Konishi M, Watanabe M
Department of Physiology, The Jikei University School of Medicine, Tokyo, Japan.
J Gen Physiol. 1998 Apr;111(4):505-19. doi: 10.1085/jgp.111.4.505.
The steady state relation between cytoplasmic Ca2+ concentration ([Ca2+]i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca2+ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca2+]i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150). In the first series of experiments, [Ca2+]i and isometric force were simultaneously measured during high K+ depolarization. Slow changes in [Ca2+]i and force induced by 15-30 mM K+ appeared to be in equilibrium, as instantaneous [Ca2+]i versus force plot tracked the common path in the rising and relaxation phases of K+ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, was used to decrease the rate of decline of [Ca2+]i after tetanic stimulation. The decay time courses of both [Ca2+]i and force were dose-dependently slowed by TBQ up to 5 micro M; the instantaneous [Ca2+]i- force relations were nearly identical at >/=1 micro M TBQ, suggesting that the change in [Ca2+]i was slow enough to reach equilibrium with force. The [Ca2+]i-force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2-3.9 and [Ca2+]i for half activation (Ca50) of 1.5-1.7 micro M. However, if fura dextran reacts with Ca2+ with a 2.5-fold greater Kd as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150), Ca50 would be 3.7-4.2 micro M. We also studied the [Ca2+]-force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca50 were estimated to be 3.3 and 1.8 microM, respectively. Although uncertainties remain about the precise levels of [Ca2+]i, we conclude that the steady state force is a 3rd to 4th power function of [Ca2+]i, and Ca50 is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.
在青蛙完整的骨骼肌纤维中研究了细胞质钙离子浓度（[Ca2+]i）与力之间的稳态关系。将完整的单收缩纤维注射右旋糖酐偶联的钙离子指示剂呋喃丹，利用先前在通透化肌纤维中估算的校准参数（小西和渡边，1995年。《普通生理学杂志》106:1123 - 1150）将呋喃丹的荧光信号转换为[Ca2+]i。在第一系列实验中，在高钾去极化期间同时测量[Ca2+]i和等长力。15 - 30 mM钾离子诱导的[Ca2+]i和力的缓慢变化似乎处于平衡状态，因为瞬时[Ca2+]i与力的关系图在钾离子挛缩的上升和松弛阶段跟踪了共同路径。在第二系列实验中，使用肌浆网钙离子泵抑制剂2,5 - 二叔丁基对苯二酚（TBQ）来降低强直刺激后[Ca2+]i的下降速率。TBQ使[Ca2+]i和力的衰减时间进程剂量依赖性地减慢，直至5微摩尔；在TBQ浓度≥1微摩尔时，瞬时[Ca2+]i - 力关系几乎相同，这表明[Ca2+]i的变化足够缓慢，能够与力达到平衡。从这两种类型实验获得的[Ca2+]i - 力数据与希尔曲线一致，希尔系数为3.2 - 3.9，半激活时的[Ca2+]i（Ca50）为1.5 - 1.7微摩尔。然而，如果呋喃丹与钙离子反应的解离常数（Kd）如先前从动力学拟合估算的那样大2.5倍（小西和渡边，1995年。《普通生理学杂志》106:1123 - 1150），Ca50将为3.7 - 4.2微摩尔。我们还在类似实验条件下研究了去膜纤维中的[Ca2+] - 力关系。平均希尔系数和Ca50分别估计为3.3和1.8微摩尔。尽管关于[Ca2+]i的精确水平仍存在不确定性，但我们得出结论，稳态力是[Ca2+]i的三次到四次幂函数，并且在完整青蛙肌纤维中Ca50处于低微摩尔范围，这与从去膜纤维获得的结果合理一致。
