Phosphorescence quenching method for measurement of intracellular PO2 in isolated skeletal muscle fibers

Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623 Values of skeletal muscle intracellular P O 2 during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method for measurement of intracellular P O 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°C. The chamber was placed on the stage of an inverted microscope configured for epi-illumination. A solution containing palladium- meso -tetra (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 P O 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 P O 2 by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied from 40 to 70 µs at an extracellular P O 2 of 159 Torr to 650-700 µs at 0 Torr. The phosphorescent lifetimes for the varied P O 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 µs) for the phosphor within the intracellular environment. This technique demonstrates a novel method for determining intracellular P O 2 in isolated single skeletal muscle fibers. oxygen probe; porphyrin; microinjection