2+uptake by exposure to different cytoplasmic Ca 2+ concentrations ([Ca 2+ ] i = 1, 2 and 5 μm) resulted in a dose-dependent increase of NO production by mitochondria when l-arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca 2+ uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of l-arginine, mitochondrial NO production during stimulation of Ca 2+ uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit l-arginine availability resulted in 50% inhibition of Ca 2+ -induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N -(4-amino-5[aminoethyl]aminopentyl)-N -nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor l-N5 -(1-iminoethyl)ornithine (l-NIO) or iNOS inhibitor N -(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca 2+ -induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of l-arginine, mitochondrial Ca 2+ uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and MnTBAP, and reversed by l-arginine supplementation. In the presence of the mtNOS cofactor (6R)-5,6,7,8,-tetrahydrobiopterin (BH 4 ; 100 μm) mitochondrial ROS generation and PTP opening decreased while mitochondrial NO generation slightly increased. These data demonstrate that mitochondrial Ca 2+ uptake activates mtNOS and leads to NO-mediated protection against opening of the mitochondrial PTP, provided sufficient availability of l-arginine and BH 4 . In conclusion, our data show the importance of l-arginine and BH 4 for cardioprotection via regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS.

Transient Ca(2+)/polyP-mediated mPTP opening during ischaemia may serve to protect cells against cytosolic Ca(2+) overload, whereas ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.

No abstract

In vascular endothelial cells, elevation of cytosolic free calcium concentration ([Ca2+]i) causes activation of nitric oxide synthase (NOS) and release of nitric oxide (NO). The goal of the study was to characterize the interplay between [Ca2+]i and NO production in this cell type. Simultaneous measurements of [Ca2+]i and intracellular NO concentration ([NO]i) in cultured bovine vascular endothelial cells (CPAE cell line) with the fluorescent indicators fura‐2 and DAF‐2, respectively, revealed that Ca2+ influx following agonist‐induced intracellular Ca2+ store depletion (capacitative Ca2+ entry, CCE) represents the preferential Ca2+ source for the activation of the Ca2+‐calmodulin‐dependent endothelial NOS (eNOS). Exposure to the NO donor sodium nitroprusside (SNP) showed that high NO levels suppressed CCE and had an inhibitory effect on Ca2+ extrusion by the plasmalemmal Ca2+‐ATPase. This inhibitory effect on CCE was mimicked by the membrane‐permeant cGMP analogue 8‐bromo‐cGMP, but was reversed by the NO scavenger haemoglobin and prevented by the inhibitor of the NO‐sensitive guanylate cyclase ODQ. Brief exposure to SNP reduced the peak of ATP‐induced Ca2+ release from the endoplasmic reticulum (ER) and accelerated Ca2+ reuptake into the ER. Prolonged incubation with SNP resulted in enhanced Ca2+ loading of the ER, as revealed by direct measurements of store content with the ER‐entrapped low‐affinity Ca2+ indicator mag‐fura‐2. The results suggest that in vascular endothelial cells, NO synthesis is under autoregulatory control that involves NO‐dependent [Ca2+]i regulation. Via cGMP‐dependent inhibition of CCE and acceleration of Ca2+ sequestration into the ER, NO can lower [Ca2+]i and therefore exert an autoregulatory negative feedback on its own Ca2+‐dependent synthesis.

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.