Biochemical and stereological analysis of rat liver mitochondria in different thyroid states

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.     

CORTISONE-INDUCED ALTERATIONS IN MITOCHONDRIAL FUNCTION AND STRUCTURE

The effects of cortisone treatment on oxygen consumption, oxidative phosphorylation, and fine structure of rat liver mitochondria have been studied. Male rats weighing 125 g were treated for 6 days with 5 mg of cortisone acetate or isotonic saline. On the 7th day, sections of liver were excised and processed for light and electron microscopy. Mitochondrial respiration and oxidative phosphorylation were studied with mitochondria isolated from these livers. Cortisone treatment is responsible for a 14–40% decrease in the amount of oxygen consumed per mg of mitochondrial protein when succinate, α-ketoglutarate, or β-hydroxybutyrate are used as substrates, or with ascorbate and N,N,N¹,N¹-tetramethyl p-phenylenediamine as electron donors. In addition, oxidative phosphorylation is uncoupled with a lowering of the P:O ratios. Randomly selected liver cells have been analyzed by quantitative morphometric techniques. The average mitochondrial volume is increased fourfold in the peripheral and midzonal regions with a commensurate decrease in the number of mitochondria per cell. These alterations are present throughout the hepatic lobule, but are most marked in midzonal cells. The total mitochondrial volume per cell and the per cent of the total cytoplasmic volume occupied by mitochondria remains relatively unaltered, as does the total amount of cristae surface per cell. While the mitochondria are enlarged, they are not "swollen." The relationships between the steroid hormone treatment and the alterations in mitochondrial function and structure are discussed.     

A morphologic and morphometric study of the mitochondria in several hepatoma cell lines and in isolated hepatocytes.

Some characteristics of the mitochondria of hepatocytes and of three hepatoma cell lines have been compared. By means of stereologic analysis of electron micrographs of cross-sections through cells the volume of mitochondria per unit volume of cell cytoplasm and the surface areas of the mitochondrial envelope and cristae membranes have been measured. The relative mitochondrial volume in the cytoplasm decreases with increasing growth rate but the surface area of outer and cristae membranes per unit volume of mitochondria is not altered. The internal organization of hepatoma mitochondria, however, differs distinctly from that of normal liver mitochondria as evident from electron micrographs; the hepatoma cells contain mitochondria in which parallel cristae appear to cross the whole mitochondrial profile unlike the irregular, short cristae seen in normal liver mitochondria. Furthermore, in the fast-growing hepatoma cells the mitochondrial matrix appears less dense than in the hepatocyte. Hepatoma cells contain less organized rough endoplasmic reticulum than normal liver cells and the spatial relationship of the mitochondria to the rough cisternae, seen in the hepatocyte, is absent in the fast-growing hepatoma cell lines. It is concluded that hepatoma cells have fewer mitochondria than normal liver cells, but that the organelles have a normal content of inner membranes.     

On the problem of counting and sizing mitochondria: A general reappraisal based on ultrastructural studies of mammalian lymphocytes

The authors ask the question whether the parameters "numbers" and "volume" are suitable for the morphometric analysis of mitochondria. In several types of cell, irregularity of mitochondrial shape makes it technically difficult, if not impossible, to obtain reliable stereological estimates of mean organelle volume or number per unit volume. Of more fundamental concern is whether number of mitochondria per cell is of any real value as a structural correlate of respiratory potential and hence as a measure of cell function. Alternative parameters might serve better for this purpose. Though the problem is illustrated by reference to quantitative studies of lymphocytes, it is also pertinent to the investigation of many other cell types.     

Modification of mitochondria in cultivated hepatocytes as a function of the protein content of the medium]

Embryonic liver cells of chicken have been cultivated in medium more or less supplemented with serum. With a rich medium mitochondria become larger and often ramify. Their fundamental structure remains normal. It is only a hypertrophic condition correlated to an increase of mitochondrial material synthesis, limited to liver mitochondria.     

Free energy of the accumulation of a fluorescent cation probe within lymphocyte mitochondria

Fluorescent probe-cation 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM) can be accumulated in mitochondria of rat thymus lymphocytes. A method is proposed for microfluorometric measuring of DSM concentrations ratio inside the thymocyte mitochondria and in the external medium. This method requires no knowledge about the cell and mitochondria volume, the number of accumulated probe and formation of ionic gradients in the cell by carriers. DSM concentration in energisized mitochondria of the thymocyte exceeds its concentration in the external medium by 10(4) times. This corresponds to the free energy accumulated by DSM equaling 5.2 +/- 0.2 kcal/mole. If such work on the probe accumulation is performed by the electrostatic fields on the plasma and mitochondrial cell membranes, the sum of the potentials of these two fields is 230 +/- 10 mV.     

