PULMONARY ENDOTHELIAL THIAZINE UPTAKE - SEPARATION OF CELL-SURFACE REDUCTION FROM INTRACELLULAR REOXIDATION

The objective of this study was to further evaluate the hypothesis tha t the accumulation of thiazine dyes, such as methylene blue, by cultur ed bovine pulmonary arterial endothelial cells involves reduction on t he cell surface, followed by diffusion of the Lipophilic reduced form of the dye into the cells and intracellular reoxidation to the relativ ely membrane-impermeant hydrophilic form. The specific question addres sed was whether inhibition of methylene blue uptake by cyanide and azi de is via inhibition of extracellular reduction or inhibition of intra cellular reoxidation. We used the cell membrane-impermeant ferricyanid e ion as a secondary electron acceptor to measure the extracellular re duction of methylene blue independently from its uptake by the cells. In addition, toluidine blue O, incorporated into an acrylamide polymer so that it could not permeate the cells in either its reduced or oxid ized forms, was used to examine the effects of cyanide and azide on th e extracellular reduction. Microscopic observations of the effect of t he inhibitors on the intracellular accumulation of methylene blue were also made. The results indicate that the reduction and intracellular sequestration are separate processes and that, in doses that inhibited intracellular reoxidation, and therefore uptake and sequestration, ne ither cyanide nor azide had an inhibitory effect on extracellular redu ction. The intracellular distribution of the observable oxidized form of the dye was consistent with oxidation of the reduced dye within sub cellular organelles. The demonstration that extracellular reduction an d intracellular sequestration are separate events is consistent with t he hypothesized sequence of events.