Dichroic components of Arsenazo III and dichlorophosphonazo III signals in skeletal muscle fibres

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Abstract
Absorbance changes were measured following stimulation of single muscle fibers injected with the metallochromic indicator dye Arsenazo III. Two dye-related signals can be clearly resolved: an early, transient isotropic signal that appears to be due to the formation of Ca2+:dye complex, and a slower, transient signal that is dichroic in nature. The time course of the dichroic signal is the same at all wavelengths employed, suggesting that a single underlying process is involved. The wavelength dependence of the magnitude of the signal is similar to that obtained for dye absorbance in a resting fiber. At 570 nm, near the isobestic wavelength for changes in H+:dye, Mg2+:dye and Ca2+:dye, the dichroic signals is near maximal. The absorbance change with 0.degree. light is positive and about twice as large as the change with 90.degree. light, which is negative. The dichroic signal arises from dye molecules which change their orientation in the radially symmetric muscle fiber. The direction of the change is for the dye''s transition moment to become more aligned with the fiber axis during activity. During a train of 10 action potentials, the (isotropic) Ca2+ transient increases in magnitude 3-fold, whereas the dichroic waveform reaches a plateau value only 30-40% larger than the single twitch value. Replacing H2O in Ringers with D2O causes a slight reduction in the Ca2+ signal, reduces the dichroic signal to 0.4 times normal and reduces tension to 0.1 times normal. Qualitatively similar reductions accompanied an increase in osmolality of H2O Ringer from 1 .times. 2.5 .times. normal. Dichroic signals are also observed in fibers injected with dichlorophosphonazo III. These are similar in many respect to the Arsenazo III dichroic signals. With Arsenazo III, the dichroic signal probably arises from a reorientation of some dye molecules which are bound to one of the oriented structures in muscle. The reorientation lags the Ca2+ transient and may be due to a change which occurs in the oriented structure itself. The Arsenazo III dichroic signal can be fitted by assuming that Ca2+ ions bind to receptor sites and that this binding induces the required change in the oriented structure. The hypothetical receptors have a Kd for Ca2+ equal to 0.1-1 times the peak value of myoplasmic free [Ca2+] during a twitch and an off rate constant equal to 10-30 s-1 at 15.degree. C.