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

• Published in: American Journal of Physiology: Cell Physiology Vol. 279; no. 4; pp. C1058 - C1066
• Main Authors: Zuo, Li, Christofi, Fievos L, Wright, Valerie P, Liu, Cynthia Yu, Merola, A. John, Berliner, Lawrence J, Clanton, Thomas L
• Format: Journal Article
• Language: English
• Published: United States 01.10.2000
• Subjects: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt - pharmacology; Animals; Antioxidants - pharmacology; Catalase - metabolism; Cell Nucleus - metabolism; Cytochrome c Group - metabolism; Cytoplasm - metabolism; Diaphragm - metabolism; Ethidium; Extracellular Space - metabolism; Fluorescence; Heat Stress Disorders - metabolism; Intracellular Fluid - metabolism; Male; Muscle Contraction - physiology; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species - metabolism; Respiratory Muscles - metabolism; Superoxide Dismutase - metabolism; Superoxide Dismutase - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.2000.279.4.c1058

1  Department of Internal Medicine, Pulmonary and Critical Care Medicine, 2  Biophysics Program, and 3  Departments of Anesthesiology, 4  Medical Biochemistry, and 5  Chemistry, Ohio State University, Columbus, Ohio 43210 Skeletal muscles are exposed to increased temperatures during intense exercise, 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 used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42°C) increased ET fluorescence by 24 ± 4%, whereas in control (37°C), fluorescence decreased by 8 ± 1% compared with baseline ( P  < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular 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 cytochrome c ·   g dry wt 1 · ml 1 were reduced in heat compared with 22 ± 13 nmol · g 1 · ml 1 in controls ( P  < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock. cytochrome c ; ethidium; laser scan confocal fluorescence imaging; exercise