INTRINSIC CYTOSOLIC CALCIUM BUFFERING PROPERTIES OF SINGLE-RAT CARDIAC MYOCYTES
INTRINSIC CYTOSOLIC CALCIUM BUFFERING PROPERTIES OF SINGLE-RAT CARDIAC MYOCYTES
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DOI：
10.1016/s0006-3495(94)80652-6
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发表时间：
1994-10-01
影响因子：
3.4
通讯作者：
BERS, DM
中科院分区：
文献类型：
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作者：
BERLIN, JR;BASSANI, JWM;BERS, DM
在电压钳位大鼠心室肌细胞中测定细胞内被动Ca 2+缓冲。用indo-1（K+盐）加载细胞至估计的胞质浓度为44 +/- 5 μ M（平均值+/- SEM，n = 5），并且可接近的细胞体积估计为24.5 +/- 3.6 μ l。分别用毒胡萝卜素和无钠溶液处理，抑制肌浆网（SR）Ca-ATP酶和肌膜Na-Ca交换的Ca 2+转运。细胞外[Ca 2 +]维持在10 mM，在一些实验中，线粒体解偶联剂“1799 "用于评估线粒体Ca 2+摄取的程度。为了进行单细胞滴定，通过一系列去极化电压钳脉冲从-40 mV到+10 mV逐渐增加细胞内Ca 2+（[Ca 2 +]（i））。通过Ca电流的积分计算每个脉冲的总Ca 2+增益，然后作为脉冲期间[Ca 2 +]（i）的快速变化的函数进行分析。在[Ca 2 +]（i）为0.1 - 2 μ M的范围内，整个细胞缓冲被很好地描述为单一集总的Michaelis-Menten型物质，表观解离常数K-D为0.63 +/- 0.07 μ M（n = 5），结合能力B-max为162 +/- 15 μ mol/l细胞H2O。对归因于胞质indo-1的缓冲进行校正，得到固有的胞质Ca 2+缓冲参数K-D = 0.96 +/- 0.18 μ M，B-max = 123 +/- 18 μ mol/l细胞H2O。以这种方式测量的快速Ca 2+缓冲与已知快速Ca缓冲液的特征（例如，肌钙蛋白C、钙调蛋白和SR Ca-ATPase），但仅为平衡时测量的总Ca 2+缓冲的约一半。包括慢钙缓冲液，如肌钙蛋白C和肌球蛋白上的Ca/Mg位点，可以解释与本实验中测量的Ca电流同相的快Ca 2+缓冲和平衡Ca 2+缓冲之间的差异。目前的数据表明，在收缩过程中，[Ca ~（2+）]（i）从0.1 μ M迅速上升到1 μ M，需要向胞质溶胶中加入约50 μ M的Ca ~（2+）。
Intracellular passive Ca2+ buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 mu M (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler ''1799'' was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+](i)) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+](i) during the pulse. In the range of [Ca2+](i) from 0.1 to 2 mu M, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, K-D, of 0.63 +/- 0.07 mu M (n = 5) and a binding capacity, B-max, of 162 +/- 15 mu mol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of K-D = 0.96 +/- 0.18 mu M and B-max = 123 +/- 18 mu mol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+](i) from 0.1 to 1 mu M during a contraction requires approximately 50 mu M Ca2+ to be added to the cytosol.