Changes in the myocardial ultrastructure of the perfused rabbit heart under hypoxia

Ultrastructure of rabbit heart left ventricle isolated according to Langendorf was examined under different conditions: in intact animals, during ligation of the coronary artery, and hypoxic heart perfusion. The intact perfused heart showed unmarked exo- and intracellular edema and moderate swelling of the mitochondria. During hypoxic perfusion, marked swelling and destruction of the mitochondria were noted. During ligation of the coronary artery, the heart was characterized by a high degree of mitochondrial heterogenicity. The correlation of the data obtained allowed one to reveal the myocardial adaptive-accommodative mechanism, (intermittent mitochondrial activity) that makes it possible to maintain the heart bioenergetics during coronary artery occlusion at a permanently high level.     

THE OXIDATION OF EXOGENOUS AND ENDOGENOUS CYTOCHROME C IN MITOCHONDRIA : A Biochemical and Ultrastructural Study

The effect of the nonionic detergent Lubrol on the oxidation of endogenous and exogenous cytochrome c by cytochrome oxidase in intact and fragmented mitochondria was studied. Mitochondria and mitochondrial fragments from liver, kidney, heart, and skeletal muscle have been used. Negatively stained preparations of intact mitochondria showed the particles of Fernández-Morán on the matrix side of their inner membrane system: under these conditions, the oxidation rate of externally added cytochrome c was very high, and it was stimulated very poorly by Lubrol. Mechanical fragmentation of liver mitochondria yielded vesicles with a smooth external profile: also under these conditions, the oxidation of externally added cytochrome c was very high, and poorly stimulated by Lubrol. The oxidation of endogenous cytochrome c was also unaffected by Lubrol. On the other hand, fragmentation of heart and skeletal muscle mitochondria yielded vesicles having numerous particles of Fernández-Morán on their external profiles. Under these conditions, the oxidation of exogenous cytochrome c was low and was markedly stimulated by Lubrol. On the contrary, no activation of the oxidation of endogenous cytochrome c was induced by the detergent. The results indicate a difference in the permeability properties of the two faces of the inner mitochondrial membrane: a permeability barrier for cytochrome c is suggested to exist at the inner face.     

Accumulation of defective mitochondria through delayed degradation of damaged organelles and its possible role in the ageing of post-mitotic and dividing cells

The mitochondrial theory of ageing proposes that an accumulation of defective mitochondria is a major contributor to the cellular deterioration that underlies the ageing process. The plausibility of the mitochondrial theory depends critically upon the population dynamics of intact and mutant mitochondria in different cell types. Earlier work suggested that mutant mitochondria might have a replication advantage but failed to account for the fact that mutants accumulate faster in post-mitotic than in dividing cells. We describe a new mathematical model that allows for damaged mitochondria to replicate more slowly, which accommodates experimental evidence of impaired energy generation and a reduced proton gradient in defective mitochondria. However, this is compensated for by a slower degradation rate of damaged mitochondria than intact ones, as suggested by de Grey (1997), which gives damaged mitochondria a selective advantage and leads to a clonal expansion of damaged mitochondria. This theoretical result is important because it agrees with evidence that, during ageing, single muscle fibres are taken over by one or only a few types of mtDNA mutants. The model also shows that cell division can rejuvenate and stabilize the mitochondrial population, consistent with data that post-mitotic tissues accumulate mitochondrial damage faster than mitotically active tissues.     

Demonstration of the existence of an organo-specific NADH dehydrogenase in heart mitochondria

Experimental evidence is presented showing the existence of an NADH-consuming enzyme in heart mitochondria, in addition to the NADH--ubiquinone oxidase of complex I. In contrast to the latter, the novel enzyme is accessible from the extramitochondrial space. Removal of the outer membranes from intact mitochondria had no influence on exogenous NADH consumption, indicating its location at the cytosolic face of the inner membrane. The enzyme could be solubilized from this membrane and purified by sedimentation through preformed sucrose gradients. Liver mitochondria exhibited no oxidation of external NADH, suggesting that the enzyme is organo-specific. The "exogenous NADH dehydrogenase" of heart mitochondria was found to introduce reducing equivalents into the respiratory chain before the rotenone block, indicating that the enzyme is associated with complex I. The enzyme was also demonstrated to be involved in electron flow from the respiratory chain to exogenous electron acceptors, including NAD+. This permitted us to elicit the existence of an energy-dependent reversed electron flow from complex II to complex I. The redox shuttle established by the novel enzyme could be of significance for the regulation of cellular NADH and the metabolic activation of foreign compounds such as adriamycin.     

Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin.

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.     

The action of digitonin on rat liver mitochondria. Phospholipid content

1. The amount and types of phospholipid and the fatty acid composition of the various phospholipids were examined in intact rat liver mitochondria, in mitochondria devoid of their outer membrane (preparation A) and in very small pieces derived from the disruption of the inner-membrane complexes (preparation B). The latter two preparations were obtained by digitonin treatment and carry out oxidative phosphorylation. 2. The ratio mug.atoms of phospholipid P/mg. of protein was 0.163 for intact mitochondria, decreased to 0.118 on removal of the outer membrane and increased markedly to 0.292 on disruption of the inner-membrane complex. 3. Examination of the various types of phospholipid present showed that the molar proportions cardiolipin:phosphatidylcholine:phosphatidylethanolamine were approx. 1:6:6 for intact mitochondria and 1:3:3 for preparations A and B. 4. There was a correlation between the recovery of cardiolipin and adenosine triphosphatase activity in the conversion of intact mitochondria into preparations A and B. 5. The fatty acid contents of the various types of phospholipid purified by thin-layer chromatography were identical in all three preparations. Our results show a considerably higher content of arachidonic acid and lower content of oleic acid for phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol than have previously been reported for mitochondrial phospholipids.     

Leaky beta-oxidation of a trans-fatty acid: incomplete beta-oxidation of elaidic acid is due to the accumulation of 5-trans-tetradecenoyl-CoA and its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine in the matrix of rat mitochondria

The degradation of elaidic acid (9-trans-octadecenoic acid), oleic acid, and stearic acid by rat mitochondria was studied to determine whether the presence of a trans double bond in place of a cis double bond or no double bond affects beta-oxidation. Rat mitochondria from liver or heart effectively degraded the coenzyme A derivatives of all three fatty acids. However, with elaidoyl-CoA as a substrate, a major metabolite accumulated in the mitochondrial matrix. This metabolite was isolated and identified as 5-trans-tetradecenoyl-CoA. In contrast, little or none of the corresponding metabolites were detected with oleoyl-CoA or stearoyl-CoA as substrates. A kinetic study of long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase revealed that 5-trans-tetradecenoyl-CoA is a poorer substrate of LCAD than is 5-cis-tetradecenoyl-CoA, while both unsaturated acyl-CoAs are poor substrates of very long-chain acyl-CoA dehydrogenase when compared with myristoyl-CoA. Tetradecenoic acid and tetradecenoylcarnitine were detected by gas chromatography/mass spectrometry and tandem mass spectrometry, respectively, when rat liver mitochondria were incubated with elaidoyl-CoA but not when oleoyl-CoA was the substrate. These observations support the conclusion that 5-trans-tetradecenoyl-CoA accumulates in the mitochondrial matrix, because it is less efficiently dehydrogenated by LCAD than is its cis isomer and that the accumulation of this beta-oxidation intermediate facilitates its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine thereby permitting a partially degraded fatty acid to escape from mitochondria. Analysis of this compromised but functional process provides insight into the operation of beta-oxidation in intact mitochondria.     

Regulation of the mitochondrial adenosine 5'-triphosphatase in situ during ischemia and in vitro in intact and sonicated mitochondria from slow and fast heart-rate hearts

