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PubMed2006

Mitochondrial activity is modulated by TNFalpha and IL-1beta in normal human chondrocyte cells.

OBJECTIVE: Pro-inflammatory cytokines play an important role in osteoarthritis (OA). In osteoarthritic cartilage, chondrocytes exhibit an alteration in mitochondrial activity. This study analyzes the effect of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) on the mitochondrial activity of normal human chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase, as well as by mitochondrial membrane potential (Deltapsim) and adenosine triphosphate (ATP) synthesis. Bcl-2 family mRNA expression and protein synthesis were analyzed by RNase protection assay (RPA) and Western-blot, respectively. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and apoptosis by 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) stain. Glycosaminoglycans were quantified in supernatant by a dimethyl-methylene blue binding assay. RESULTS: Compared to basal cells, stimulation with TNFalpha (10 ng/ml) and IL-1beta (5 ng/ml) for 48 h significantly decreased the activity of complex I (TNFalpha=35% and IL-1beta=35%) and the production of ATP (TNFalpha=18% and IL-1beta=19%). Both TNFalpha and IL-1beta caused a definitive time-dependent decrease in the red/green fluorescence ratio in chondrocytes, indicating depolarization of the mitochondria. Both cytokines induced mRNA expression and protein synthesis of the Bcl-2 family. Rotenone, an inhibitor of complex I, caused a significant reduction of the red/green ratio, but it did not reduce the viability of the chondrocytes. Rotenone also increased Bcl-2 mRNA expression and protein synthesis. Finally, rotenone as well as TNFalpha and IL-1beta, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. CONCLUSION: These results show that both TNFalpha and IL-1beta regulate mitochondrial function in human articular chondrocytes. Furthermore, the inhibition of complex I by both cytokines could play a key role in cartilage degradation induced by TNFalpha and IL-1beta. These data could be important for understanding of the OA pathogenesis.

Adenosine Triphosphate↗
PubMed2006

Enhanced sensitivity of DJ-1-deficient dopaminergic neurons to energy metabolism impairment: role of Na+/K+ ATPase.

DJ-1 gene mutations lead to an inherited form of early-onset parkinsonism. The function of DJ-1 is unclear, though a neuroprotective role has been postulated. Electrophysiological recordings were made of striatal and dopaminergic nigral neurons both of wild-type (WT) and DJ-1-knockout (DJ-1(-/-)) mice. We assessed the responses of dopaminergic cells to combined oxygen and glucose deprivation (OGD), and to the mitochondrial toxin rotenone. OGD induced a membrane hyperpolarization in nigral neurons from WT mice. Similarly, rotenone hyperpolarized neurons and then a depolarization occurred. In DJ-1(-/-) mice, the OGD-induced hyperpolarization was significantly enhanced. Moreover, rotenone caused a shorter hyperpolarization followed by an irreversible depolarization. To evaluate the involvement of Na+/K+ ATPase, we tested ouabain, a Na+/K+ ATPase inhibitor, on two distinct neuronal subtypes. Compared to WT mice, in dopaminergic neurons from DJ-1(-/-) mice, ouabain induced rapid and irreversible membrane potential changes. Notably, this effect was observed at concentrations that were unable to produce membrane potential shifts on striatal spiny neurons, both from WT and DJ-1(-/-) mice. These findings suggest that DJ-1 loss-of-function enhances vulnerability to energy metabolism alterations, and that nigral neurons are particularly sensitive to Na+/K+ ATPase impairment.

Animals↗
PubMed2004

The modulation of action potential generation by calcium-induced calcium release is enhanced by mitochondrial inhibitors in mudpuppy parasympathetic neurons.

