Use of metallochromic dyes to measure changes in myoplasmic calcium during activity in frog skeletal muscle fibres

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Abstract
Changes in transmission of quasi-monochromatic light were measured in singly dissected, dye-injected twitch fibers following a single propagated action potential. The records indicate changes in dye-related absorbance, .DELTA.A. The different components of dye-related signals in fibers injected with either Arsenazo III, Antipyrylazo III or Dichlorophosphonazo III are described. Fibers injected with Arsenazo III can show 2 kinds of changes in dye-related absorbance, an early isotropic change and a later dichroic change. The isotropic signal is transient in nature; it starts to develop before tension, reaches a peak in .apprx. 10 ms and is nearly over by 0.1 s (16.degree. C). This signal is largest at 650-660 nm and indicates that the peak .DELTA.A varies .apprx. linearly with dye concentration between 0.2 and 0.7 mM. The wavelength dependence of the peak amplitude can be qualitatively fitted by the Ca2+-difference spectrum determined from cuvette calibration measurements. There may be a small maintained (0.4-0.5 s) absorbance change of a few percent of the peak value at 650-660 nm, possibly reflecting a maintained increase in myoplasmic pH or free [Mg2+]. In a fiber injected with .apprx. 0.5 mM-Antipyrylazo III, there were 2 kinds of dye-related absorbance signals, both of which were isotropic. There was no signal that was obviously dichroic. The earlier signal was similar in time course to the early isotropic Ca2+ signal which was measured with Arsenazo III and its magnitude followed the wavelength dependence of the Ca2+-difference spectrum determined from cuvette calibration measurements. The wavelength dependence of the later absorbance change was similar to either the H+ or Mg2+-difference spectrum. The direction of this late signal (0.2 s after stimulus) would correspond to an increase in either myoplasmic pH or free [Mg2+]. Records of the absorbance change at all wavelengths can be fitted by a linear combination of the Ca2+ waveform and the H+/Mg2+ waveform. Fibers injected with Dichlorophosphonazo III showed 3 dye-related absorbance changes. There was an early isotropic signal, a later dichroic signal and a 2nd isotropic signal. The wavelength dependence of the 1st part of the early signal is similar to the Ca2+-difference spectrum; the wavelength dependence of the 2nd isotropic signal is similar to the H+- or Mg2+-difference spectrum. As was the case with Arsenazo III and Antipyrylazo III, the direction of the 2nd signal at late times would correspond to an increase in either pH or free [Mg2+]. Replacing H2O with D2O resulted in a marked diminution of the dichroic signal. In D2O linear combinations of 2 basic isotropic waveforms were sufficient to account for the absorbance changes measured at all wavelengths. With all 3 metallochromic dyes, the time course of the early isotropic signal is similar to that of the 2nd component of the intrinsic birefringence signal, at least to time of peak. On the assumption that this birefringence signal bears a unique temporal relationship to the myoplasmic free [Ca2+] waveform, at least to time of peak, the similarity suggests that all 3 dyes track free [Ca2+] with similar speed. There are 2 dye-related isotropic absorbance signals seen with Arsenazo III, Antipyrylazo III and Dichlorophosphonazo III. One has an early, transient time course and appears to be due to the formation of Ca2+:dye complex in response to a transient increase in myoplasmic free [Ca2+]. The other signal persists after the free [Ca2+] transient has decayed. This appears to be due to a change in H+:dye or Mg2+:dye complex, such as would occur if there were a small maintained increase in myoplasmic pH or free [Mg2+].