Intact single twitch fibres from frog muscle were mounted at long sarcomere spacing (3.5-4.2 microns) on an optical bench apparatus for the measurement of absorbance and fluorescence signals following the myoplasmic injection of either or both of the Ca2+ indicator dyes Fura-2 and Antipyrylazo III. Dye-related signals were measured at 16-17 degrees C in fibres at rest and stimulated electrically to give a single action potential or brief train of action potentials. 2. The apparent diffusion constant of Fura-2 in myoplasm, Dapp, was estimated from Fura-2 fluorescence measured as a function of time and distance from the site of dye injection. On average (N = 7), Dapp was 0.36 x 10(-6) cm2 s-1, a value nearly 3-fold smaller than expected if all the Fura-2 was freely dissolved in the myoplasmic solution. The small value of Dapp is explained if approximately 60-65% of the Fura-2 molecules were bound to relatively immobile sites in myoplasm. 3. In resting fibres the fraction of Fura-2 in the Ca2+-bound form was estimated to be small, on average (N = 11) 0.06 of total dye. However, because of the large fraction of Fura-2 not freely dissolved in myoplasm, and the indirect method employed for estimating Ca2+-bound dye, calibration of the resting level of myoplasmic free Ca2+ ([Ca2+]) from the fraction of Ca2+-bound dye was not considered reliable. 4. In response to a single action potential, large changes in Fura-2 fluorescence (delta F) and absorbance (delta A) were detected, which had identical time courses. As expected, the directions of these transients corresponded to an increase in Ca2+-dye complex. For wavelengths, lambda, between 380 and 460 nm, peak delta A(lambda) was closely similar to the Ca2+-dye difference spectrum for Fura-2 determined in in vitro calibrations. Beer's law was used to calibrate the concentration of Ca2+-dye complex formed during activity (delta[CaFura-2]) from the delta A(lambda) signal. Peak delta[CaFura-2] was found to vary between 0.01 and 0.4 mM, depending on the total concentration of injected Fura-2 ([Fura-2T]), which ranged as high as 0.9 mM. 5. In fibres in which peak delta[CaFura-2] was less than 0.06 mM, delta[CaFura-2] had a limiting minimal half-width of 50-60 ms. However, as peak delta[CaFura-2] increased (up to 0.3-0.4 mM), delta[CaFura-2] half-width became markedly prolonged (up to 150-200 ms), indicative of a strong buffering action of large concentrations of Fura-2 on the underlying [Ca2+] transient (delta[Ca2+]).(ABSTRACT TRUNCATED AT 400 WORDS)
