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PubMed2000

cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures.

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.

3T3 Cells↗
PubMed2000

Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells.

The Ndi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells (Seo, B. B., Kitajima-Ihara, T., Chan, E. K., Scheffler, I. E., Matsuno-Yagi, A., and Yagi, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9167-9171) and human embryonal kidney 293 (HEK 293) cells (Seo, B. B., Matsuno-Yagi, A., and Yagi, T. (1999) Biochim. Biochem. Acta 1412, 56-65) and that the Ndi1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I). To extend the potential use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1). With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1. The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells. Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1-transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated. The Ndi1 protein has a great potential as a molecular remedy for complex I defects, and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.

Dependovirus↗
PubMed2000

Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria. evidence for the slow active/inactive transition.

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg(2+) was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg(2+). After exposure to elevated temperature (37 degrees C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg(2+). The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.

Alamethicin↗
PubMed2001

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Binding Sites↗
PubMed2003

Regional loss of the mitochondrial membrane potential in the hepatocyte is rapidly followed by externalization of phosphatidylserines at that specific site during apoptosis.

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to approximately 30% and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATP-consuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme.

Adenosine Triphosphate↗
PubMed2003

Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition.

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50-200 microm H(2)O(2) caused the activation of caspase-3 beginning after 2-3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H(2)O(2)-induced caspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H(2)O(2) or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss.

Apoptosis↗
PubMed2004

Beta-synuclein regulates Akt activity in neuronal cells. A possible mechanism for neuroprotection in Parkinson's disease.

Recent studies have shown that the neurodegenerative process in disorders with Lewy body formation, such as Parkinson's disease and dementia with Lewy bodies, is associated with alpha-synuclein accumulation and that beta-synuclein might protect the central nervous system from the neurotoxic effects of alpha-synuclein. However, the mechanisms are unclear. The main objective of the present study was to investigate the potential involvement of the serine threonine kinase Akt (also known as protein kinase B) signaling pathway in the mechanisms of beta-synuclein neuroprotection. For this purpose, Akt activity and cell survival were analyzed in synuclein-transfected B103 neuroblastoma cells and primary cortical neurons. Beta-synuclein transfection resulted in increased Akt activity and conferred protection from the neurotoxic effects of rotenone. Down-regulation of Akt expression resulted in an increased susceptibility to rotenone toxicity, whereas transfection with a lentiviral vector encoding for beta-synuclein was protective. The effects of beta-synuclein on the Akt pathway appear to be by direct interaction between these molecules and were independent of upstream signaling molecules. Taken together, these results indicate that the mechanisms of beta-synuclein neuroprotection might involve direct interactions between beta-synuclein and Akt and suggest that this signaling pathway could be a potential therapeutic target for neurological conditions associated with parkinsonism and alpha-synuclein aggregation.

Cell Line↗
PubMed2004

Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Neither the route of electron transport nor the sites or mechanism of superoxide production in mitochondrial complex I has been established. We examined the rates of superoxide generation (measured as hydrogen peroxide production) by rat skeletal muscle mitochondria under a variety of conditions. The rate of superoxide production by complex I during NADH-linked forward electron transport was less than 10% of that during succinate-linked reverse electron transport even when complex I was fully reduced by pyruvate plus malate in the presence of the complex III inhibitor, stigmatellin. This asymmetry was not explained by differences in protonmotive force or its components. However, when inhibitors of the quinone-binding site of complex I were added in the presence of ATP to generate a pH gradient, there was a rapid rate of superoxide production by forward electron transport that was as great as the rate seen with reverse electron transport at the same pH gradient. These observations suggest that quinone-binding site inhibitors can make complex I adopt the highly radical-producing state that occurs during reverse electron transport. Despite complete inhibition of NADH: ubiquinone oxidoreductase activity in each case, different classes of quinone-binding site inhibitor (rotenone, piericidin, and high concentrations of myxothiazol) gave different rates of superoxide production during forward electron transport (the rate with myxothiazol was twice that with rotenone) suggesting that the site of rapid superoxide generation by complex I is in the region of the ubisemiquinone-binding sites and not upstream at the flavin or low potential FeS centers.

Animals↗
PubMed2006

Reactive oxygen and targeted antioxidant administration in endothelial cell mitochondria.

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H(2)O(2). In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H(2)O(2) fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.

Animals↗
PubMed1990

A sensitive method for measuring ATP-formation in rat muscle mitochondria.

