ABSORBENCY SIGNALS FROM RESTING FROG SKELETAL-MUSCLE FIBERS INJECTED WITH THE PH INDICATOR DYE, PHENOL RED

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(λ) and A90(λ), were estimated as a function of wavelength λ (450 nm ≤ λ ≤ 640 nm) with light polarized parallel (0°) and perpendicular (90°) to the fiber axis, respectively. At all λ, A0(λ) was slightly greater than A90(λ), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "is tropic" signal, [A0(λ) + 2A90(λ)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by ~ 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCI. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye’s apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 × 10-6 cm2 s-1 (at 16°C), can be explained if ~80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the is tropic absorbance of phenol red was fitted by in vitro calibration spectra, pHi, p was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer’s solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations. © 1990, Rockefeller University Press., All rights reserved.