Pulmonary endothelial thiazine uptake: separation of cell surface reduction from intracellular reoxidation

Abstract

The objective of this study was to further evaluate the hypothesis that the accumulation of thiazine dyes, such as methylene blue, by cultured bovine pulmonary arterial endothelial cells involves reduction on the cell surface, followed by diffusion of the lipophilic reduced form of the dye into the cells and intracellular reoxidation to the relatively membrane-impermeant hydrophilic form. The specific question addressed was whether inhibition of methylene blue uptake by cyanide and azide is via inhibition of extracellular reduction or inhibition of intracellular reoxidation. We used the cell membrane-impermeant ferricyanide ion as a secondary electron acceptor to measure the extracellular reduction 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 oxidized forms, was used to examine the effects of cyanide and azide on the extracellular reduction. Microscopic observations of the effect of the 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, neither cyanide nor azide had an inhibitory effect on extracellular reduction. The intracellular distribution of the observable oxidized form of the dye was consistent with oxidation of the reduced dye within subcellular organelles. The demonstration that extracellular reduction and intracellular sequestration are separate events is consistent with the hypothesized sequence of events.

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

“…However, the clinical use of MB has been hampered by its propensity for rapid chemical alteration when systemically applied: MB in the biological environment is usually converted by accepting electrons from NADH/NADPH, and unfortunately, the formed colorless leukomethylene blue (LMB) has negligible photodynamic activity [11][12][13]. Recent studies suggested the presence of a transmembrane thiazine dye reductase at the cell surface as the initial factor for MB reduction [14][15][16]. Once within the cells, MB also can be reduced via NADH/NADPH dehydrogenases.…”

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…In such cases, inhibitors are used to provide discriminating data relevant to these processes, and kinetic modeling is used to interpret such data and obtain values for kinetic parameters descriptive of the individual processes involved. Using this overall strategy, we have studied the impact of pulmonary endothelial cells on a variety of redox-active and other substances (4,12,13,38,40,41). The studies have revealed the wide range of pulmonary endothelial redox enzymes and other processes capable of influencing the metabolism, redox status, and disposition of compounds carried in the blood.…”

Reduction and uptake of methylene blue by human erythrocytes

“…It was proposed that MB ϩ freely diffuses across the erythrocyte membrane and is rapidly reduced to MBH, which is then trapped in the cell (36). On the basis of their studies in pulmonary arterial endothelial cells, however, Merker and colleagues (3,26,27,30) concluded that MB ϩ is reduced at the exofacial cell surface. The resulting MBH diffuses into the cell, where it is oxidized to MB ϩ , which is then trapped in the cell.…”