title: Endothelial cell oxidant generation during K(+)-induced membrane depolarization.
abstract: We tested the hypothesis that membrane depolarization may initiate oxidant generation in the endothelial cell. Depolarization was produced in bovine pulmonary arterial endothelial cells (BPAEC) in monolayer culture with varying external K+, or with glyburide (10 microM), tetraethylammonium (TEA, 10 mM), gramicidin (1 microM), or nigericin (2 microM). Evaluation of bisoxonol fluorescence of BPAEC indicated concentration-dependent depolarization by high K+ (2% change in fluorescence/mV change in membrane potential in the 5.9-48 mM range of K+) and essentially complete depolarization with glyburide. Generation of oxidants was assessed with o-phenylenediamine dihydrochloride (o-PD) oxidation in the presence of horseradish peroxidase (HRP). There was a time-dependent increase in o-PD oxidation with 24 mM K+, nigericin, and gramicidin over 2 hours compared with control. In 1 hour o-PD oxidation increased 2.8-fold for 24 mM and 3.7-fold for 48 mM K+ compared with control. Catalase reduced 24 mM K(+)-induced o-PD oxidation by 50%, while Cu/Zn-superoxide dismutase (SOD) abolished the increase. Oxidation of o-PD was reduced by 57% in the absence of HRP in the system. With K+ channel blockade, o-PD oxidation increased 3.8-fold with glyburide and 4.6-fold with TEA compared with control. These data indicate formation of H2O2 and possibly other oxidants with depolarization and suggest involvement of K(+)-channels in this process.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
authors.forename: A B
authors.initials: AB
authors.lastname: al-Mehdi
authors.forename: H
authors.initials: H
authors.lastname: Ischiropoulos
authors.forename: A B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D006096:Gramicidin / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D016877:Oxidants / Q000378:metabolism*; D010655:Phenylenediamines / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology*; D026902:Potassium Channel Blockers; D013858:Thiobarbiturates
chemical_list: D005456:Fluorescent Dyes; D016877:Oxidants; D010655:Phenylenediamines; D026902:Potassium Channel Blockers; D013858:Thiobarbiturates; D006096:Gramicidin; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol; C034193:1,2-diaminobenzene; D009550:Nigericin; D011188:Potassium