The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89: 41-81). Like arsenazo 111, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18°C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca: indicator complexation and, for indicator, concentrations 50.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 µM at 18°C. If only freely diffusible indicator can react, the estimate is 42 pM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42-AM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief, its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-µM transient and 0.93 for the 42-µM transient. During a I00-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J. R., R. Rudel, and S. R. Taylor. 1978. Journal of Physiology. 277:291-323; Lopez, J. R., L. A. Wanck, and S. R. Taylor. 1981. Science. 214:79-82).
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