Optical measurements of intracellular pH and magnesium in frog skeletal muscle fibres

Abstract

Single twitch fibers were isolated from frog muscle and mounted in a chamber which was positioned on an optical bench. The fibers were immobilized by high stretch (sarcomere spacing 3.9-4.3 .mu.m) and by placement on a pedestal. Their optical properties were determined by illuminating a 35- to 65-.mu.m-diameter spot with quasimonochromatic light of intensity I0 and measuring the intensity I of the transmitted light. Since the main purpose of the experiments was to draw inferences from the absorbance spectra of different indicator dyes injected into the fibers, all results were expressed in terms of absorbance A calculated from the equation I = I0 10-A. Changes in absorbance .DELTA.A were calculated from the differential form of the equation .**GRAPHIC**. The absorbance of a normal, noninjected fiber was, on average, equal to 0.03 at 570 nm and varied approximately inversely with wavelength between 450 and 750 nm. The earliest change in absorbance following an action potential was a small, transient increase which was followed by a larger decrease. The decrease in fiber absorbance varied from 0.5 .times. 10-4-3 .times. 10-4 U. Resting myoplasmic pH was determined by comparing the absorbance spectrum from fibers injected with Phenol Red with that obtained from calibrating solutions in cuvettes. The muscle measurements were corrected for the intrinsic absorbance of fiber without dye. the average value of pH in 2 fibers was 6.9. The change in absorbance following an action potential in these highly stretched fibers was small. In 1 experiment the change, if due to pH alone, corresponded to an increase in pH of 0.004 peak and 0.002 maintained (relative to a resting level of 6.9). The maintained signal can be satisfactorily explained by the known amount of phosphocreatine hydrolysis. Estimates of myoplasmic free [Mg2+] were made using 3 metallochromic indicator dyes. A different estimate was obtained with each dye as indicated below. Since these dyes are sensitive to pH, as well as [Mg2+], the estimate depends on the assumed value of intracellular pH: Arsenazo III, [Mg2+] = 1.2 mM (pH 6.9) and 0.5 mM (pH 7.1); Dichlorophosphonazo III, [Mg2+] = 0.3 mM (pH 6.9) and 0.2 mM (pH 7.1); Arsenazo I, [Mg2+] = 6 mM (pH 6.9) and 3 mM (pH 7.1). This variability probably means that at least 2, and possibly all 3, dyes behave differently inside muscle fibers than they do in calibrating solutions. The most likely explanation is that dye, once injected, can bind to cellular contents and that this alters its properties. Changes in absorbance of fibers injected with Arsenazo I, a dye 3 times more sensitive to Mg2+ than to Ca2+, were used to determine whether changes in free [Mg2+] occur following an action potential. The observed changes were small and could be due to a small increase in pH of the magnitude measured with Phenol Red and/or free [Mg2+]. In terms of a change in free [Mg2+], the results set an upper limit of 2%. Apparently, neither intracellular pH nor free [Mg2+] changes appreciably in highly stretched fibers. Changes in these 2 quantities can be neglected in analyzing the relatively large, 650- to 660-nm Ca2+ signal in fibers injected with the Ca2+ (but also pH and Mg2+)-sensitive indicator dye, Arsenazo III.