The fluorometric technique for measuring the levels of reduced pyridine nucleotides was used to study oxidative metabolism in isolated rabbit papillary muscle at 23°C. The 100% standard level of tissue fluorescence was defined as that measured for muscles resting in oxygenated 10 mM pyruvate solution. This level increased 15% with anoxia and decreased 45% with stimulation in substrate-free solution. Thus, about one-half of the standard tissue fluorescence was metabolically labile and this labile fraction is suggested to be mitochondrial in origin. Decreased tissue fluorescence following mechanical activity was identified with increased oxidation of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) owing to stimulation by adenosine diphosphate (ADP), released during activity, of mitochondrial respiration. The kinetics of the fluorescence transients were slowed fourfold by removal of pyruvate. This effect was not significantly reversed by addition of 10 mM glucose. The time integrals of the fluorescence transients were linearly related to the amounts of mechanical activity in the presence, but not in the absence, of pyruvate. A positive correlation was observed between the steady-state peak tension at constant stimulus rate and the resting level of reduction of pyridine nucleotides in various media. The fluorometric results are interpreted to be indicative of the steady and transient states established by the substrate dehydrogenases and the respiratory chain during oxidative phosphorylation in mitochondria.

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