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PubMed1999

Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I).

Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q1 reductase activities of bovine heart submitochondrial particles (SMP) with an apparent Ki of 1x10-5 M (pH 8.0, 25 degrees C). The NADH-hexammineruthenium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the inhibitor concentrations below 0.15 mM. The succinate-supported aerobic reverse electron transfer is less sensitive to the inhibitor (Ki=5x10-5 M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD+ reduction when the nucleotide is added to tightly coupled SMP oxidizing succinate aerobically. Also similar to rotenone, Triton X-100 partially protects Complex I against the thermally induced deactivation and partially activates the thermally deactivated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastically decreased in the presence of Triton X-100 which indicates a competition between these two inhibitors for a common specific binding site. In contrast to rotenone, the inhibitory effect of Triton X-100 is instantly reversed upon dilution of the reaction mixture. The NADH-Q1 reductase activity of SMP is inhibited non-competitively by added Q1 whereas a simple competition between Q1 and the inhibitor is seen for isolated Complex I. The results obtained show that Triton X-100 is a specific inhibitor of the ubiquinone reduction by Complex I and are in accord with our previous findings which suggest that different reaction pathways operate in the forward and reverse electron transfer at this segment of the mammalian respiratory chain.

Animals↗
PubMed2000

3,4-Dihydroxyphenylacetaldehyde potentiates the toxic effects of metabolic stress in PC12 cells.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic metabolite formed by the oxidative deamination of dopamine. This aldehyde is mainly oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH), but is also partly reduced to 3, 4-dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase (ARs). In a previous study, we found that rotenone, a complex I inhibitor, induced a rapid accumulation of DOPAL and DOPET in the medium of cultured PC12 cells. Here, we examined the potential role of DOPAL in the toxicity induced by complex I inhibition in PC12 cells and compared the effects of rotenone on concentrations of DOPAL and DOPET to those of MPP(+). DOPAL and DOPET levels were increased by rotenone but decreased by MPP(+). Inhibition of ALDH by daidzein reduced the formation of DOPAC and increased the accumulation of DOPAL. Inhibition of ARs (with AL1576) diminished DOPET formation and elevated DOPAL concentrations. Combined inhibition of ALDH and ARs markedly elevated DOPAL concentrations while diminishing DOPET and DOPAC levels. The elevation of DOPAL levels induced by combined inhibition of ALDH and ARs had no effect on cell viability. However, combined inhibition of ALDH and ARs potentiated rotenone-induced toxicity. Both the potentiation of toxicity and the increase in DOPAL levels were blocked by inhibition of monoamine oxidase with clorgyline indicating that accumulation of DOPAL was responsible for the potentiated rotenone-induced toxicity following combined inhibition of ALDH and ARs. Since complex I dysfunction is reported to be involved in the pathogenesis of Parkinson's disease, DOPAL potentiation of the deleterious effects of complex I inhibition may contribute to the specific vulnerability of dopaminergic neurons to injury.

1-Methyl-4-phenylpyridinium↗
PubMed2003

Rapid reduction of ATP synthesis and lack of free radical formation by MPP+ in rat brain synaptosomes and mitochondria.

MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1997

Metabolic inhibition enhances selective toxicity of L-DOPA toward mesencephalic dopamine neurons in vitro.

Recent in vitro studies have described the toxicity of levodopa (L-DOPA) to dopamine (DA) neurons. We investigated whether metabolic inhibition with rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, may enhance the toxicity of L-DOPA toward DA neurons in mesencephalic cultures. The uptakes of DA and GABA were determined to evaluate the functional and morphological integrity of DA and non-DA neurons, respectively. Pretreatment with rotenone significantly augmented the toxic effect of L-DOPA on DA neurons. Interestingly, prior metabolic inhibition with rotenone rendered DA cells susceptible to a dose (5 microM) of L-DOPA that otherwise exhibited no toxic effect. DA uptake was more intensely attenuated than GABA uptake after the combined exposure to rotenone and L-DOPA. This was confirmed by cell survival estimation showing that tyrosine hydroxylase-positive DA cells are more vulnerable to the sequential exposure to the drugs than total cells. The selective toxic effect of L-DOPA on rotenone-pretreated DA neurons was significantly blocked by antioxidants, but not antagonists of NMDA or non-NMDA glutamate receptors. This indicates that oxidative stress play a central role in mediating the selective damage of DA cells in the present experimental paradigm. Our results raise the possibility that long-term L-DOPA treatment could accelerate the progression of degeneration of DA neurons in patients with Parkinson's disease where potential energy failure due to mitochondrial defects has been demonstrated to take place.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2003

Production of reactive oxygen species by mitochondria: central role of complex III.

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.

Animals↗
PubMed2001

Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor.

