FURA-2 CALCIUM TRANSIENTS IN FROG SKELETAL-MUSCLE FIBERS
FURA-2 CALCIUM TRANSIENTS IN FROG SKELETAL-MUSCLE FIBERS
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DOI：
10.1113/jphysiol.1988.sp017244
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发表时间：
1988-09-01
影响因子：
5.5
通讯作者：
HOLLINGWORTH, S
中科院分区：
文献类型：
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作者：
BAYLOR, SM;HOLLINGWORTH, S
将完整的蛙肌纤维以长肌节间距(3.5-4.2µm)固定在光学台上，用于测量肌浆内注射钙离子指示剂Fura-2和安替比拉偶III的吸光度和荧光信号。染料相关信号在16-17度测量。在静止和电刺激的纤维中产生单个动作电位或简短的一系列动作电位。Fura-2在肌浆中的表观扩散常数DAPP是通过测量Fura-2荧光随时间和距离染料注射部位的函数来估算的。平均(N=7)DAPP为0.36次。10-6cm2 S-1，如果所有的Fura-2都自由溶解在肌浆液中，这个值几乎是预期的3倍。如果大约60%-65%的Fura-2分子结合到肌浆中相对静止的位置，就可以解释DAPP的小价值。在静息纤维中，Fura-2以钙离子结合的形式估计很少，平均(N=11)占总染料的0.06。然而，由于Fura-2的很大一部分不能自由溶解在肌浆中，并且采用间接方法估计钙结合染料，因此从钙结合染料组分中校准肌浆游离钙([Ca~(2+)])是不可靠的。对单个动作电位的响应，检测到Fura-2荧光(ΔF)和吸光度(ΔA)的巨大变化，这两个变化具有相同的时间进程。正如预期的那样，这些瞬变的方向与钙离子-染料复合体的增加相对应。观察到的Fura-2对肌质网钙离子释放的影响符合这样的假设，即游离[Ca~(2+)]水平的升高以快速的时间尺度反馈抑制SR钙的持续释放，而Fura-2通过对[Ca~(2+)]的缓冲作用抑制这种负反馈。此外，由于Fura-2对肌浆内[Ca~(2+)]的缓冲作用并不抑制SR的Ca~(2+)释放，因此实验反对信使[Ca~(2+)]瞬间(即Ca~(2+)诱导的Ca~(2+)释放)在正常的兴奋-收缩偶联序列中起生理作用的观点。
Intact single twitch fibres from frog muscle were mounted at long sarcomere spacing (3.5-4.2 .mu.m) on an optical bench apparatus for the measurement of absorbance and fluorescence signals following the myoplasmic injection of either or both of the Ca2+ indicator dyes Fura-2 and antipyrylazo III. Dye-related signals were measured at 16-17.degree. C in fibres at rest and stimulated electrically to give a single action potential or brief train of action potentials. The apparent diffusion constant of Fura-2 in myoplasm, Dapp, was estimated from Fura-2-fluorescence measured as a function of time and distance from the site of dye injection. On average (N = 7), Dapp was 0.36 .times. 10-6 cm2 s-1, a value nearly 3-fold small than expected if all the Fura-2 was freely dissolved in the myoplasmic solution. The small value of Dapp is explained if approximately 60-65% of the Fura-2 molecules were bound to relatively immobile sites in myoplasm. In resting fibres the fraction of Fura-2 in the Ca2+-bound form was estimated to be small, on average (N = 11) 0.06 of total dye. However, because of the large fraction of Fura-2 not freely dissolved in myoplasm, and the indirect method employed for estimating Ca2+-bound dye, calibration of the resting level of myoplasmic free Ca2+ ([Ca2+]) from the fraction of Ca2+-bound dye was not considered reliable. In response to a single action potential, large changes in Fura-2 fluorescence (.DELTA.F)and absorbance (.DELTA.A) were detected, which had identical time courses. As expected, the directions of these transients corresponded to an increase in Ca2+-dye complex. The observed effects of Fura-2 on SR Ca2+ release are consistent with the hypothesis that a rising level of free [Ca2+] feeds back on a rapid time scale to inhibit continued SR Ca2+ release, and that Fura-2, by its buffering action on .DELTA.[Ca2+], suppresses this negative feed-back. Moreover, since buffering of myoplasmic [Ca2+] by Fura-2 did not inhibit SR Ca2+ release, the experiments argue against the idea that messenger [Ca2+] transients (i.e. Ca2+-induced Ca2+ release'') play a physiological role in the normal excitation-contraction coupling sequence.