Endothelial redox status and transplasma membrane electron transport

To examine the relationship between endothelial cell transplasma membrane electron transport[Am J. Physiol. 269(13):L78-L84.1995] and endothelial cell redox status, we studied extracellular endothelial cell reduction of the electron acceptor methylene blue (MB) and intracellular NAD(P)H during changes in cellular redox status. Bovine pulmonary arterial endothelial cells were grown to confluence on microcarrier beads, placed in a chromatography column (cell-column) and perfused with a buffered salt solution. A bolus of FITC-Dextran and MB was injected into the cell-column and the concentration of each dye in the column effluent was determined by absorbance measurements. MB reduction by endothelial cells was quantified by measuring the loss of blue color relative to the non-reactive FITC-Dextran reference indicator Redox status of the cells was measured using NAD(P)H fluorescence. Addition of 2mM potassium cyanide (KCN) to the perfusate resulted in an increase in MB reduction from 56.4 ± 1.6 to 68.7 ± 2.2% and a 14 ± 1% increase in NAD(P)H fluorescence. This positive correlation between cellular NAD(P)H and MB reduction suggests that the redox status of the cell may control transplasma membrane electron transport in endothelial cells.