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

1. Changes in transmission of quasi-monochromatic light were measured in singly dissected, dye-injected twitch fibres following a single propagated action potential. The records, after correction for the intrinsic transmission signal, indicate changes in dye-related absorbance, DeltaA. This paper describes the different components of dye-related signals in fibres injected with either Arsenazo III, Antipyrylazo III or Dichlorophosphonazo III.2. Fibres injected with Arsenazo III can show two kinds of changes in dye-related absorbance, an early isotropic change and a later dichroic change. The isotropic signal, which is the main subject of this paper, is transient in nature; it starts to develop before tension, reaches a peak in about 10 msec and is nearly over by 0.1 sec (16 degrees C). This signal is largest at 650-660 nm and measurements in this range indicate that the peak DeltaA varies approximately linearly with dye concentration between 0.2 and 0.7 mM. The wavelength dependence of the peak amplitude can be qualitatively fitted by the Ca(2+)-difference spectrum determined from cuvette calibration measurements. There may be a small maintained (0.4-0.5 sec) absorbance change of a few percent of the peak value at 650-660 nm, possibly reflecting a maintained increase in myoplasmic pH or free [Mg(2+)].3. In a fibre injected with approximately 0.5 mM-Antipyrylazo III, there were two 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 Ca(2+) signal which was measured with Arsenazo III, and its magnitude followed the wavelength dependence of the Ca(2+)-difference spectrum determined from cuvette calibration measurements. By contrast, the wavelength dependence of the later absorbance change was similar to either the H(+) or Mg(2+)-difference spectrum. The direction of this late signal (0.2 sec after stimulus) would correspond to an increase in either myoplasmic pH or free [Mg(2+)]. Records of the absorbance change at all wavelengths can be fitted by a linear combination of the Ca(2+) waveform and the H(+)/Mg(2+) waveform.4. Fibres injected with Dichlorophosphonazo III showed three dye-related absorbance changes. There was an early isotropic signal, a later dichroic signal and a second isotropic signal. The wavelength dependence of the first part of the early signal is similar to the Ca(2+)-difference spectrum whereas the wavelength dependence of the second isotropic signal is similar to the H(+)- or Mg(2+)-difference spectrum. As was the case with Arsenazo III and Antipyrylazo III, the direction of the second signal at late times would correspond to an increase in either pH or free [Mg(2+)]. Replacing H(2)O with D(2)O resulted in a marked diminution of the dichroic signal. In D(2)O, linear combinations of two basic isotropic waveforms were sufficient to account for the absorbance changes measured at all wavelengths.5. With all three metallochromic dyes, the time course of the early isotropic signal is similar to that of the second 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 [Ca(2+)] waveform, at least to time of peak, the similarity suggests that all three dyes track free [Ca(2+)] with similar speed.6. The conclusion from the experiments is that there are, in general, two 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 Ca(2+): dye complex in response to a transient increase in myoplasmic free [Ca(2+)]. The other signal persists after the free [Ca(2+)] transient has decayed. This appears to be due to a change in H(+): dye or Mg(2+): dye complex, such as would occur if there were a small maintained increase in myoplasmic pH or free [Mg(2+)].