Previously, we demonstrated that outward currents activated by calcium-induced calcium release (CICR) opposed depolarization-induced action potential (AP) generation in dissociated mudpuppy parasympathetic neurons [J Neurophysiol 88 (2002) 1119]. In the present study, we tested whether AP generation by depolarizing current ramps could be altered by dissipating the mitochondrial membrane potential and thus interrupting mitochondrial Ca2+ buffering. Exposure to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 microM) alone or in combination with the mitochondrial ATP synthase inhibitor oligomycin (8 microg/ml), increased the latency to AP generation. Exposure to the electron transport chain inhibitor rotenone (10 microM) alone or in combination with oligomycin (8 microg/ml) similarly increased the latency to AP generation. CCCP and oligomycin or rotenone and oligomycin treatment caused rhodamine 123 loss from mitochondria within a few minutes, confirming that the mitochondrial membrane potential was dissipated during drug exposure. Oligomycin alone had no effect on the latency to AP generation and did not cause loss of rhodamine 123 from mitochondria. The increase in latency induced by CCCP and oligomycin was similar when recordings were made with either the perforated patch or standard whole cell patch recording configuration. Exposure to the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (1 microM), decreased the latency to AP generation. In cells pretreated with thapsigargin to eliminate CICR, CCCP and oligomycin had no effect on AP latency. Pretreatment with iberiotoxin (IBX; 100 nM), an inhibitor of large conductance, calcium- and voltage-activated potassium channels, reduced the extent of the CCCP- and oligomycin-induced increase in latency to AP generation. These results indicate that treatment with CCCP or rotenone to dissipate the mitochondrial membrane potential, a condition which should minimize sequestration of Ca2+ by mitochondria, facilitated the Ca(2+)-induced Ca2+ release activation of IBX-sensitive and IBX-insensitive conductances that regulate AP generation.

Action Potentials↗
PubMed2003

Mitochondrial effects with ceramide-induced cardiac apoptosis are different from those of palmitate.

The objective of this study was to evaluate whether ceramide, palmitate, and inhibitors of mitochondrial electron transport chain shared similar effects on the mitochondria of intact cardiomyocytes in order to determine the likelihood that ceramide and palmitate utilize similar mitochondrial mechanisms or pathways to apoptosis. In embryonic chick cardiomyocytes, ceramide, 100 microM for 24h, induced a 42.9+/-5.8% increase in cell death assessed by the MTT assay, and a significant (P<0.01) 3.9+/-0.6-fold increase in apoptosis assessed by propidium iodide staining of permeabilized cells. Mitochondrial potential (delta psi (m)), as demonstrated microscopically and by flow cytometry of cardiomyocytes stained with a J-aggregate dye, was markedly and significantly reduced by ceramide, palmitate, and two different inhibitors of the mitochondrial electron transport chain-rotenone and antimycin A. In contrast, the effect on mitochondria as assessed by CMX-Ros oxidation was dramatically different, as palmitate, rotenone, and antimycin A each produced a reduction, while ceramide increased CMX-Ros fluorescence. Further ceramide-induced cardiomyocyte apoptosis and loss of delta psi (m) operated through a cyclosporine-insensitive pathway similar to rotenone and antimycin A but distinct from palmitate which induced apoptosis though a cyclosporine-sensitive mechanism in these cells. These data suggest that ceramide acts on the mitochondria of intact cells through a cyclosporine-insensitive mechanism likely from a combination of actions including production of mitochondrial oxidants. The discordant findings between ceramide and palmitate suggest that palmitate-induced cell death is not primarily mediated by de novo ceramide synthesis.

Animals↗
PubMed2001

Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium.

The compound 1-methyl-4-phenylpyridinium (MPP) is a selective inhibitor of mitochondrial complex I, and is widely used in model systems to elicit neurochemical alterations that may be associated with Parkinson's disease. In the present study treatment of human neuroblastoma SH-SY5Y cells with MPP resulted in a time- and concentration-dependent activation of the apoptosis-associated cysteine protease caspase-3, and caused morphological changes characteristic of apoptosis. To test if the activation state of the cell survival-promoting phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway affects MPP-induced caspase-3 activation, PI3K was inhibited with LY294002, or activated with insulin-like growth factor-1. MPP-induced caspase-3 activation was increased by inhibition of PI3K, and decreased by stimulation of PI3K, indicative of anti-apoptotic signaling by the PI3K/Akt pathway. To test if glycogen synthase kinase-3beta (GSK3beta), a pro-apoptotic kinase that is inhibited by Akt, is involved in regulating MPP-induced apoptosis, overexpression of GSK3beta and lithium, a selective inhibitor of GSK3beta, were used to directly alter GSK3beta activity. MPP-induced caspase-3 activity was increased by overexpression of GSK3beta. Conversely, the GSK3beta inhibitor lithium attenuated MPP-induced caspase-3 activation. To test if these regulatory interactions applied to other mitochondrial complex I inhibitors, cells were treated with rotenone. Rotenone-induced activation of caspase-3 was enhanced by inhibition of PI3K or increased GSK3beta activity, and was attenuated by inhibiting GSK3beta with lithium. Overall, these results indicate that inhibition of GSK3beta provides protection against the toxic effects of agents, such as MPP and rotenone, that impair mitochondrial function.