1. The mechanisms responsible for sensing hypoxia and initiating hypoxic pulmonary vasoconstriction (HPV) are unclear. We therefore examined the roles of the mitochondrial electron transport chain (ETC) and glycolysis in HPV of rat small intrapulmonary arteries (IPAs). 2. HPV demonstrated a transient constriction (phase 1) superimposed on a sustained constriction (phase 2). Inhibition of complex I of the ETC with rotenone (100 nM) or complex III with myxothiazol (100 nM) did not cause vasoconstriction in normoxia, but abolished both phases of HPV. Rotenone inhibited the hypoxia-induced rise in intracellular Ca(2+) ([Ca(2+)](i)). Succinate (5 mM), a substrate for complex II, reversed the effects of rotenone but not myxothiazol on HPV, but did not affect the rise in NAD(P)H fluorescence induced by hypoxia or rotenone. Inhibition of cytochrome oxidase with cyanide (100 microM) potentiated phase 2 constriction. 3. Phase 2 of HPV, but not phase 1, was highly correlated with glucose concentration, being potentiated by 15 mM but abolished in its absence, or following inhibition of glycolysis by iodoacetate or 2-deoxyglucose. Glucose concentration did not affect the rise in [Ca(2+)](i) during HPV. 4. Depolarisation-induced constriction was unaffected by hypoxia except in the absence of glucose, when it was depressed by approximately 50 %. Depolarisation-induced constriction was depressed by rotenone during hypoxia by 23 +/- 4 %; cyanide was without effect. 5. Hypoxia increased 2-deoxy-[(3)H]glucose uptake in endothelium-denuded IPAs by 235 +/- 32 %, and in mesenteric arteries by 218 +/- 38 %. 6. We conclude that complex III of the mitochondrial ETC acts as the hypoxic sensor in HPV, and initiates the rise in smooth muscle [Ca(2+)](i) by a mechanism unrelated to changes in cytosolic redox state per se, but more probably by increased production of superoxide. Additionally, glucose and glycolysis are essential for development of the sustained phase 2 of HPV, and support an endothelium-dependent Ca(2+)-sensitisation pathway rather than the rise in [Ca(2+)](i).

Animals↗
PubMed2004

The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells.

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN(-)) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca(2+), which was inhibited on removal of extracellular Ca(2+). Under current clamp conditions rotenone and CN(-) caused a rapid membrane depolarization and elevation of [Ca(2+)](i). Voltage clamping cells to -70 mV substantially attenuated this rise in [Ca(2+)](i). Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K(+) currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K(+) current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN(-) or rotenone or the mitochondrial uncoupler p-trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.

Animals↗
PubMed2005

Inhibition of complex I of the electron transport chain causes O2-. -mediated mitochondrial outgrowth.

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O(2)(-)* production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O(2)(-)* production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY(581/591). Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O(2)(-)* to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O(2)(-)* production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O(2)(-)* production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O(2)(-)* to prevent alterations in mitochondrial morphology.

Cells, Cultured↗
PubMed1990

Differential effects of respiratory inhibitors on glycolysis in proximal tubules.

The effects of inhibition of mitochondrial energy production at various points along the respiratory chain on glycolytic lactate production and transport function were examined in a suspension of purified rabbit renal proximal tubules. Paradoxically, partial blockage at site 3 by hypoxia (1% O2) induced lactate production, whereas total site 3 blockage by anoxia (0% O2) failed to stimulate glycolysis. Compared with anoxia, hypoxic tubules exhibited greater preservation of ATP and K+ contents during O2 deprivation and more fully recovered oxidative metabolism and transport function during reoxygenation. The mitochondrial site 1 inhibitor rotenone and the uncoupler carbonyl cyanide-p-trifluorome-thoxyphenylhydrazone (FCCP) were equipotent stimuli for lactate production, whereas the site 2 inhibitor antimycin A failed to stimulate glycolysis despite a 90% inhibition of O2 consumption. Compared with antimycin A, treatment with rotenone or FCCP resulted in less cell injury [measured by lactate dehydrogenase (LDH) release] and greater preservation of cell K+ and ATP contents. 2-Deoxyglucose blocked lactate production by 50% in the presence of rotenone and increased LDH release, suggesting that glycolytic ATP is partially protective. Addition of ouabain during rotenone treatment reduced lactate production by 50%, indicating that glycolytic ATP can be used to fuel the Na pump when mitochondrial ATP production is inhibited. We conclude that 1) proximal tubules can generate lactate during inhibition of oxidative metabolism by hypoxia, rotenone, or FCCP; 2) mitochondrial inhibition is not obligatorily linked to activation of glycolysis, since neither anoxia nor antimycin A stimulate lactate production; 3) when ATP can be produced through anaerobic glycolysis it serves to protect cell viability and transport function during respiratory inhibition.