The characterization of mitochondrial permeability transition in clonal pancreatic beta-cells. Multiple modes and regulation

Mitochondrial permeability transition (MPT), which contributes substantially to the regulation of normal mitochondrial metabolism, also plays a crucial role in the initiation of cell death. It is known that MPT is regulated in a tissue-specific manner. The importance of MPT in the pancreatic beta-cell is heightened by the fact that mitochondrial bioenergetics serve as the main glucose-sensing regulator and energy source for insulin secretion. In the present study, using MIN6 and INS-1 beta-cells, we revealed that both Ca(2+)-phosphate- and oxidant-induced MPT is remarkably different from other tissues. Ca(2+)-phosphate-induced transition is accompanied by a decline in mitochondrial reactive oxygen species production related to a significant potential dependence of reactive oxygen species formation in beta-cell mitochondria. Hydroperoxides, which are indirect MPT co-inducers active in liver and heart mitochondria, are inefficient in beta-cell mitochondria, due to the low mitochondrial ability to metabolize them. Direct cross-linking of mitochondrial thiols in pancreatic beta-cells induces the opening of a low conductance ion permeability of the mitochondrial membrane instead of the full scale MPT opening typical for liver mitochondria. Low conductance MPT is independent of both endogenous and exogenous Ca(2+), suggesting a novel type of nonclassical MPT in beta-cells. It results in the conversion of electrical transmembrane potential into DeltapH instead of a decrease in total protonmotive force, thus mitochondrial respiration remains in a controlled state. Both Ca(2+)- and oxidant-induced MPTs are phosphate-dependent and, through the "phosphate flush" (associated with stimulation of insulin secretion), are expected to participate in the regulation in beta-cell glucose-sensing and secretory activity.     

Higher efficiency of the liver phosphorylative system in diabetic Goto-Kakizaki (GK) rats.

Liver mitochondrial bioenergetics of Goto-Kakizaki (GK) rats (a model of non-insulin dependent diabetes mellitus) reveals a Delta Psi upon energization with succinate significantly increased relatively to control animals. The repolarization rate following ADP phosphorylation is also significantly increased in GK mitochondria in parallel with increased ATPase activity. The increase in the repolarization rate and ATPase activity is presumably related to an improved efficiency of F(0)F(1)-ATPase, either from a better phosphorylative energy coupling or as a consequence of an enlarged number of catalytic units. Titrations with oligomycin indicate that diabetic GK liver mitochondria require excess oligomycin pulses to completely abolish phosphorylation, relative to control mitochondria. Therefore, accepting that the number of operational ATP synthase units is inversely proportional to the amount of added oligomycin, it is concluded that liver mitochondria of diabetic GK rats are provided with extra catalytic units relative to control mitochondria of normal rats. Other tissues (kidney, brain and skeletal muscle) were evaluated for the same bioenergetic parameters, confirming that this feature is exclusive to liver from diabetic GK rats.     

The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.

Cellular manifestations of aging are most pronounced in postmitotic cells, such as neurons and cardiac myocytes. Alterations of these cells, which are responsible for essential functions of brain and heart, are particularly important contributors to the overall aging process. Mitochondria and lysosomes of postmitotic cells suffer the most remarkable age-related alterations of all cellular organelles. Many mitochondria undergo enlargement and structural disorganization, while lysosomes, which are normally responsible for mitochondrial turnover, gradually accumulate an undegradable, polymeric, autofluorescent material called lipofuscin, or age pigment. We believe that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material), but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological 'garbage'). A possible factor limiting the effectiveness of mitochondial turnover is the enlargement of mitochondria which may reflect their impaired fission. Non-autophagocytosed mitochondria undergo further oxidative damage, resulting in decreasing energy production and increasing generation of reactive oxygen species. Damaged, enlarged and functionally disabled mitochondria gradually displace normal ones, which cannot replicate indefinitely because of limited cell volume. Although lipofuscin-loaded lysosomes continue to receive newly synthesized lysosomal enzymes, the pigment is undegradable. Therefore, advanced lipofuscin accumulation may greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells.     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

Giant mitochondria distinct from enlarged mitochondria in secretory and ciliated cells of gerbil trachea and bronchioles

Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.     

