Abstract
Mitochondrial ATP synthesis is tightly coupled to oxygen consumption in a highly regulated process; however, unavoidable byproducts in the form of reactive oxygen species (ROS) are formed. An important regulator of this process is calcium, but the precise relationship between calcium and free radical production is obscure. Therefore, we measured ROS production under a variety of experimental conditions that mimic the pathological environmental setting. The Oroborous oxygraph system was used to simultaneously measure ROS emission and the oxygen consumption rate from isolated cardiac guinea pig mitochondria energized with different substrates and challenged with different calcium concentrations. The Amplex Red assay was used to quantify ROS emission rates. The substrates used utilized a mixture of the carbohydrate and fatty acid dependent metabolic pathways. Two respiration buffers, one lactobionate‐based and the other potassium chloride‐based, were used to quantify the differences in respiration and ROS emission rates. Relative to the potassium chloride‐based respiration buffer, the lactobionate‐based buffer resulted in higher ADP‐stimulated respiration rates and higher ROS emission rates for each substrate combination. On average, the presence of calcium depressed ROS emission rates during the leak respiration, sodium/calcium cycling, and ADP‐stimulated states. The lower respiration rates in the potassium chloride‐based buffer is likely due to the presence of inhibitory levels of chloride. The high chloride levels also appear to depress ROS emission. Overall, these data refute the hypothesis that calcium stimulates ROS emission from isolated mitochondria. Studies are ongoing to further characterize ROS emission from cardiac guinea pig mitochondria and elucidate the relationship between calcium and ROS emission.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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