1-Methyl-4-phenylpyridinium↗
PubMed1999

Functional consequences of the 3460-bp mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy.

Complex I is the largest of the mitochondrial respiratory chain proteins, and contains subunits encoded by both mitochondrial and nuclear genomes. Leber's hereditary optic neuropathy has been clearly linked to mutations of mitochondrial DNA complex I genes, and variable complex I functional defects have been reported. We have confirmed an approximate 60% defect in mitochondrial NADH CoQ1 reductase activity in cultured fibroblasts bearing the 3460-bp G to A mutation within the ND1 gene. However complex I-linked ATP synthesis was found to be normal in these fibroblasts. A 60% rotenone-induced decrease in complex I activity was shown to reduce ATP synthesis in normal fibroblasts, indicating that this level of complex I activity was below the threshold required to affect ATP synthesis. Although 3460 LHON mitochondria were less sensitive to rotenone inhibition, this did not explain the decreased complex I activity as the rotenone insensitive activity was not increased, nor did the inhibitor diphenyleneiodonium inhibit the NADH CoQ1 reductase activity to a greater extent. Decreased NADH cytochrome c reductase activity in cybrids homoplasmic for the 3460 LHON mtDNA mutation confirmed that the decrease in complex I activity was not specific to the assay used and was not caused by inhibitory effects of ubiquinone analogues used in the NADH CoQ1 reductase assay. These findings have important implications for our understanding of complex I dysfunction in the pathogenesis of 3460 Leber's hereditary optic neuropathy.

Adenosine Triphosphate↗
PubMed2002

The fetal and neonatal brain protein neuronatin protects PC12 cells against certain types of toxic insult.

The protein neuronatin is expressed in the nervous system of the fetus and neonate at a much higher level than in the adult. Its function is unknown. As a result of variable splicing, neuronatin mRNA exists in two forms, alpha and beta. Wild type PC12 cells express neuronatin-alpha. We have isolated a PC12 variant, called 1.9, that retains many of the neuron-like properties of wild type PC12 cells, but it does not express neuronatin and it exhibits markedly increased sensitivity to the toxic effects of nigericin, rotenone and valinomycin. Pretreatment of the 1.9 cells with alpha-methyltyrosine, which inhibits dopamine synthesis, had little effect on the cells' sensitivity to nigericin, rotenone or valinomycin indicating that dopamine-induced oxidative stress was not involved in the toxicity of these compounds. However, flattened cell subvariants of the 1.9 cells, which do not have any neuron-specific characteristics, did not exhibit increased sensitivity to nigericin indicating that some neuronal characteristic of the 1.9 cells contributed to the toxicity of nigericin. After the neuronatin-beta gene was transfected into and expressed in the 1.9 cells, they regained wild type PC12 levels of resistance to nigericin, rotenone and valinomycin. These studies suggest that the function of neuronatin during development could be to protect developing cells from toxic insult occurring during that period.

Animals↗
PubMed2001

Interleukin 1-induced cyclooxygenase and nitric oxide synthase gene expression in the rat dorsal root ganglia is modulated by antioxidants.

