Inhibitory effects of S-nitrosoglutathione on cell proliferation and DNA synthesis: possible role of glyoxalase I inactivation

We investigated the inhibitory effects of S-nitrosoglutathione (GSNO) on cell proliferation, DNA synthesis and several enzymatic activities using spontaneously immortalized human endothelial cells (ECV304). Proliferation of ECV304 was inhibited by GSNO in a dose-dependent manner (125-1000 microM). DNA synthesis was decreased 2 h after addition of GSNO to cells and was markedly repressed from 20 h after the addition. The activity of ribonucleotide reductase, a rate-limiting enzyme for DNA synthesis, was unchanged in GSNO-treated cells. GSNO inhibited less than 40% of mitochondrial respiration activity, and the membrane potential and cellular levels of ATP were not significantly decreased by GSNO. GSNO had no inhibitory effect on activities of glutathione peroxidase, glutathione S-transferase and glutathione reductase. However, glyoxalase I (Glo I) activity was decreased to 20% of the control level within 60 min, and was consistently repressed during exposure to GSNO for 20 h. A membrane-permeable Glo I inhibitor, S-bromobenzylglutathione diethylester, inhibited proliferation of ECV304 cells, while methylglyoxal (MG), a toxic metabolite generated during glycolysis and a substrate for Glo I, failed to inhibit the cell growth even at 100 microM. Glo I in several mammalian cell lines was inactivated by GSNO with a pI shift. Although we failed to detect accumulation of MG under conditions of Glo I inactivation, these results suggest that the inhibitory effects of GSNO on cell proliferation and DNA synthesis might be at least partly due to inactivation of Glo I.     

Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues

Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration. We then discuss their interactions with phospholipid liposomes, studied with differential scanning calorimetry, spin labelling techniques and UV-Vis spectrophotometry, in order to obtain information on drug distribution and the modifications they impose on lipid bilayer. The results of the experiments performed on mitochondria and model membranes prove an interesting correlation between the effects of the molecules on both systems.     

Control of skeletal muscle mitochondria respiration by adenine nucleotides: differential effect of ADP and ATP according to muscle contractile type in pigs

Skeletal muscle exhibits considerable variation in mitochondrial content among fiber types, but it is less clear whether mitochondria from different fiber types also present specific functional and regulatory properties. The present experiment was undertaken on ten 170-day-old pigs to compare functional properties and control of respiration by adenine nucleotides in mitochondria isolated from predominantly slow-twitch (Rhomboideus (RM)) and fast-twitch (Longissimus (LM)) muscles. Mitochondrial ATP synthesis, respiratory control ratio (RCR) and ADP-stimulated respiration with either complex I or II substrates were significantly higher (25-30%, P<0.05) in RM than in LM mitochondria, whereas no difference was observed for basal respiration. Based on mitochondrial enzyme activities (cytochrome c oxidase [COX], F0F1-ATPase, mitochondrial creatine kinase [mi-CK]), the higher ADP-stimulated respiration rate of RM mitochondria appeared mainly related to a higher maximal oxidative capacity, without any difference in the maximal phosphorylation potential. Mitochondrial K(m) for ADP was similar in RM (4.4+/-0.9 microM) and LM (5.9+/-1.2 microM) muscles (P>0.05) but the inhibitory effect of ATP was more marked in LM (P<0.01). These findings demonstrate that the regulation of mitochondrial respiration by ATP differs according to muscle contractile type and that absolute muscle oxidative capacity not only relies on mitochondrial density but also on mitochondrial functioning per se.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.     

Therapeutic doses of menadione reduce the rotenone-induced inhibition of respiration and membrane potential generation in mitochondria

Menadione restores the rotenone-inhibited respiration of diaphragm muscle pieces in approximately the same degree as the respiration of heart mitochondria, i.e., to 30-40%. The respiration of heart mitochondria induced by 2-5 microM menadione (after its inhibition by rotenone) is partly coupled with ATP synthesis whose rate is much lower than that of oxidation of NAD-dependent substrates. The effects of menadione and mitochondrial energetics inhibitors on lymphocyte respiration and rhodamine 123 fluorescence in individual lymphocytes and their suspensions were compared. Menadione (2--5 microM) increased the rotenone + oligomycin suppressed delta psi m in lymphocytes. At 5-40 microM menadione did not act as an uncoupler and had little effect on the uncoupled lymphocyte respiration. All these effects were observed at menadione concentrations close to therapeutic ones. Vicasol, a water-soluble analog of menadione, exerted a similar effect.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.     

Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria

Chicken liver mitochondria consumed O2 at an accelerated rate when supplied with low concentrations of hydrogen sulfide. Maximum respiration occurred in 10 microM sulfide, and continued more slowly up to concentrations as high as 60 microM. Sulfide oxidation was coupled to adenosine triphosphate (ATP) synthesis, as shown by firefly luciferase luminescence and by measurement of the mitochondrial membrane electrochemical gradient. Synthesis of ATP required low, steady-state concentrations of sulfide (< 5 microM), which were maintained by use of a syringe pump. The ratio of consumed O2 to sulfide changed at low sulfide and O2 concentrations, indicating alternative metabolic reactions and products. In low concentrations of sulfide, presumably most similar to physiological, the O2/sulfide ratio was 0.75. This is the first report of sulfide oxidation linked to ATP synthesis in any organism not specifically adapted to a sulfide-rich environment. We suggest that this may be a widespread mitochondrial trait, and that it is consistent with the hypothesis that mitochondria originated from sulfide-oxidizing symbionts.     

Huperzine A protects isolated rat brain mitochondria against β-amyloid peptide

Our previous work in cells and animals showed that mitochondria are involved in the neuroprotective effect of huperzine A (HupA). In this study, the effects of HupA on isolated rat brain mitochondria were investigated. In addition to inhibiting the Aβ_25-35 (40 μM)-induced decrease in mitochondrial respiration, adenosine 5′-triphosphate (ATP) synthesis, enzyme activity, and transmembrane potential, HupA (0.01 or 0.1 μM) effectively prevented Aβ-induced mitochondrial swelling, reactive oxygen species increase, and cytochrome c release. More interestingly, administration of HupA to isolated mitochondria promoted the rate of ATP production and blocked mitochondrial swelling caused by normal osmosis. These results indicate that HupA protects mitochondria against Aβ at least in part by preserving membrane integrity and improving energy metabolism. These direct effects on mitochondria further extend the noncholinergic functions of HupA.     

Inhibitors of the slow stage of proton transfer in the link connecting respiration with mitochondrial phosphorylation]

Electrophilic agents--derivatives of carbonic acids--are found to inhibit respiration, ATP synthesis and reverse electrone transport in intact mitochondria. The inhibition of respiration and ATPase was observed in intact mitochondria at 3 and 3u states (by Chance). Inhibitors concentrations, which caused 50% inhibition, were approximately the same. Sharp decrease of the effect of electrophilic inhibitors on respiration and ATPase activity in mitochondria and submitochondrial particles with substantially impaired coupling system was observed. The following conclusions are drawn on the basis of the data obtained: 1) electrophilic inhibitor attack the coupling site of respiration and ATP synthesis in mitochondria; 2) the reaction of the proton transport from the respiration proton pump to ATP synthetase is one of the slowest steps of the process of ATP-synthesis in mitochondria. A scheme of working the coupling system is suggested which includes the step of proton lateral diffusion.     

Uncoupling of oxidative phosphorylation: different effects of lipophilic weak acids and electrogenic ionophores on the kinetics of ATP synthesis

Previous studies from this laboratory have shown that the kinetics of ATP synthesis by bovine heart submitochondrial particles (SMP) are modulated by the coupled rate of respiration between two extremes of Vmax and apparent Km's for ADP and Pi [Matsuno-Yagi, A., & Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038; Hekman, C., Matsuno-Yagi, A., & Hatefi, Y. (1988) Biochemistry 27, 7559-7565]. Thus, with ADP as the variable substrate, ATP synthesis occurred with Vmax = 200 nmol of ATP min-1 (mg of protein)-1 at 30 degrees C and an apparent KmADP = 2-4 microM at low rates of respiration, and with Vmax = 11,000 nmol of ATP min-1 (mg of protein)-1 at 30 degrees C and an apparent KmADP = 120-160 microM at high rates of respiration. At intermediate respiration rates, it was necessary to introduce a third intermediate KmADP for best fit of the kinetic data, indicating that transition from one kinetic extreme to the other is not abrupt and involves intermediate kinetic states of the ATP synthase complexes. The present paper shows that uncouplers affect the kinetics of ATP synthesis by SMP in two ways. When used at moderate concentrations, electrogenic ionophores such as gramicidin D or valinomycin plus nigericin decreased the Vmax for ATP synthesis without changing the contributions of the low, intermediate, and high KmADP to the overall rate of ATP synthesis. By contrast, potent lipophilic weak acid uncouplers, such as FCCP, CCCP, S-13, and SF6847, decreased Vmax and converted the kinetics of ATP synthesis toward high KmADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat heart mitochondria release adenosine

Isolated rat heart mitochondria release adenosine under specific conditions. Lowest adenosine release occurs at 4 degrees C while highest release occurs in the presence of pyruvate + malate or rotenone at 30 degrees C. The release is attenuated during state 3 respiration, in the presence of atractyloside or in the presence of 1799. Oligomycin only partially decreases adenosine release. Release is unaffected by 200 microM Ca++ and is independent of oxygen concentration as low as 2 microM. The data are consistent with the hypothesis that adenosine is released from mitochondria via the adenine nucleotide transporter and the release is regulated by the intramitochondrial ATP to ADP ratio.     