In the present study we examined the regulation of the cardiac muscle mitochondrial ATPase both in situ and in vitro in intact and sonicated mitochondria from rabbit, pigeon, and rat. We chose to study these three species because each is representative of one of the three classes into which all species thus far studied may be placed with respect to the in situ activity of their cardiac muscle mitochondrial ATPase inhibitor and with respect to the amount of ATPase inhibitor present in their cardiac muscle mitochondria (1). Class A species (rabbit) contain a full complement of ATPase inhibitor and show a marked ATPase inhibition during ischemia. Class B species (pigeon) also contain a full complement of inhibitor but exhibit only a low level of ATPase inhibition in situ. Class C species (rat) contain only low levels of inhibitor and, like class B species, don't appear to utilize the inhibitor they possess during ischemia in situ. We found that, while hearts from all three species developed a marked cytosolic acidosis during ischemia, only rabbit exhibited a marked ATPase inhibition in situ. In in vitro experiments in which matrix pH values close to 6.2 and delta psi values close to zero were measured in intact mitochondria from all three species, matrix pH appeared to be an important factor regulating ATPase inhibition in rabbit, but it had little effect upon ATPase--inhibitor interaction in pigeon and rat despite the lack of membrane potential. However, a pH-dependent further release of ATPase inhibitor was observed in sonicated pigeon heart mitochondria only. This latter observation suggests that, while slow heart-rate heart mitochondria appear to be designed for ATPase down regulation during ischemia by inhibitor binding to the ATPase, fast heart-rate heart mitochondria appear to be designed primarily for ATPase up regulation by a further release of inhibitor from the enzyme.     

Activation of NAD-linked malic enzyme in intact plant mitochondria by exogenous coenzyme A

O2 uptake by potato and cauliflower bud mitochondria oxidizing malate was progressively inhibited as the pH of the external medium was increased, in response to accumulation of oxaloacetate. Adding 0.5 mM coenzyme A to the medium reversed this trend by stimulating intramitochondrial NAD-linked malic enzyme at alkaline pH. In intact potato mitochondria, coenzyme A stimulation of malic enzyme was not observed when the external pH was above 7.5; in cauliflower mitochondria, coenzyme A stimulated even at pH 8. This difference in the response of intact mitochondria was attributed to an inherent difference in the properties of malic enzyme from the two tissues. Malic enzyme solubilized from potato mitochondria was inactive at pH values above 7.8, while that from cauliflower mitochondria retained its activity at pH 8 in the presence of coenzyme A. In potato mitochondria, coenzyme A stimulation of O2 uptake at alkaline pH was only observed when NAD+ was also provided exogenously. The results show that coenzyme A can be taken up by intact mitochondria and that pH, NAD+, and coenzyme A levels in the matrix act together to regulate malate oxidation.     

Interaction of cytochrome c with mitochondrial proteins and cybacrone-dextran

Interaction of cytochrome c with electron carriers in intact and damaged (with destroyed outer membrane) rat liver mitochondria was studied. It was shown that the increase in ionic strength causes changes in the respiration rate of damaged mitochondria due to the reduction of the cytochrome c affinity for its binding sites in the organelles. This suggests that cytochrome c concentration in the intermembrane space of intact mitochondria is increased by salts, whereas the increase in ionic strength has a slight influence on the rates of succinate oxidase and external rotenone-insensitive NADH-oxidase of intact mitochondria. At low ionic strength values, the Michaelis constant (KM) value of external NADH-oxidase for cytochrome c exceeds by one order of magnitude that for succinate oxidase, while the maximal activity of these two systems is nearly the same. The increase in ionic strength causes an increase in the KM value for both oxidases. Interaction of cytochrome c with mitochondrial proteins was modelled by cytochrome c interaction with cibacron-dextran anions. It was concluded that the ionic strength-sensitive electrostatic interactions play a decisive role in cytochrome c binding to electron carriers in mitochondrial membranes. However, cytochrome c content and its binding parameters in intact-mitochondrial membranes prevent the latent activity of external NADH oxidase to be revealed in intact mitochondria after the increase in the ionic strength of the surrounding medium.     

Cardiolipin is the membrane receptor for mitochondrial creatine phosphokinase

Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.     

