Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers

Abstract

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by ΔA0 and ΔA90) were measured with 0° and 90° polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of ΔA were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (ΔA0 + 2 ΔA90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 ± 0.0002 (±SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 ± 0.2 ms (±SEM; n = 8) and 8.4 ± 0.5 ms (±SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (ΔA0 - ΔA90 > 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because ∼80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.