title: Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells.
abstract: In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca2+ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100-500 microM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca2+ uptake and Ca2+ extrusion rates, whereas low concentrations of Sper/NO (<100 microM) had no effect on mitochondrial Ca2+ levels ([Ca2+]mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca2+ uptake, whereas inhibition of mtNOS with 10 microM L-N(5)-(1-iminoethyl)ornithine resulted in a significant increase of [Ca2+]mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca2+-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100-500 microM) induced PTP opening, whereas lower concentrations (<100 microM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca2+ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca2+ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca2+ overload under pathological conditions where cellular [NO] can reach very high levels.
authors.affiliation: Department of Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Ave., Maywood, Illinois 60153, USA.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Dedkova
authors.forename: Lothar A
authors.initials: LA
authors.lastname: Blatter
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D007473:Ion Channels / Q000502:physiology; D008564:Membrane Potentials; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009589:Nitrogen Oxides; D009952:Ornithine / Q000031:analogs & derivatives / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D007473:Ion Channels; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009589:Nitrogen Oxides; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; C065027:N(G)-iminoethylornithine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D002118:Calcium