Interleukin 1beta induced both nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2) gene expression in dorsal root ganglion explant culture with increased NOS-2 and COX-2 activities, and corresponding increases in the production of nitric oxide and prostaglandin E(2). The proinflammatory cytokine also increased 8-isoprostaglandin F(2alpha) concentration, an index of oxidant stress-mediated production of lipid hydroperoxides/reactive oxygen species. The signaling mechanisms by which interleukin 1beta regulates NOS-2 and COX-2 genes remain obscure. Reactive oxygen species play an important role in inflammatory processes as mediators of injury, and potentially as intracellular signaling molecules in interleukin 1beta-mediated regulation of gene expression. The effects of antioxidants that act by different mechanisms on interleukin 1beta-mediated NOS-2 and COX-2 gene expression were studied in rat dorsal root ganglion explants. The oxidant scavenger pyrrolidine dithiocarbamate abolished interleukin 1beta-induced NOS-2 mRNA accumulation and decreased nitric oxide production in a concentration-dependent manner, thus indicating that this antioxidant decreased either the transcription of NOS-2 gene or the stability of NOS-2 mRNA. In contrast, pyrrolidine dithiocarbamate significantly inhibited COX-2 gene expression at the posttranscriptional level, since pyrrolidine dithiocarbamate did not affect interleukin 1beta-induced COX-2 mRNA transcripts but inhibited COX-2 protein expression and prostaglandin E(2) production. Rotenone, another antioxidant that attenuates reactive oxygen species production by inhibiting the mitochondrial electron transport system, failed to inhibit interleukin 1beta-induced NOS-2 and COX-2 mRNA-encoding transcripts. However, rotenone inhibited NOS-2 and COX-2 proteins and associated nitric oxide and prostaglandin E(2) production, respectively, suggesting a posttranscriptional target for interleukin 1beta-mediated regulation of NOS-2 and COX-2 gene expression. Furthermore, both pyrrolidine dithiocarbamate and rotenone also decreased interleukin 1beta-induced 8-isoprostaglandin F(2alpha) production. These results indicate that not only transcriptional regulation, but also posttranscriptional events are involved in a redox-sensitive regulation of interleukin 1beta-induced NOS-2 and COX-2 gene expression in the dorsal root ganglia. Overall, interleukin 1beta-induced oxidant stress appears to regulate NOS-2 and COX-2 gene expression primarily at the level of protein translation. By implicating reactive oxygen species production in interleukin-1beta receptor-activated molecular signaling in the dorsal root ganglia, our data suggest a possible novel target for intervention in cytokine-mediated inflammatory processes.

Animals↗
PubMed2003

The mitochondrial complex I inhibitor annonacin is toxic to mesencephalic dopaminergic neurons by impairment of energy metabolism.

The death of dopaminergic neurons induced by systemic administration of mitochondrial respiratory chain complex I inhibitors such as 1-methyl-4-phenylpyridinium (MPP(+); given as the prodrug 1-methyl-1,2,3,6-tetrahydropyridine) or the pesticide rotenone have raised the question as to whether this family of compounds are the cause of some forms of Parkinsonism. We have examined the neurotoxic potential of another complex I inhibitor, annonacin, the major acetogenin of Annona muricata (soursop), a tropical plant suspected to be the cause of an atypical form of Parkinson disease in the French West Indies (Guadeloupe). When added to mesencephalic cultures for 24 h, annonacin was much more potent than MPP(+) (effective concentration [EC(50)]=0.018 versus 1.9 microM) and as effective as rotenone (EC(50)=0.034 microM) in killing dopaminergic neurons. The uptake of [(3)H]-dopamine used as an index of dopaminergic cell function was similarly reduced. Toxic effects were seen at lower concentrations when the incubation time was extended by several days whereas withdrawal of the toxin after a short-term exposure (<6 h) arrested cell demise. Unlike MPP(+) but similar to rotenone, the acetogenin also reduced the survival of non-dopaminergic neurons. Neuronal cell death was not excitotoxic and occurred independently of free radical production. Raising the concentrations of either glucose or mannose in the presence of annonacin restored to a large extent intracellular ATP synthesis and prevented neuronal cell demise. Deoxyglucose reversed the effects of both glucose and mannose. Other hexoses such as galactose and fructose were not protective. Attempts to restore oxidative phosphorylation with lactate or pyruvate failed to provide protection to dopaminergic neurons whereas idoacetate, an inhibitor of glycolysis, inhibited the survival promoting effects of glucose and mannose indicating that these two hexoses acted independently of mitochondria by stimulating glycolysis. In conclusion, our study demonstrates that annonacin promotes dopaminergic neuronal death by impairment of energy production. It also underlines the need to address its possible role in the etiology of some atypical forms of Parkinsonism in Guadeloupe.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Anomalous structure-activity relationships of 13-homo-13-oxarotenoids and 13-homo-13-oxadehyrorotenoids.

