Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers.

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Abstract
Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted .DELTA.pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation, and fura-2, to minitor the associated change in the myoplasmic free calcium concentration (.DELTA.[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter .DELTA.pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2+-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by .DELTA.[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate .DELTA.[Ca2+]. The experiments show that .DELTA.pHapp was increased at the larger fura-2 concentrations; moreover, the increase in .delta.pHapp was approximately in proportion to the increase in .DELTA.[CaT]. At all fura-2 concentrations, the time course of .DELTA.pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of .delta.[CaT]. These properties of .DELTA.pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol, Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of .DELTA.pHapp with that of .DELTA.[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.