USE OF FURA RED AS AN INTRACELLULAR CALCIUM INDICATOR IN FROG SKELETAL-MUSCLE FIBERS
USE OF FURA RED AS AN INTRACELLULAR CALCIUM INDICATOR IN FROG SKELETAL-MUSCLE FIBERS
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DOI：
10.1016/s0006-3495(93)81564-9
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发表时间：
1993-06-01
影响因子：
3.4
通讯作者：
BAYLOR, SM
中科院分区：
文献类型：
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作者：
KUREBAYASHI, N;HARKINS, AB;BAYLOR, SM
Fura红是一种在可见波长处具有吸收带的荧光Ca 2+指示剂，将其注射到已拉伸至长肌节长度（约3.8 μ m）并浸泡在“高Ca 2+”林格氏液（[Ca 2 +] = 11.8 mM）中的完整的单肌纤维中。根据呋喃红在肌浆中的缓慢扩散系数，0.16（+/-0.01，SEM）× 10（-6）cm 2 s-1（N = 5; 16 ℃），估计约85%的指示剂分子与大分子量的肌肉成分结合。结合似乎使指示剂对Ca 2+（K（D））的表观解离常数提高3- 4倍，估计在肌浆中为1.1-1.6 μ M。Fura red的肌浆吸收光谱用于估计f（r），即静息时Ca 2+结合形式的Fura red分子的分数。在3根被认为受到微量注射损伤最小的纤维中，f（r）估计为0.15（+/-0.01）。因此，静息肌浆游离[Ca 2 +]（[Ca 2 +]r）估计为0.19-0.28 μ M。对于正常林格氏溶液（[Ca 2 +] = 1.8 mM）中的纤维，在较短的肌节长度（约2.7 μ m），并含有非干扰浓度的指示剂（小于或等于0.2 mM），[Ca 2 +]r估计为0.18-0.27 μ M。在6根纤维中，R，指示剂的荧光比率信号（等于用420 nm激发测量的发射强度除以用480 nm激发测量的发射强度），在休息时和电刺激后测量，并与从相同纤维区域进行的吸光度测量进行比较。分析表明，R（MIN）和R（MAX）（如果所有指示剂分子分别处于无Ca 2+和Ca 2+结合状态，则测量的R值）在肌浆中比在缺乏肌肉蛋白的校准溶液中小得多。分析和讨论了由R估算[Ca ~（2+）]r的几种方法。
Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 mum) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (+/-0.01, SEM) X 10(-6) cm2 s-1 (N = 5; 16-degrees-C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (K(D)), which is estimated to be 1.1-1.6 muM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate f(r), the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, f(r) was estimated to be 0.15 (+/-0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 muM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 mum), and containing a nonperturbing concentration of indicator (less-than-or-equal-to 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 muM. This range is higher than estimated previously in frog fibers with other techniques.In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that R(MIN) and R(MAX) (the values of R that would be measured if all indicator molecules were in the Ca2+-free and Ca2+-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.