Mitochondrial hexokinase and cardioprotection of the intact heart

The interaction of hexokinase with mitochondria has emerged as a powerful mechanism in protecting many cell types against cell death. However, the role of mitochondrial hexokinase (mitoHK) in cardiac ischemia-reperfusion injury has as of yet received little attention. In this review we examine whether increased binding of hexokinase to the mitochondrion is also an integral component of cardioprotective signalling. We discuss observations in cardiac mitochondrial activation that directed us to the hypothesis of hexokinase cellular redistribution with reversible, cardioprotective ischemia, summarize the data showing that many cardioprotective interventions, such as ischemic preconditioning, insulin, morphine and volatile anesthetics, increase mitochondrial hexokinase binding within the intact heart, and discuss similarities between mitochondrial hexokinase association and ischemic preconditioning. Although most data indicate that mitochondrial hexokinase may indeed be an integral part of cardioprotection, a definitive proof for a causal relation between the amount of mitoHK and cardiac ischemia-reperfusion injury in the intact heart is eagerly awaited. When such relationship is indeed observed, the association of hexokinase with mitochondria will offer an opportunity to develop new therapies to combat ischemic cardiac diseases.     

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.     

Change in the mitochondria of the left heart ventricle in the intact rabbit during the course of the day (based on scanning electron microscopic data)

The myocardium of the left ventricle of intact rabbit was investigated by scanning electron microscopy during 24 hours, namely at 0, 6 a.m., noon and 6 p.m. It was discovered that the mitochondria reach the maximal volume at 6 p.m. and the minimal at 6 a.m. The qualitative differences in the mitochondria at different periods of the day are described. A correlation was established between the swelling of the mitochondria and some indices of heart function.     

Evidence for the role of malic enzyme in the rapid oxidation of malate by cod heart mitochondria

Mitochondria isolated from the heart of cod (Gadus morrhua callarias) oxidized malate as the only exogenous substrate very rapidly. Pyruvate only slightly increased malate oxidation by these mitochondria. This is in contrast with the mitochondria isolated from rat and rabbit heart which oxidized malate very slowly unless pyruvate was added. Arsenite and hydroxymalonate (an inhibitor of malic enzyme) inhibited the respiration rate of mitochondria isolated from cod heart, when malate was the only exogenous substrate. Inhibition caused by hydroxymalonate was reversed by the addition of pyruvate. In the presence of arsenite, malate was converted to pyruvate by cod heart mitochondria. Cod heart mitochondria incubated in the medium containing Triton X-100 catalyzed the reduction of NADP+ in the presence of L-malate and Mn2+ at relatively high rate (about 160 nmoles NADPH formed/min/mg mitochondrial protein). The oxidative decarboxylation of malate was also taking place when NADP+ was replaced by NAD+ (about 25 nmol NADH formed per min per mg mitochondrial protein). These results suggest that the mitochondria contain both NAD+- and NADP+-linked malic enzymes. These two activities were eluted from DEAE-Sephacel as two independent peaks. It is concluded that malic enzyme activity (presumably both NAD+- and NADP+-linked) is responsible for the rapid oxidation of malate (as the only external substrate) by cod heart mitochondria.     

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves

The presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. Intact mitochondria and peroxisomes had no latent APX activity, and this remained in the membrane fraction after solubilization assays with 0.2 M KCl. Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Glutathione reductase had a high latency in mitochondria and peroxisomes and was present in the soluble fractions of both organelles. In intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles.     

Tissue-specific modulation of the mitochondrial calcium uniporter by magnesium ions

This paper analyzes the kinetics of the Ca2+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Ca2+ concentrations near the steady-state value (1-4 microM). Heart mitochondria exhibit the lowest activity, and physiological Mg2+ concentrations inhibit the mitochondrial Ca2+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Ca2+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment.     

Phosphorus nuclear magnetic resonance of perfused salivary gland

The effect of Sr2+ on the set point for external Ca2+ was studied in rat heart and liver mitochondria with the aid of a Ca2+-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca2+ influx on the Ca2+ uniporter and efflux by various mechanisms. We studied the Ca2+-Na+ exchange pathway in heart mitochondria and the delta psi-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr2+ was found to shift the set points towards lower external Ca2+ both in heart mitochondria under conditions of Ca2+-Na+ exchange and in liver mitochondria under conditions that should promote opening of the delta psi-modulated pathway. The effect on the set point was found to be due to inhibition of Ca2+ efflux by Sr2+ taken up by the mitochondria, while Sr2+ efflux was too slow to be measurable.     

Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.     

Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.