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

Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological condit ions. Because the degree of muscle dysfunction observed in some of these co nditions appears to be both free radical dependent and modulated by extrace llular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radi cals may be dependent on extracellular calcium concentrations. For this rea son, we compared formation of reactive oxygen species (ROS; i.e., free radi cals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli fo r 10 min, train rate 0.25 trains/s) incubated in organ baths filled with ph ysiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM ) calcium levels. Generation of ROS was assessed by measuring the conversio n of hydroethidine to ethidium. We found ROS generation with contraction va ried 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 (1 9.30 +/- 4.50 ng/mg) studies (P < 0.001). Control, noncontracting diaphragm s (in 2.5 mM calcium) had little ROS production (3.40 +/- 0.80 ng/mg; P < 0 .001). To further investigate this issue, we added nimodipine (20 mu M), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 +/- 0.8 5 ng ethidium/mg tissue). These data indicate that ROS generation by the co ntracting diaphragm is strongly influenced by extracellular calcium concent rations and may be dependent on calcium transport through L-type calcium ch annels.