Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells.
Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]mt). Stimulation of mitochondrial Ca2+ uptake by exposure to different cytoplasmic Ca2+ concentrations (1, 2, and 5 microM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]mt was sensitive to the NOS antagonist l-N5-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca2+ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl-beta-cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca2+ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002199:Capillary Permeability; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D021941:Caveolae / Q000502:physiology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008665:Metalloporphyrins / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010766:Phosphorylation; D013481:Superoxides / Q000037:antagonists & inhibitors; D014475:Uncoupling Agents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D007093:Imidazoles; D008665:Metalloporphyrins; D014475:Uncoupling Agents; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; D013481:Superoxides; D009569:Nitric Oxide; C065027:N(G)-iminoethylornithine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C031938:calmidazolium; D009952:Ornithine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium