ASCORBATE-MEDIATED TRANSPLASMA MEMBRANE ELECTRON-TRANSPORT IN PULMONARY ARTERIAL ENDOTHELIAL-CELLS

Pulmonary endothelial cells are capable of reducing certain electron a ccepters at the luminal plasma membrane surface. Motivation for studyi ng this phenomenon comes in part from the expectation that it may be i mportant both as an endothelial antioxidant defense mechanism and in r edox cycling of toxic free radicals. Pulmonary arterial endothelial ce lls in culture reduce the oxidized forms of thiazine compounds that ha ve been used as electron acceptor probes for studying the mechanisms o f transplasma membrane electron transport. However, they reduce anothe r commonly studied electron acceptor, ferricyanide, only very slowly b y comparison. In the present study, we examined the influence of ascor bate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid ( DHAA)] on the ferricyanide and thiazine reductase activities of the bo vine pulmonary arterial endothelial cell surface. The endothelial cell s were grown on microcarrier beads so that the reduction of ferricyani de and methylene blue could be studied colorimetrically in spectrophot ometer cuvettes and in flow-through cell columns. The ferricyanide red uctase activity could be increased 80-fold by adding DHAA to the mediu m, with virtually no effect on methylene blue reduction. The DHAA effe ct persisted after the DHAA was removed from the medium. AA also stimu lated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rate s of uptake by the cells. The results are consistent with the hypothes is that AA is an intracellular electron donor for an endothelial plasm a membrane ferricyanide reductase and that the stimulatory effect of D HAA is the result of increasing intracellular AA. Adding sufficient DH AA to markedly increase extracellular ferricyanide reduction had littl e effect on the plasma membrane methylene blue reductase activity sugg esting that pulmonary arterial endothelial cells have at least two sep arate transplasma membrane electron transport systems.