Cube resin, used as an insecticide/miticide and piscicide, contains in decreasing amounts rotenone (1), deguelin (2), the 6a,12a-dehydro derivatives of rotenone (3) and deguelin (4), and the newly-discovered 13-homo-13-oxa-6a,12a-dehydro analogs [referred to as oxadehydrorotenone (5) and -deguelin (6)]. These six rotenoids were compared for potency as inhibitors of NADH:ubiquinone oxidoreductase activity and for organismal toxicity to mosquito larvae, goldfish, and mice and cytotoxicity in three mammalian cell lines (Hepa 1C1C7, MCF 7, and NB 41A3). Although rotenoids 3-6 contribute very little to the overall activity of cube resin, there were two surprising aspects to the structure-activity relationships. First, 1 was 7-15-fold more active than 2 in the cytotoxicity assays of 4-day duration but not in the other systems. This difference in cytotoxicity is not due to specificity at the oxidoreductase target but instead to more extensive cytochrome P450-dependent (piperonyl butoxide-sensitive) detoxification of 2 than of 1. Second, the observed potency increase on conversion of dehydrorotenone to either rotenone or oxadehydrorotenone suggests that combining both structural changes to form cis-13-homo-13-oxarotenone (8) might result in maximal activity. Accordingly, 5 was reduced with diisobutylaluminum hydride to the trans-isomer 7 and then epimerized with aqueous pyridine to the cis-isomer 8 of the same configuration as 1. Surprisingly, 8 was much less active than 1. This is rationalized on the basis of conformational changes in the B/C ring system and decreasing dihedral angle (determined by X-ray crystallography and/or molecular modeling) between the A and D rings that follow the potency order, i.e., rotenoids 1 and 2 > oxadehydrorotenoids 5 and 6 > trans- and cis-oxarotenoids 7 and 8 > dehydrorotenoids 3 and 4. Thus, the novel oxarotenoids and oxadehydrorotenoids help define the conformation optimal for NADH:ubiquinone oxidoreductase inhibition and toxicity.

Aedes↗
PubMed2000

Calcium sequestering ability of mitochondria modulates influx of calcium through glutamate receptor channel.

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca2+ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca2+ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca2+]i followed by a slow and persistently rising phase. The rapid increase in [Ca2+]i was mainly due to influx of Ca2+ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca2+ in the extracellular medium elicited a small transient rise in [Ca2+]i which can be attributed to the mobilization of Ca2+ from IP3 sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca2+ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca2+ uniporter. These results imply that mitochondria sequester the Ca2+ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca2+ caused permeability transition and released the sequestered Ca2+ which is manifested as slow rise in [Ca2+]i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca2+]i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca2+]i response. Combined treatment with oligomycin and rotenone further diminished the [Ca2+]i response and also abolished the CCCP mediated rise in [Ca2+]i. However, rotenone alone had no effect on glutamate induced [Ca2+]i response. These changes in glutamate-induced [Ca2+]i response could not be explained on the basis of deficient mitochondrial Ca2+ sequestration or ATP dependent Ca2+ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca2+ buffering processes leading to elevation of [Ca2+]i. In contrast our results showed repression of Ca2+ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca2+ sequestering ability of mitochondria, the glutamate induced Ca2+ influx could be impeded. To validate this, influx of Mn2+ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca2+ sequestration may desensitize the NMDA receptor operated channel.

Adenosine Diphosphate↗
PubMed1992

In vitro metabolic inhibition of the human lymphocyte: influence on the expression of interleukin-2 receptors.

