ROLE OF CA2+ CHANNEL IN CARDIAC EXCITATION-CONTRACTION COUPLING IN THE RAT - EVIDENCE FROM CA2+ TRANSIENTS AND CONTRACTION
ROLE OF CA2+ CHANNEL IN CARDIAC EXCITATION-CONTRACTION COUPLING IN THE RAT - EVIDENCE FROM CA2+ TRANSIENTS AND CONTRACTION
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DOI：
10.1113/jphysiol.1991.sp018385
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发表时间：
1991-01-01
影响因子：
5.5
通讯作者：
MORAD, M
中科院分区：
文献类型：
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作者：
CLEEMANN, L;MORAD, M
1. 光学方法用于同时测量全细胞电压夹紧大鼠心室肌细胞的空载细胞缩短和细胞内Ca2+瞬态。红光（> 670 nm）用于线性光电二极管阵列测量细胞缩短。以Fura-2 （K(d) = 140 nM）和Mag-Fura-2 （K(d) = 44 mu- m）作为Ca2+指示剂，荧光激发波长为340 nM和410 nM，发射波长为510 nM .2。当0.4 mM-Fura-2从电压钳式移液器的尖端扩散到细胞内时，每隔6 s重复测量显示，细胞内Ca2+瞬态的上升速率和峰值没有降低，只有很小的细胞缩短抑制，这表明Fura-2.3的缓冲能力没有显著改变调节Ca2+释放的分子机制。通过暴露于咖啡因或反复短暂（20 ms）电压钳去极化，肌浆网（SR）的Ca2+被耗尽的实验证实，SR是激活剂Ca2+的主要来源。Mag-Fura-2（1或5 mM）用于记录[Ca2+]i瞬态的初始快速发展，但用该染料测量的Ca2+瞬态的后期时间过程被细胞缩短引起的运动伪影所掩盖。Fura-2和magg -Fura-2均表明，从-80 mV保持电位退极化至0 mV会导致[Ca2+]i瞬态发展，延迟3-9 ms，并在8-19 ms后达到峰值。两种Ca2+指标也表明，Ca2+瞬态接近峰值的速度随着夹膜电位的升高而减慢。Ca2+信号（Fura-2）和细胞缩短的电压依赖性都是钟形的，并且在质量上与同时测量的Ca2+电流的电压依赖性相似。这是在-40和-80 mv的保持电位下观察到的。比较不同膜电位下Ca2+电流、I（Ca）、胞内Ca2+瞬态（Fura-2）和细胞缩短的时间关系表明，在去极化25 ms时测得的Ca2+瞬态与此时间之前测得的Ca2+电流积分密切相关。另一方面，细胞缩短在大约100毫秒后达到峰值，并与随后的Ca2+活性测量结果相关。与-20 mV相比，+ 30 mV时Fura-2瞬态发展较慢，这反映在Ca2+电流向内的时间过程中，+ 30 mV时Fura-2瞬态电流比-20 mV时更小，但持续时间更长。同时，Ca2+瞬态的初始延迟在+ 30 mv时比在-20 mv时短。从+ 100到-40 mV再极化引起的Ca2+瞬态（Fura-2）的初始延迟比从-40到0 mV的去极化引起的Ca2+瞬态更短。当去极化箝位脉冲的持续时间（例如0 mV）减少到10-20 ms以下时，Fura-2瞬态的幅度减小。类似地，Ca2+瞬态的发展可以通过进一步去极化到100 mV.11而过早终止。我们得出结论，同时测量细胞缩短和细胞内Ca2+瞬态提供了电压箝位大鼠心室肌细胞兴奋-收缩耦合的详细数据。我们的研究结果支持这样的假设，即Ca2+从SR的释放是在Ca2+通道的Ca2+内流的直接、连续控制下的。
1. Optical methods were used to measure simultaneously unloaded cell shortening and intracellular Ca2+ transients in whole-cell voltage clamped rat ventricular myocytes. Red light ( > 670 nm) was used to measure cell shortening with a linear photodiode array. The dyes Fura-2 (K(d) = 140 nM) and Mag-Fura-2 (K(d) = 44-mu-M) were used as Ca2+ indicators with fluorescence excitation at 340 and 410 nm and emission at 510 nm.2. Repeated measurements at 6 s intervals as 0.4 mM-Fura-2 diffused into the cell from the tip of the voltage clamp pipette showed no decrease in the rate of rise and peak value of the intracellular Ca2+ transient and only a small suppression of cell shortening, suggesting that the molecular mechanisms regulating the Ca2+ release were not significantly altered by the buffering capacity of the Fura-2.3. Experiments in which the sarcoplasmic reticulum (SR) was depleted of Ca2+ either by exposure to caffeine or by repeated brief (20 ms) voltage clamp depolarizations confirm that the SR is the major source of activator Ca2+.4. Mag-Fura-2 (1 or 5 mM) was used to register the initial rapid development of the [Ca2+]i transient but the later time course of the Ca2+ transients measured with this dye was obscured by motion artifacts resulting from cell shortening.5. Both Fura-2 and Mag-Fura-2 showed that depolarization to 0 mV from a holding potential of -80 mV resulted in a [Ca2+]i transient which developed with a delay of 3-9 ms and approached its peak value in an additional 8-19 ms. Both Ca2+ indicators also showed that the Ca2+ transient approached its peak value more slowly as the clamped membrane potential was made increasingly more positive.6. The voltage dependencies of the Ca2+ signal (Fura-2) and cell shortening were both bell-shaped and were qualitatively similar to the voltage dependence of Ca2+ current simultaneously measured. This was observed with holding potentials of both -40 and -80 mV.7. Comparison of the temporal relation of the Ca2+ current, I(Ca), and intracellular Ca2+ transient (Fura-2) and cell shortening at different membrane potentials showed that Ca2+ transient measured 25 ms into the depolarization correlated closely to the integral of the Ca2+ current measured prior to this time. Cell shortening, on the other hand, peaked about 100 ms later and correlated with measurements of the Ca2+ activity at the later time.8. The slower development of the Fura-2 transient at + 30 mV than at -20 mV was mirrored in the time course of the inward Ca2+ current which was smaller but longer lasting at + 30 mV than at -20 mV. At the same time the initial delay of the Ca2+ transient was shorter at + 30 than at -20 mV.9. The initial delay was briefer for Ca2+ transients (Fura-2) evoked by repolarization from + 100 to -40 mV than for Ca2+ transients evoked by depolarization from -40 to 0 mV.10. The amplitude of the Fura-2 transient was reduced as the duration of a depolarizing clamp pulse (e.g. to 0 mV) was reduced to values below 10-20 ms. Similarly the development of the Ca2+ transient could be prematurely terminated by further depolarization to 100 mV.11. We conclude that simultaneous measurements of both cell shortening and intracellular Ca2+ transients provide detailed data on excitation-contraction coupling in voltage clamped rat ventricular myocytes. Our results support the hypothesis that the release of Ca2+ from the SR is under direct, continous control of the Ca2+ influx through the Ca2+ channel.