Increased superoxide production during fatigue in the perfused rat diaphragm

This study test the hypothesis that a temporal relationship exists between the production of superoxide anion (O₂^-) and the contractile activity of perfused rat diaphragm. O₂^- levels were determined minute to minute by measuring the reduction of cytochrome c in the perfusate as the diaphragms were subjected to various levels of contractile activity. After equilibrating at low contractile rates (one 500 ms 80 Hz train/min), diaphragms were fatigued by increasing their contractile activity for 5 min (one 500 ms 80 Hz train/s) and then allowed to recover for 30 min (one 500 ms 80 Hz train/min). During equilibration, diaphragms did not produce O₂^- above the background level measured in the presence of superoxide dismutase (SOD). Within the first minute of fatigue-inducing stimulation, however, the rate of O₂^- production increased to 0.70 ± 0.17 nmol/min and remained elevated until the recovery period when production returned towards baseline. SOD blocked this stimulation-related increase of O₂^- . Tension (± SOD) fell to 12% of the control value during the fatigue-inducing stimulation. During recovery the contractile response returned to 51% of control, indicating long-lasting effects on the contractile machinery. SOD did not limit fatigue or improve recovery, probably because it is a large protein that cannot cross cell membranes and protect the cells by scavenging O₂^- at its site of production.