We have investigated the effects of energy depletion in human lymphocytes on the expression of the membrane-bound interleukin-2 receptor (IL-2R) and the release of soluble IL-2R. Concanavalin-A (Con-A) and IL-2-transformed lymphocytes were incubated with sodium fluoride, inhibiting the Embden-Meyerhof pathway, rotenone and 2,4-dinitrophenol (2,4-DNP), which are known to disturb mitochondrial high energy production. Rotenone was found to be the most potent inhibitor, with a half maximal effect at 10 nmol/L. 2,4-DNP and sodium fluoride, although not as potent as rotenone, showed marked inhibitory effects in higher concentrations, with half maximal effects at 50 mumol/L and 5 mmol/L respectively. No difference in the inhibition pattern for membrane-bound IL-2R compared with sIL-2R was observed. It is concluded that intracellular synthesis, transport and subsequent membrane insertion or release of receptors important for immunoregulation require high energy phosphate compounds and are sensitive to disturbances in intracellular energy levels.

2,4-Dinitrophenol↗
PubMed1999

Dual actions of the metabolic inhibitor, sodium azide on K(ATP) channel currents in the rat CRI-G1 insulinoma cell line.

1. The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP-sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI-G1) cell line using a combination of whole cell and single channel recording techniques. 2. Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI-G1 cells with a concomitant increase in K+ conductance. This is due to activation of K(ATP) channels as the sulphonylurea tolbutamide (100 microM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 microM) or oligomycin (2 microM) did not hyperpolarize CRI-G1 cells or increase K+ conductance. 3. In cell-attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in K(ATP) channel activity, an action readily reversible by tolbutamide (100 microM). Application of sodium azide (3 mM), in the presence of Mg-ATP, to the intracellular surface of excised inside-out patches also increased K(ATP) channel activity, in a reversible manner. 4. In contrast, rotenone (10 microM) or oligomycin (2 microM) did not increase K(ATP) channel activity in either cell-attached, in the absence of glucose, or inside-out membrane patch recordings. 5. Addition of sodium azide (3 mM) to the intracellular surface of inside-out membrane patches in the presence of Mg-free ATP or the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) inhibited, rather than increased, K(ATP) channel activity. 6. In conclusion, sodium azide, but not rotenone or oligomycin, directly activates K(ATP) channels in CRI-G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide.

Adenosine Triphosphate↗
PubMed2005

Estrogen-induced G1/S transition of G0-arrested estrogen-dependent breast cancer cells is regulated by mitochondrial oxidant signaling.

We previously reported that 17-beta-estradiol (E2)-induced mitochondrial reactive oxygen species (mtROS) act as signaling molecules. The purpose of this study was to investigate the effects of E2-induced mtROS on cell cycle progression. E2-induced cell growth was reduced by antioxidants N-acetyl-L-cysteine (NAC), catalase, and the glutathione peroxidase mimic ebselen. Flow cytometry showed that mitochondrial blockers of protein synthesis (chloramphenicol), transcription and replication (ethidium bromide), and function (rotenone, rhodamine 6G) blocked E2-induced G1 to S transition. Reduction of E2-induced DNA synthesis in the presence of mitochondrial blockers occurred without influencing the level of ATP. Additionally, the mitochondrial blockers inhibited the E2-induced expression of early cell cycle genes such as cyclins D1, D3, E1, E2, and B2. NAC or rotenone reduced E2-induced cyclin D1 expression. Furthermore, E2-induced binding of AP-1 and CREB to the TRE and CRE response sequences, respectively, in the promoter of cyclin D1 was inhibited by NAC or rotenone. In addition, E2-induced expression of PCNA, PRC1, and bcl-2 were inhibited by mitochondrial blockers. These data indicate that E2-induced mtROS are involved in the regulation of early G1-phase progression. Since neither antioxidants nor mitochondrial blockers used in this study are reported to bind the estrogen receptor (ER), our findings suggest that E2-induced mtROS modulates G1 to S transition and some of the early G1 genes through a nongenomic, ER-independent signaling pathway. Thus, our results suggest (1) a new paradigm that estrogen-induced mitochondrial oxidants control the early stage of cell cycle progression and (2) provide the basis for the discovery of novel antioxidant-based drugs or antioxidant gene therapies for the prevention and treatment of estrogen-dependent breast cancer.

Base Sequence↗
PubMed1972

Proton translocation coupled to quinone reduction by reduced nicotinamide--adenine dinucleotide in rat liver and ox heart mitochondria.

