Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle

Skeletal muscles are exposed to increased temperatures during intense exerc ise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was use d to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degre es C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degree s C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001) . The superoxide scavenger Tiron (10 mM) abolished the rise in intracellula r fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml ) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cyt ochrome c.g dry wt(-1) . ml(-1) were reduced in heat compared with 22 +/- 1 3 nmol.g(-1).ml(-1) in controls (P < 0.001). SOD decreased cytochrome c red uction in heat to control levels. The results suggest that heat stress stim ulates intracellular and extracellular superoxide production, which may con tribute to the physiological responses to severe exercise or the pathology of heat shock.