Thyroid hormone effects on the State IV proton motive force in rat liver mitochondria

In order to further investigate the mechanisms regulating the control of mitochondrial respiration by thyroid hormone, the proton motive force was measured during State IV respiration in liver mitochondria isolated from euthyroid, hyperthyroid, hypothyroid and T₃-treated hypothyroid rats. The proton motive force was significantly higher in the hyperthyroid group due to an increased pH. The proton motive force of hypothyroid mitochondria was lower than controls due to a decreased membrane potential. The proton motive force for the T3-treated hypothyroid group did not differ from the euthyroid group due to negating changes in the pH gradient and the membrane potential. The intramitochondrial volume was decreased in the hyperthyroid group and unchanged in the other groups. The results indicate that the thyroid status alters the proton motive force in State IV through individual changes in the pH and membrane potential components of the force. The component that changes in hyperthyroid mitochondria is different from that changing in hypothyroid mitochondria.     

Mitofusin-2 maintains mitochondrial structure and contributes to stress-induced permeability transition in cardiac myocytes

Mitofusin-2 (Mfn-2) is a dynamin-like protein that is involved in the rearrangement of the outer mitochondrial membrane. Research using various experimental systems has shown that Mfn-2 is a mediator of mitochondrial fusion, an evolutionarily conserved process responsible for the surveillance of mitochondrial homeostasis. Here, we find that cardiac myocyte mitochondria lacking Mfn-2 are pleiomorphic and have the propensity to become enlarged. Consistent with an underlying mild mitochondrial dysfunction, Mfn-2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration. The absence of Mfn-2 is associated with a marked delay in mitochondrial permeability transition downstream of Ca(2+) stimulation or due to local generation of reactive oxygen species (ROS). Consequently, Mfn-2-deficient adult cardiomyocytes are protected from a number of cell death-inducing stimuli and Mfn-2 knockout hearts display better recovery following reperfusion injury. We conclude that in cardiac myocytes, Mfn-2 controls mitochondrial morphogenesis and serves to predispose cells to mitochondrial permeability transition and to trigger cell death.     

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Control of mitochondrial volume by mitochondrial metabolic water

It is well-known that mitochondrial volume largely controls mitochondrial functioning. We investigate whether metabolic water produced by oxidative phosphorylation could be involved in mitochondrial volume regulation. We modulated the generation of this water in liver mitochondria and assess their volume by two independent techniques.In liver mitochondria, the mitochondrial volume was specifically decreased when no water was produced independently of energetic parameters and uncoupling activity. In all other conditions associated with water generation, there was no significant change in mitochondrial metabolic volume.Altogether these data demonstrate that mitochondrial volume is regulated, independently of energetic status, by the mitochondrial metabolic water that acts as a signal.     

Adult rat cardiomyocytes cultured in creatine-deficient medium display large mitochondria with paracrystalline inclusions, enriched for creatine kinase

In adult regenerating cardiomyocytes in culture, in contrast to fetal cells, mitochondrial creatine kinase (Mi-CK) was expressed. In the same cell, two populations of mitochondria, differing in shape, in distribution within the cell and in content of Mi-CK, could be distinguished. Immunofluorescence studies using antibodies against Mi-CK revealed a characteristic staining pattern for the two types of mitochondria: giant, mostly cylindrically shaped, and, as shown by confocal laser light microscopy, randomly distributed mitochondria exhibited a strong signal for Mi-CK, whereas small, "normal" mitochondria, localized in rows between myofibrils, gave a much weaker signal. Transmission EM of the giant mitochondria demonstrated paracrystalline inclusions located between cristae membranes. Immunogold labeling with anti-Mi-CK antibodies revealed a specific decoration of these inclusions for Mi-CK. Addition of 20 mM creatine, the substrate of Mi-CK, to the essentially creatine-free culture medium caused the disappearance of the giant cylindrically shaped mitochondria as well as of the paracrystalline inclusions, accompanied by an increase of the intracellular level of total creatine. Replacement of creatine in the medium by the creatine analogue and competitor beta-guanidinopropionic acid caused the reappearance of the enlarged mitochondria. It is believed that the accumulation of Mi-CK within the paracrystalline inclusions, similar to those observed in certain myopathies, represents a compensatory effect of the cardiomyocytes to cope with a metabolic stress situation caused by low intracellular total creatine levels.     

Energetics of swelling in isolated hepatocytes: A comprehensive study

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.