Measurements were made of the stoicheiometry of proton-translocation coupled to NAD(P)H oxidation by several quinones (duroquinone, ubiquinone(0), ubiquinone(1), ubiquinone(2)) in mitochondria from rat liver and ox heart. Observed stoicheiometries of protons translocated per mol of NADH oxidized (-->H(+)/2e(-) ratios; Mitchell, 1966) ranged from 0.75 (rat liver mitochondria with ubiquinone(1)) to 1.55 (ox heart mitochondria with ubiquinone(1) or ubiquinone(2)). Only the rotenone-sensitive pathway of NADH oxidation by quinone was able to support proton translocation. Correction of the observed -->H(+)/2e(-) ratios for the loss of reducing equivalents to the rotenone-insensitive pathway increased their value to approx. 2.0. It is concluded that the rotenone-sensitive NADH- ubiquinone reductase activity of the respiratory chain may be organized in the mitochondrial membrane as a proton-translocating oxidoreduction loop. The number of such loops between NADH and ubiquinone is one, and not two, as initially proposed by Mitchell (1966).

Animals↗
PubMed2002

Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state.

Several lines of evidence indicate that mitochondrial reactive oxygen species (ROS) generation is the major source of oxidative stress in the cell. It has been shown that ROS production accompanies cytochrome c release in different apoptotic paradigms, but the site(s) of ROS production remain obscure. In the current study, we demonstrate that loss of cytochrome c by mitochondria oxidizing NAD(+)-linked substrates results in a dramatic increase of ROS production and respiratory inhibition. This increased ROS production can be mimicked by rotenone, a complex I inhibitor, as well as other chemical inhibitors of electron flow that act further downstream in the electron transport chain. The effects of cytochrome c depletion from mitoplasts on ROS production and respiration are reversible upon addition of exogenous cytochrome c. Thus in these models of mitochondrial injury, a primary site of ROS generation in both brain and heart mitochondria is proximal to the rotenone inhibitory site, rather than in complex III. ROS production at complex I is critically dependent upon a highly reduced state of the mitochondrial NAD(P)(+) pool and is achieved upon nearly complete inhibition of the respiratory chain. Redox clamp experiments using the acetoacetate/L-beta-hydroxybutyrate couple in the presence of a maximally inhibitory rotenone concentration suggest that the site is approx. 50 mV more electronegative than the NADH/NAD(+) couple. In the absence of inhibitors, this highly reduced state of mitochondria can be induced by reverse electron flow from succinate to NAD(+), accounting for profound ROS production in the presence of succinate. These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe-S centre N-1a.

3-Hydroxybutyric Acid↗
PubMed2002

NADH oxidation and NAD+ reduction catalysed by tightly coupled inside-out vesicles from Paracoccus denitrificans.

Tightly coupled inside-out vesicles were prepared from Paracoccus denitrificans cells (SPP, sub-Paracoccus particles) and characterized kinetically. The rate of NADH oxidation, catalysed by SPP, increases 6-8 times on addition of gramicidin. The vesicles are capable of catalysing Delta micro H+-dependent reverse electron transfer from quinol to NAD+. The kinetic parameters of the NADH-oxidase and the reverse electron transfer carried out by membrane-bound P. denitrificans complex I were estimated and compared with those of the mitochondrial enzyme. The data demonstrate that catalytic properties of the dinucleotide-binding site of the bacterial and mitochondrial complex I are almost identical, pointing out similar organization of the site in mammals and P. denitrificans. Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme. The inhibitor is capable of suppressing the NADH oxidation reaction only at micromolar concentrations, while the activity of mitochondrial enzyme is suppressed by nanomolar concentrations of rotenone. In contrast to the mitochondrial enzyme, rotenone, even at concentrations as high as 10 micro m, does not inhibit the reverse, Delta micro H+-dependent NAD+-reductase reaction on SPP.

Animals↗
PubMed2003

Cytosol-mitochondria transfer of reducing equivalents by a lactate shuttle in heterotrophic Euglena.

To assess the expression and physiological role of the mitochondrial NAD(+)-independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the d- and l-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three- to fourfold from latency to the stationary phase. Intracellular levels of D- and L-lactate were high (5-40 mm) from the start of the culture and increased (20-80 mm) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactate-grown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle).

Adenosine Triphosphate↗