would suggest that, in these cells, another trigger for SR Ca release (in addition to ICa,L) might be present. We conclude that rat ventricular myocytes, produced using a standard isolation technique and under standard recording conditions, can show either a bell-shaped or a sigmoidal voltage dependence of the Fura-2 transient.
我们采用全细胞膜片钳方法，研究了从大鼠心脏分离出的心室肌细胞中L型钙电流（ICa,L）和细胞内钙（Cai）瞬变的电压依赖性。使用Fura-2监测细胞内钙，实验在36摄氏度下进行。我们通过使用基于铯的内部透析液来消除干扰性钾电流，从而测量ICa,L。ICa,L峰值幅度的电压依赖性呈钟形：ICa,L在+10 mV时最大，并在更正电位时下降。当在最初的25毫秒内对ICa,L进行积分以估计钙内流的大小时，其电压依赖性与ICa,L峰值非常相似。在所有细胞中，5 microM的ryanodine（一种肌浆网，SR的阻滞剂）消除了阶段性Fura-2瞬变，表明Fura-2瞬变提供了SR钙释放量的指标。对于测量Cai瞬变的实验，我们使用基于钾的内部透析液来维持正常的兴奋-收缩偶联。在30%-40%的细胞中，我们发现Fura-2瞬变具有钟形电压依赖性。这表明，在这些细胞中，钙诱导钙释放的主要触发机制可能是通过ICa,L的钙内流。在其余60%-70%的细胞中，Fura-2瞬变的电压依赖性不是钟形。Fura-2瞬变在+10 mV的脉冲时达到最大值，并且在比此更正电位时瞬变幅度没有明显下降。在Fura-2瞬变电压依赖性非钟形的细胞中，高达+140 mV的脉冲会引发阶段性Fura-2瞬变。由于该电位超过了钙的能斯特电位，因此在该电位下不太可能存在通过ICa,L的触发钙内流。这表明，在这些细胞中，可能存在另一种SR钙释放的触发因素（除了ICa,L之外）。我们得出结论，使用标准分离技术并在标准记录条件下制备的大鼠心室肌细胞，其Fura-2瞬变的电压依赖性可能呈钟形或S形。
