We have used phosphorus nuclear magnetic resonance (31P NMR) to study muscular fatigue in anaerobic amphibian muscle. In this paper the biochemical and energetic changes that result from a series of tetani are related to the decrease in rate constant (1/tau) for the final, exponential, phase of relaxation. 2. Using 31P NMR we have measured the concentrations of phosphocreatine (PCr), inorganic phosphate (Pi) and ATP as well as the internal pH. From our measurements we have calculated [creatine], [free ADP], the free-energy change (more precisely, the affinity A = -dG/d xi) for ATP hydrolysis and the rates of lactic acid production and of ATP hydrolysis. 3. We have found that 1/tau, the rate constant of relaxation, is correlated with each of the following, independently of the pattern of stimulation: isometric force production, all of the measured or calculated metabolite levels, pH and dG/d xi. 4. There is a clear dependence upon the pattern of stimulation of the relation between 1/tau and each of the following: total duration of the experiment, number of contractions, rate of lactic acid production and rate of ATP hydrolysis. 5. The rate of relaxation is linearly related to [PCr], [creatine], [Pi] and dG/d xi. It is nonlinearly related to isometric force, [ATP], [H+] and rate of ATP hydrolysis. 6. We conclude that the change in 1/tau, like that of isometric force, depends upon metabolic factors, and not upon any independent changes in the activation or deactivation of contraction. We suggest that 1/tau may depend upon the free-energy change for ATP hydrolysis which in turn may be related to the rate of Ca2+ uptake into the sarcoplasmic reticulum.