Adenosine Triphosphate↗
PubMed2001

The endosomal compartment is an insulin-sensitive recruitment site for GLUT4 and GLUT1 glucose transporters in cardiac myocytes.

In nonstimulated cardiomyocytes, the glucose transporter GLUT4 is confined to intracellular vesicles forming at least two populations: a storage pool enriched in GLUT4 (pool 1) and an endosomal pool containing both GLUT4 and GLUT1 (pool 2). We have now studied the dynamics of these pools in response to insulin or the mitochondrial inhibitor rotenone in rat cardiomyocytes. Rotenone recruited GLUT4 and GLUT1 to the cell surface from endosomal pool 2 without affecting pool 1. Kinetic experiments were consistent with rotenone acting on an intracellular compartment that is in close connection with the plasma membrane. In contrast, insulin caused rapid, complete depletion of GLUT4 from pool 1 and reduced the GLUT1 content of pool 2 by approximately 50%, whereas, surprisingly, no net decrease in GLUT4 occurred in this pool. Subsequent insulin withdrawal resulted in slow replenishment of pool 2 with GLUT1 and of pool 1 with GLUT4. When pool 1 was still largely depleted of GLUT4, a second insulin challenge did reduce GLUT4 in pool 2 and stimulated glucose transport to the same extent as the first insulin treatment. In conclusion, the storage pool is the primary source of GLUT4 in response to insulin, but not to rotenone. In addition, the endosomal compartment is an important recruitment site of both GLUT1 and GLUT4 when the storage pool is either unaffected (rotenone) or depleted (by a previous insulin challenge). GLUT4 mobilized by insulin from the storage pool may pass through an intermediary (possibly endosomal) compartment on its way to the cell surface.

Animals↗
PubMed2005

Partial mitochondrial inhibition causes striatal dopamine release suppression and medium spiny neuron depolarization via H2O2 elevation, not ATP depletion.

Mitochondrial dysfunction is a potential causal factor in Parkinson's disease. We show here that acute exposure to the mitochondrial complex I inhibitor rotenone (30-100 nM; 30 min) causes concentration-dependent suppression of single-pulse evoked dopamine (DA) release monitored in real time with carbon-fiber microelectrodes in guinea pig striatal slices, with no effect on DA content. Suppression of DA release was prevented by the sulfonylurea glibenclamide, implicating ATP-sensitive K+ (KATP) channels; however, tissue ATP was unaltered. Because KATP channels can be activated by hydrogen peroxide (H2O2), as well as by low ATP, we examined the involvement of rotenone-enhanced H2O2 generation. Confirming an essential role for H2O2, the inhibition of DA release by rotenone was prevented by catalase, a peroxide-scavenging enzyme. Striatal H2O2 generation during rotenone exposure was examined in individual medium spiny neurons using fluorescence imaging with dichlorofluorescein (DCF). An increase in intracellular H2O2 levels followed a similar time course to that of DA release suppression and was accompanied by cell membrane depolarization, decreased input resistance, and increased excitability. Extracellular catalase markedly attenuated the increase in DCF fluorescence and prevented rotenone-induced effects on membrane properties; membrane changes were also largely prevented by flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thus, partial mitochondrial inhibition can cause functional DA denervation via H2O2 and KATP channels, without DA or ATP depletion. Furthermore, amplified H2O2 levels and TRP channel activation in striatal spiny neurons indicate potential sources of damage in these cells. Overall, these novel factors could contribute to parkinsonian motor deficits and neuronal degeneration caused by mitochondrial dysfunction.

Adenosine Triphosphate↗
PubMed1995

A selective toxicity toward cultured mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic metabolism impairment and NMDA receptor activation.

Numerous observations strongly support the hypothesis that dopaminergic neurons could be particularly vulnerable to an impairment of their energetic metabolism. In order to demonstrate the existence of such a selective vulnerability, the toxic effects of rotenone, an inhibitor of complex I of the respiratory chain, and of glutamate, which is very likely involved in the neurotoxicity induced by an energetic stress, were analyzed on cultured mouse mesencephalic neurons. Toxicity toward dopaminergic and GABAergic neurons was compared by measuring the residual uptakes of dopamine and GABA. Exposure to 5 nM rotenone for 6 hr or to a low concentration of glutamate (100 microM) for 1 hr did not lead to a high selective toxic effect on dopaminergic neurons. In contrast, dopaminergic neurons were three times less resistant to the sequential exposure to rotenone and glutamate than GABAergic neurons. A particular resistance of mesencephalic GABAergic neurons to the synergistic toxic effects of rotenone and glutamate was ruled out since two other neuronal types, the striatal cholinergic and GABAergic neurons, displayed the same weak vulnerability as the mesencephalic GABAergic neurons. This selective toxic effect of glutamate on rotenone-pretreated dopaminergic neurons was blocked by either AMPA or NMDA receptor antagonists and mimicked by combined treatment with AMPA and NMDA, or by NMDA alone when the medium was deprived of Mg2+ ions. Moreover, this NMDA-selective neurotoxicity was critically dependent on the presence of a physiological extracellular sodium concentration, since the use of choline chloride instead of sodium chloride had a protective effect on dopaminergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

A rat model of Parkinsonism shows depletion of dopamine in the retina.