A sensitive method for the measurement of the ATP production rate in isolated skeletal muscle mitochondria is presented. Mitochondrial suspensions were prepared by differential centrifugation from approximately 80 mg of soleus muscle. ATP production rates were measured luminometrically, utilizing a reagent based upon firefly luciferase, which emits light proportional to the ATP concentration. In a group of 10 rats the ATP production rates were measured with the following substrate combinations: pyruvate + malate, palmitoyl-L-carnitine + malate, alpha-ketoglutarate, succinate + rotenone and succinate alone. The variance of the method including tissue preparation, protein determination and the luminometric determination of ATP production was estimated to be 10-14% for the various substrates. Compared to values in the literature, the present results show a good agreement for the substrates pyruvate + malate and palmitoyl-L-carnitine + malate, but lower rates were obtained in our study for alpha-ketoglutarate and succinate + rotenone. The advantage of the luminometric method is its high sensitivity. Only 30-40 mg of tissue is required for a complete determination, compared to 1-2 g for a similar assay of oxygen consumption. The method is intended for use in human subjects and will facilitate studies of mitochondrial respiration both in patients of different age groups and in healthy subjects.

Adenosine Triphosphate↗
PubMedDate not supplied

Complex I and Parkinson's disease.

Complex I of the mammalian electron transfer chain is composed of at least 43 protein subunits, of which 7 are encoded by mtDNA. It catalyzes the transfer of electrons from NADH to ubiquinone and translocates protons from the mitochondrial matrix to the intermembrane space. It may also play direct roles in the mitochondrial permeability transition and in cell death pathways. Despite the limitations of current complex I assays, biochemical studies have suggested the presence of a mild, systemic defect of complex I in Parkinson's disease (PD). Recent experimental work has modeled this abnormality using rotenone to systemically inhibit complex I. Chronic rotenone exposure accurately recapitulated the pathological, biochemical, and behavioral features of PD. Thus, relatively subtle complex I abnormalities--either genetic or acquired--may be central to the pathogenesis of PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Mechanism of dihydrolipoate stimulation of the mitochondrial permeability transition: effect of different respiratory substrates.

The stimulation of the mitochondrial permeability transition (MPT) by dihydrolipoate (DHLA) was studied in rat liver mitochondria in the presence of different respiratory substrates. The Ca2+ threshold for the induction of MPT was lowest for pyruvate, followed by 2-hydroxybutyrate, 2-oxoglutarate, glutamate plus malate, and succinate plus rotenone, both in the presence and absence of DHLA. DHLA was not able to induce MPT in the absence of Ca2+, in the presence of cyclosporin A, or rotenone with pyridine nucleotide-dependent substrates. The difference in sensitivity of MPT to DHLA with various substrates was correlated with the redox state of pyridine nucleotides but not the redox state of glutathione. These findings demonstrate that DHLA induced MPT pore opening through the P-site thiol. The similarities between the effect of DHLA and that of production of reactive oxygen species found in model experiments suggest that DHLA stimulates MPT by production of reactive oxygen species that exhaust the antioxidant defence.

Animals↗
PubMed1999

ATP dependence of the ICl,swell channel varies with rate of cell swelling. Evidence for two modes of channel activation.

Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4-5 min with solutions containing (microM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25-35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15-55%/min in the absence of intracellular ATP, current activation was slow (0.3-0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4-6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4-6 pA/pF per min) in the absence of ATP when cells were swollen at rates >/=65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.

Adenosine Triphosphate↗
PubMed2003

Metallothionein attenuates 3-morpholinosydnonimine (SIN-1)-induced oxidative stress in dopaminergic neurons.

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other subcortical nuclei associated with a widespread occurrence of Lewy bodies. The causes of cell death in Parkinson's disease are still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative stress have been proposed. We have examined 3-morpholinosydnonimine (SIN-1)-induced apoptosis in control and metallothionein-overexpressing dopaminergic neurons, with a primary objective to determine the neuroprotective potential of metallothionein against peroxynitrite-induced neurodegeneration in Parkinson's disease. SIN-1 induced lipid peroxidation and triggered plasma membrane blebbing. In addition, it caused DNA fragmentation, alpha-synuclein induction, and intramitochondrial accumulation of metal ions (copper, iron, zinc, and calcium), and enhanced the synthesis of 8-hydroxy-2-deoxyguanosine. Furthermore, it down-regulated the expression of Bcl-2 and poly(ADP-ribose) polymerase, but up-regulated the expression of caspase-3 and Bax in dopaminergic (SK-N-SH) neurons. SIN-1 induced apoptosis in aging mitochondrial genome knockout cells, alpha-synuclein-transfected cells, metallothionein double-knockout cells, and caspase-3-overexpressed dopaminergic neurons. SIN-1-induced changes were attenuated with selegiline or in metallothionein-transgenic striatal fetal stem cells. SIN-1-induced oxidation of dopamine to dihydroxyphenylacetaldehyde was attenuated in metallothionein-transgenic fetal stem cells and in cells transfected with a mitochondrial genome, and enhanced in aging mitochondrial genome knockout cells, in metallothionein double-knockout cells and caspase-3 gene-overexpressing dopaminergic neurons. Selegiline, melatonin, ubiquinone, and metallothionein suppressed SIN-1-induced down-regulation of a mitochondrial genome and up-regulation of caspase-3 as determined by reverse transcription-polymerase chain reaction. The synthesis of mitochondrial 8-hydroxy-2-deoxyguanosine and apoptosis-inducing factors were increased following exposure to 1-methyl-4-phenylpyridinium ion or rotenone. Pretreatment with selegiline or metallothionein suppressed 1-methyl-4-phenylpyridinium ion-, 6-hydroxydopamine-, and rotenone-induced increases in mitochondrial 8-hydroxy-2-deoxyguanosine accumulation. Transfection of aging mitochondrial genome knockout neurons with mitochondrial genome encoding complex-1 or melanin attenuated the SIN-1-induced increase in lipid peroxidation. SIN-1 induced the expression of alpha-synuclein, caspase-3, and 8-hydroxy-2-deoxyguanosine, and augmented protein nitration. These effects were attenuated by metallothionein gene overexpression. These studies provide evidence that nitric oxide synthase activation and peroxynitrite ion overproduction may be involved in the etiopathogenesis of Parkinson's disease, and that metallothionein gene induction may provide neuroprotection.

