Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III.

Abstract

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 . Journa l 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 jM 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-AM transient and 0.93 for the 42-EtM 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.

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

“…A similar but more marked reduction in SR Ca release has been inferred in other experiments in which [Ca] transients were measured with low affinity indicators in intact fibers (Baylor et al, 1983) and in cut fibers equilibrated with solutions that contained only 0.1 mM EGTA (Maylie et al, 1987c). Fig.…”

“…Previous estimates of the amount of Ca released by an action potential in cut fibers have been made with small concentrations of low affinity Ca indicators and only 0.1 mM EGTA in the end-pool solutions: 333 ~M (SEM, 8 IxM) with antipyrylazo III (high Ca calibration, Table VII in Maylie et al, 1987c), 317 IxM with tetramethylpurpurate (SEM, 5 ~M) (page 168 in Maylie et al, 1987a) and 294 IxM (SEM, 12 ~M) with PDAA (page 321 in Pape et al, 1993). These values are all somewhat smaller than the mean value in our experiments, 375 ixM.…”

Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III.

“…The signals show an early transient absorbance change, seen most clearly in the 520-, 550-, and 720-nm traces. The wavelength dependence of this component matches the Ca-difference spectrum (Maylie et al, 1987c). A second component of the antipyrylazo III absorbance change, seen most clearly in the 590-and 600-nm traces, does not recover towards baseline in the recording period shown in Fig.…”

“…The average concentration of antipyrylazo III inside the fiber at the optical recording site was 0.31 mM (range, 0.26-0.34 mM). The Ca signals recorded from these fibers were similar to those listed in Table IV of Maylie et al (1987c): the average half-width of the indicator-related AA(720) signal was 10.6 ms (range, 8.9-12.4 ms), the average peak amplitude of the AA (720) average value of AA(590)/A(550), -3.12 x 10 -s, is similar to values reported for intact fibers, -2.5 to -3.1 x 10 -3 (from the fiber used in Fig. 11 of Baylor et al, 1982b) and -4.0 x 10 -s (from the fiber used in Fig.…”

“…This may be related to the progressive increase in duration of the Ca signal that occurs during this same period (Maylie et al, 1987c). We therefore wanted to measure the ~A(590) signal early in an experiment when the Ca signal was brief and the condition of the fiber had changed minimally from the time that saponin was applied to permeabilize the end-pool segments.…”