Extracellular calcium modulates generation of reactive oxygen species by the contracting diaphragm

Pulmonary Division, Department of Medicine, Case Western Reserve University and MetroHealth Medical Center, Cleveland, Ohio 44109 Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 ± 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18.90 ± 2.70 ng/mg) or high-calcium (19.30 ± 4.50 ng/mg) studies ( P