Animals↗
PubMedDate not supplied

Reactive oxygen and nitrogen species: weapons of neuronal destruction in models of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease whose etiology and pathogenesis remain mainly unknown. To investigate its cause and, more particularly, its mechanism of neuronal death, numerous in vivo experimental models have been developed. Currently, both genetic and toxic models of PD are available, but the use of neurotoxins such as 6-hydroxydopamine, paraquat, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and rotenone are still the most popular means for modeling the destruction of the nigrostriatal dopaminergic neurons seen in PD. These four neurotoxins, although distinct in their intimate cytotoxic mechanisms, kill dopaminergic neurons via a cascade of deleterious events that consistently involves oxidative stress. Herein, we review and compare the molecular mechanisms of 6-hydroxydopamine, paraquat, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine, and rotenone, placing the emphasis of our discussion on how reactive oxygen and nitrogen species contribute to the neurotoxic properties of these four molecules. As the reader will discover, to achieve the above stated goal, we had to not only appraise recent findings, but also revisit earlier landmark studies to provide a comprehensive view on this topic. This approach also enabled us to describe how our understanding of the mechanism of actions of certain toxins has evolved over time, which is particularly striking in the case of the quatrogenarian neurotoxin, 6-hydroxydopamine.

Animals↗
PubMed1984

The action of beta-adrenoceptor antagonists on rat heart mitochondrial function in vitro: a comparison of propranolol, timolol, and atenolol.

The effect of beta-adrenoceptor antagonists, propranolol, timolol and atenolol on functions of rat heart mitochondria in vitro was investigated. Concentrations of all three drugs less than 2 mmol X litre-1 did not affect rates of respiration of uncoupled intact mitochondria supported by substrates of alpha-oxoglutarate, pyruvate-malate or succinate. Inhibition was observed if mitochondria were broken to allow access of substrates and drugs into the mitochondria. Studies of the effect of drug concentration on NADH-supported oxygen uptake and NADH oxidase activity of submitochondrial particles produced from rat heart mitochondria showed a marked inhibition by propranolol: the effect of timolol and atenolol were much less marked and inhibition was only observed at relatively high concentrations of these drugs. Transfer of electrons through Complex I measured by the rate of reduction of ferricyanide by submitochondrial particles inhibited by rotenone and the lack of specific inhibition by propranolol of succinate supported respiration indicated that one site of drug action was between NADH dehydrogenase and its associated flavoprotein, possibly close to the site of rotenone inhibition.

Animals↗
PubMed1997

HIV type 1 Tat protein enhances activation-but not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals.

T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5-tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.

Adjuvants, Immunologic↗
PubMed2006

Obligatory role for complex I inhibition in the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder characterized by a loss of dopamine-producing neurons in the substantia nigra and corresponding motor deficits. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I of the mitochondrial respiratory chain, displacement of dopamine from vesicular stores, and formation of reactive oxygen species from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxicity is still a matter of debate. Recently, we reported that the yeast single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase (NDI1) is resistant to rotenone, a complex I inhibitor that produces a parkinsonian syndrome in rats, and that overexpression of NDI1 in SK-N-MC cells prevents the toxicity of rotenone. In this study, we used viral-mediated overexpression of NDI1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxicity of MPTP. In cell culture, NDI1 overexpression abolished the toxicity of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of NDI1 through stereotactic administration of a viral vector harboring the NDI1 gene into the substantia nigra protected mice from both the neurochemical and behavioral deficits elicited by MPTP. These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits produced by MPTP. Furthermore, combined with reports of a complex I defect in Parkinson's disease (PD) patients, the present study affirms the utility of MPTP in understanding the molecular mechanisms underlying dopaminergic neurodegeneration in PD.

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