The pulmonary endothelium carries out a variety of metabolic precesses that modulate the composition of venous blood before it enters the systemic arterial circulation. These include activation and inactivation of vasoactive substances and the oxidation and reduction of blood constituents. One mechanism by which the endothelium influences the redox status of blood constituents involves electron transport systems (reductases) within the pulmonary endothelial cell plasma membrane that transfer reducing equivalents from intracellular donors to extracellular electron acceptors. The hypothesis motivating the studies that are being carried out in the ESR Center is that trans-plasma membrane electron transport is involved in regeneration of antioxidant components of the blood and redox cycling of pulmonary toxins such as paraquat. One criterion for putative electron acceptors for the transplasma membrane electron transporters is that, after addition of the acceptor to a su spen sion of pulmonary endothelial cells, evidence of reduction of the acceptor can be found in the extracellular medium. Paraquat was chosen for our initial studies in collaboration with the ESR Center because ESR studies have previously been carried out using paraquat and endothelial cells. Paraquat was added to bovine pulmonary artery endothelial cells grown on microcarrier beads, and the mixture was incubated at 37 degrees. About 60 minutes after addition of paraquat and the free radical spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to endothelial cells, the signal indicating the presence of a radical trapped by DMPO was observed in the cell supernatant. A substantially lower intensity signal was observed when the cells were incubated with DMPO alone, in the absence of paraquat. These data show that the paraquat was reduced to the paraquat radical by the cells and that the paraquat radical reacted with molecular oxygen. The resulting oxygen radical products were trapped by the DMPO, and the trapped radical was detected in the extracellular medium. Thus, these studies demonstrate the feasibility of using EPR to study the role of transplasma membrane electron transport in the redox cycling of paraquat by endothelial cells.
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