Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells.
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.
Dedkova
Elena N
EN
Department of Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Ave., Maywood, Illinois 60153, USA.
Blatter
Lothar A
LA
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
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