title: 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.
authors.affiliation: Department of Anesthesiology, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Merker
authors.forename: R D
authors.initials: RD
authors.lastname: Bongard
authors.forename: J H
authors.initials: JH
authors.lastname: Linehan
authors.forename: Y
authors.initials: Y
authors.lastname: Okamoto
authors.forename: D
authors.initials: D
authors.lastname: Vyprachticky
authors.forename: B M
authors.initials: BM
authors.lastname: Brantmeier
authors.forename: D L
authors.initials: DL
authors.lastname: Roerig
authors.forename: C A
authors.initials: CA
authors.lastname: Dawson
mesh_terms: D000180:Acrylic Resins; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003486:Cyanides / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008751:Methylene Blue / Q000493:pharmacokinetics; D010084:Oxidation-Reduction / Q000187:drug effects; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D013843:Thiazines / Q000493:pharmacokinetics*; D014048:Tolonium Chloride / Q000031:analogs & derivatives; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D001386:Azides; D003486:Cyanides; D008751:Methylene Blue; D013843:Thiazines; D014048:Tolonium Chloride; C094878:toluidine blue O-polyacrylamide polymer; D000099166:Polyacrylamides