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Single, intact, frog muscle fibres were injected electrophoretically with a Ca2+-sensitive metallochromic dye, Arsenazo III, to a local concentration of 1.2-1.5 mmol/l. The intracellular concentration of free Mg2+, estimated photometrically in the presence of approximately millimolar Arsenazo III, was 3-4 mmol/l in fibres at rest. Ca2+-related changes in dye absorbance were characterized in vitro using 1 mM-Arsenazo III in solutions approximating the intracellular ionic environment. Isometric twitch contractions and related changes in light transmittance of dye-injected regions of fibre were recorded at 2.4 and 3.0 micron sarcomere lengths, at 15 degrees C. A method was developed for separating Ca2+ transients from larger, movement-related optical changes recorded as compound signals during fibre contraction. Decreases in twitch amplitude by about one-third following dye injection, together with the in vitro characteristics of the dye, suggested that millimolar intracellular Arsenazo III acted as a major Ca2+ buffer and inhibited the activation of contractile filaments. The onset of both the Ca2+ transient and latency relaxation occurred at virtually the same time in the twitch response and neither of those transition times was altered significantly with changes in sarcomere length from 2.4 to 3.0 micron. The amount of activation Ca2+ released in dye-injected regions of fibres following a single stimulus was about 0.3 mmol/l at 2.4 micron sarcomere length. The rate of rise and the amplitude of the Ca2+ transient were reversibly decreased with increase in sarcomere length from 2.4 to 3.0 micron. That finding is reviewed in relation to other evidence indicating length dependence of the intracellular release and distribution of activation Ca2+ up to 3.9 micron sarcomere length.

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