Phosphorescence quenching method for measurement of intracellular Po2 in isolated skeletal muscle fibers

Values of skeletal muscle intracellular Po, during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method for measurement of intracellular Po-2 in isolated single skeletal muscle fibers by using O-2-dependent quenching of a phosphorescent-O-2 probe is described. Intact single skeletal muscle fibers from Xenopus laevis were dissected from the lumbrical muscle and mounted in a glass chamber containing Ringer solution at 20 degrees C. The chamber was placed on the stage of an inverted microscope configured for epi-illumination. A solution containing palladium-mesotetra (4-carboxyphenyl) porphine bound to bovine serum albumin was injected into single fibers by micropipette pressure injection. Phosphorescence-decay curves (average of 10 rapid flashes) were recorded every 7 s from single cells (n = 24) in which respiration had been eliminated with NaCN, while the Po-2 of the Ringer solution surrounding the cell was varied from 0 to 159 Torr. For each measurement, the phosphorescence lifetime was calculated at the varied extracellular Po-2 by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied from 40 to 70 mu s at an extracellular Po-2, of 159 Torr to 650-700 ps at 0 Torr. The phosphorescent lifetimes for the varied Po-2 were used to calculate, by using the Stern-Volmer relationship, the phosphorescence-quenching constant (100 Torr(-1) s(-1)), and the phosphorescence lifetime in a zero-O-2 environment (690 mu s) for the phosphor within the intracellular environment. This technique demonstrates a novel method for determining intracellular Po-2, in isolated single skeletal muscle fibers.