Melzer W, Ríos E, Schneider M F
J Physiol. 1986 Mar;372:261-92. doi: 10.1113/jphysiol.1986.sp016008.
Transient changes in intracellular free calcium concentration (delta [Ca2+]) in response to pulse depolarizations were monitored in isolated segments of single frog skeletal muscle fibres cut at both ends and voltage clamped at a holding potential of -90 mV in a double-Vaseline-gap chamber. Calcium transients were monitored optically using the metallochromic indicator dye Antipyrylazo III (APIII), which entered the fibre by diffusion from the solution applied to the cut ends. Optical artifacts due to fibre movement were minimized or eliminated by stretching the fibres to sarcomere lengths at which there was little or no overlap of thick and thin contractile filaments. Remaining movement-independent optical changes intrinsic to the fibre and unrelated to the dye were monitored at 850 nm, where free and dye-bound APIII have no absorbance. These 850 nm signals scaled by lambda -1.2 were used to remove intrinsic components from the signals at 700 or 720 nm, wave-lengths at which the APIII absorbance increases when calcium is bound. The corrected 700 or 720 nm signals were used to calculate delta [Ca2+]. The decay of delta [Ca2+] following fibre repolarization at the termination of a depolarizing pulse was well described by a single exponential plus a constant. The exponential rate constant for the decay of delta [Ca2+] decreased and the final 'steady' level that delta [Ca2+] appeared to be approaching increased with increasing amplitude and/or duration of the depolarizing pulse. Both the decreasing decay rate and the build up of the 'steady' level can be accounted for using a two-component model for the removal of free calcium from the myoplasm. One component consists of a set number of a single type of saturable calcium binding site in the myoplasm. The second component is a non-saturable, first-order uptake mechanism operating in parallel with the saturable binding sites. The removal model parameter values were adjusted to fit simultaneously the decay of delta [Ca2+] after pulses of various amplitudes and durations in a given fibre. The basic procedure was to track delta [Ca2+] during each pulse when an undetermined calcium release was occurring, but to calculate the decay of delta [Ca2+] starting 14 ms after repolarization when release was assumed to be negligible. After appropriate selection of parameter values, the model reproduced most aspects of the decay of delta [Ca2+].(ABSTRACT TRUNCATED AT 400 WORDS)
在双凡士林间隙室中，对两端切断并在-90 mV的钳制电位下进行电压钳制的单根青蛙骨骼肌纤维的分离节段，监测其对脉冲去极化的细胞内游离钙浓度（Δ[Ca2+]）的瞬态变化。使用金属显色指示剂染料安替比拉宗III（APIII）通过光学方法监测钙瞬变，该染料通过扩散从施加于切断端的溶液进入纤维。通过将纤维拉伸至粗、细收缩丝几乎没有或没有重叠的肌节长度，可将由于纤维移动引起的光学伪像降至最低或消除。在850 nm处监测纤维固有的、与染料无关的剩余非移动性光学变化，在此波长下，游离和与染料结合的APIII均无吸光度。这些经λ-1.2缩放的850 nm信号用于从700或720 nm处的信号中去除固有成分，当钙结合时，APIII在这些波长处的吸光度会增加。经校正的700或720 nm信号用于计算Δ[Ca2+]。去极化脉冲终止时纤维复极化后，Δ[Ca2+]的衰减可用单指数加常数很好地描述。随着去极化脉冲幅度和/或持续时间的增加，Δ[Ca2+]衰减的指数速率常数降低，Δ[Ca2+]似乎趋近的最终“稳定”水平升高。衰减速率的降低和“稳定”水平的升高都可以用一个双组分模型来解释，该模型用于从肌浆中去除游离钙。一个组分由肌浆中一组固定数量的单一类型的可饱和钙结合位点组成。第二个组分是与可饱和结合位点并行运行的非饱和一级摄取机制。调整去除模型的参数值，以同时拟合给定纤维中不同幅度和持续时间脉冲后Δ[Ca2+]的衰减。基本步骤是在每次发生不确定的钙释放的脉冲期间跟踪Δ[Ca2+]，但在假设释放可忽略不计的复极化后14 ms开始计算Δ[Ca2+]的衰减。在适当选择参数值后，该模型重现了Δ[Ca2+]衰减的大部分方面。（摘要截断于400字）
