Cyanide increases reduction but decreases sequestration of methylene blue by endothelial cells

The mechanisms of endothelial cell transplasma membrane electron transport (TMET) have not been completely identified. Redox probes such as methylene blue (MB) can be useful tools, but the complexity of their disposition upon exposure to the cells can hinder interpretation. For example, MB is reduced on the cell surface by TMET, but after entering the cell in reduced form, it is reoxidized and sequestered within the cell. We developed a method to separately quantify the reduction and reoxidation rates such that it can be determined whether a metabolic inhibitor such as cyanide affects the reduction or oxidation process. MB was introduced at the inlet to a column filled with endothelial cell covered beads either as a short 12 s injection (bolus) or a long 45 min infusion (pulse), and its effluent concentration was measured as a function of time. The cells extracted 56% of the MB from the bolus, but only 41% during the pulse steady state. In the presence of cyanide, these extractions increased to 70% and decreased to 4%, respectively. Mathematical model results support the interpretation that these paradoxical effects on bolus and pulse extractions reflect the differential effects of cyanide on extracellular reduction and intracellular oxidation, i.e., cyanide increased the reduction rate from 7.3 to 13.0 cm s-1 X 10(-5) and decreased the oxidation rate from 1.09 to 0.02 cm s-1 X 10(-3). Cyanide also increased intracellular NADH by almost eight times, suggesting that TMET is sensitive to the cell redox status, i.e., NADH is a direct or indirect electron source. The cyanide-induced decrease in sequestration indicates a cyanide-sensitive intracellular oxidation mechanism. The results also demonstrate the potential utility of this approach for further evaluation of these endothelial redox mechanisms.