The retinal dopamine (DA) deficiency is an important feature of the pathogenesis in Parkinson's disease (PD) visual dysfunction. Systemic inhibition of complex I (rotenone) in rats has been proposed as a model of PD. In this study, we investigated whether systemic inhibition of complex I can induce impairment of DA-ergic cells in the retina, similar to the destruction of retinal cells found in PD patients. Rotenone (2.5mg/kg i.p., daily) was administered over 60 days. Neurochemically, rotenone treated rats showed a depletion of DA in the striatum and substantia nigra (SN). In addition, the number of retinal DA-ergic amacrine cells was significantly reduced in the rotenone treated animals. This study is the first one giving highlight towards a deeper understanding of systemic complex I inhibition (rotenone as an environmental toxin) and the connection between both, DA-ergic degeneration in the nigrostriatal pathway, and in the DA-ergic amacrine cells of the retina.

Animals↗
PubMed1999

The nuclear origin of the non-phosphorylating NADH dehydrogenases of plant mitochondria.

The oxidation of matrix and cytosolic NADH by isolated beetroot and wheat leaf mitochondria was investigated to determine whether the rotenone-insensitive NADH dehydrogenases of plant mitochondria were the products of nuclear or mitochondrial genes. After aging beetroot tissue (slicing and incubating in a CaSO4 solution), the induction of the level of matrix NADH oxidation in the presence of rotenone was greatly reduced in mitochondria isolated from tissue treated with cycloheximide, a nuclear protein synthesis inhibitor. This was also true for the oxidation of cytosolic NADH. Mitochondria isolated from chloramphenicol-treated tissue exhibited greatly increased levels of both matrix and external rotenone-insensitive NADH oxidation when compared to the increase due to the aging process alone. This increase was not accompanied by an increase in matrix NAD-linked substrate dehydrogenases such as malic enzyme nor intra-mitochondrial NAD levels. Possible explanations for this increase in rotenone-insensitive NADH oxidation are discussed. Based on these results we have concluded that the matrix facing rotenone-insensitive NADH dehydrogenase of plant mitochondria is encoded by a nuclear gene and synthesis of the protein occurs in the cytosol.

Cell Nucleus↗
PubMedDate not supplied

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures.

Adenosine Triphosphate↗
PubMed1994

NADH: ubiquinone oxidoreductase in obligate aerobic yeasts.

The strictly aerobic yeasts Candida pinus, Cryptococcus albidus, Rhodotorula minuta, Rhodotorula mucilaginosa and Trichosporon beigelii possess mitochondrial NADH dehydrogenases with significant features of the NADH:ubiquinone oxidoreductase (complex I). These species show in all growth phases and under standard cultivation conditions, NADH dehydrogenases of approximately 700 kDa, which are sensitive to rotenone, a specific inhibitor of this complex. Identical results were obtained with the weakly fermenting C. pinus. The facultatively fermenting yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus do not possess the 700 kDa-complex and are insensitive to rotenone. In S. cerevisiae, a rotenone-insensitive NADH dehydrogenase of about 500-600 kDa is detected only in stationary phase cells. As in Neurospora crassa, upon incubation of the obligately aerobic yeast R. mucilaginosa with chloramphenicol, an intermediate NADH dehydrogenase of approximately 350 kDa was formed, which was insensitive to rotenone.

Aerobiosis↗
PubMed1990

The abnormal-shaped mitochondria in thymus lymphocytes treated with inhibitors of mitochondrial energetics.

The effects of uncouplers (DNP, FCCP), oligomycin, and rotenone on the energetics and mitochondrial ultrastructure in lymphocytes have been studied. We confirmed the previous observations done on Ehrlich ascites and cardiomyocyte culture cells that uncouplers and respiratory inhibitors cause the appearance of ringlike and dumbbell-like mitochondria. It is shown that this effect does not correlate with decrease in ATP concentration, changes in oxygen consumption, or condensation of the mitochondrial matrix. FCCP (2 microM) is more effective in the induction of abnormal-form mitochondria than 240 microM DNP, oligomycin, or rotenone. Combined treatment with DNP, oligomycin, and rotenone or with DNP and rotenone produces an effect as strong as 2 microns FCCP. DNP (240 microM) and FCCP (2 microM) have a similar effect on respiration and intracellular ATP, but only the latter induces condensation of the mitochondrial matrix.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
PubMed1995

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria.

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits from Neurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits in Neurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.

Animals↗