AMP-dependence of the cyanide-insensitive pathway in the respiratory chain of Paramecium tetraurelia

The AMP-dependent stimulation of the cyanide-insensitive respiration of Paramecium mitochondria was investigated. The nucleotides exhibiting a stimulatory effect on the cyanide-insensitive oxidation of pyruvate (+ malate) in a medium supplemented with EDTA or carboxyatractyloside were, in decreasing order of efficiency, AMP, GMP, IMP, UMP and TMP. On the other hand, ADP, ATP and cyclic AMP were ineffective. In the presence of carboxyatractyloside, addition of AMP to Paramecium mitochondria incubated with pyruvate (+malate) led to an increase in membrane potential. In the absence of light, the photoactivable derivative of AMP, 3'-[4-[N-(4-azido-2-nitrophenyl)amino]butyryl]-AMP (NAP4-AMP) added to Paramecium mitochondria opposed the stimulatory effect of AMP on the cyanide-insensitive respiration; the Ki for NAP4-AMP was much lower than the Km for AMP, 0.2 microM compared with 120 microM. The ADP-stimulated respiration was not affected. Photoirradiation of Paramecium mitochondria in the presence of NAP4-AMP resulted in irreversible inhibition of the AMP-stimulated cyanide-insensitive respiration. No effect on the ADP-stimulated respiration was observed. A heatlabile cyanide-insensitive ubiquinol oxidase was extracted from Paramecium mitochondria with the detergent NN-dimethyl-N-(3-laurylamidopropyl)amine oxide. The quinol oxidase activity was slightly stimulated by AMP.     

The interactions of cobalt(II) with mitochondria from rat liver

The interactions of Co²⁺ with mitochondria have been investigated. The results indicate that Co²⁺ inhibits ATP synthesis. Further investigations into ATP synthesis mechanisms indicated that inhibition is due to the opening of a transmembrane pore. The opening of this pore causes the collapse of the high-energy intermediate where, under a pH and a potential gradient, the energy is stored and subsequently utilized to form ATP from ADP.     

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.     

Inhibition of oxygen consumption in skeletal muscle-derived mitochondria by pinacidil,diazoxide, and glibenclamide,but not by 5-hydroxydecanoate

Cell intermediary metabolism and energy production succeeds by means of mitochondria, whose activity is in relation to transmembrane potential and/or free radical production. Adenosine triphosphate (ATP)-dependent potassium channels (K_ATP) in several cell types have shown to couple cell metabolism to membrane potential and ATP production. In this study, we explore whether oxygen consumption in isolated skeletal-muscle mitochondria differs in the presence of distinct respiration substrates and whether these changes are affected by K_ATP-channel inhibitors such as glibenclamide, 5-Hydroxydecanoate (5-HD), and K_ATP channel activators (pinacidil and diazoxide). Results demonstrate a concentration-dependent diminution of respiration rate by glibenclamide (0.5–20 μM), pinacidil (1–50 μM), and diazoxide (50–200 μM), but no significant differences were found when the selective mitochondrial K_ATP-channel inhibitor (5-HD, 10–500 μM) was used. These results suggest that these K_ATP-channel agonists and antagonists exert an effect on mitochondrial respiration and that they could be acting on mito-K_ATP or other respiratory-chain components.     

Metabolic effects of carbenoxolone in rat liver

The action of carbenoxolone on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In perfused livers, carbenoxolone (200-300 microM) increased oxygen consumption, glucose production and glycolysis from endogenous glycogen. Gluconeogenesis from lactate or fructose, an energy-dependent process, was inhibited. This effect was already evident at a concentration of 25 microM. The cellular ATP levels and the adenine nucleotide content were decreased by carbenoxolone, whereas the AMP levels were increased. In isolated mitochondria, carbenoxolone stimulated state IV respiration and decreased the respiratory coefficient with the substrates beta-hydroxybutyrate and succinate. The ATPase of intact mitochondria was stimulated, the ATPase of uncoupled mitochondria was inhibited, and the ATPase of disrupted mitochondria was not altered by carbenoxolone. These results indicate that carbenoxolone acts as an uncoupler of oxidative phosphorylation and, possibly, as an inhibitor of the ATP/ADP exchange system. The inhibitory action of carbenoxolone on mitochondrial energy metabolism could be contributing to induce the mitochondrial permeability transition (MPT), a key phenomenon in apoptosis. The results of the present study can explain, partly at least, the in vivo hepatotoxic actions of carbenoxolone that were found in a previous clinical evaluation.     

Respiratory activation of 2,4-dinitrophenol-stimulated ATPase activity in plant mitochondria

A comparison has been made of cauliflower mitochondria, which have no 2,4-dinitrophenol-stimulated ATPase (EC 3,6,1,4), with corn mitochondria, which do. Unlike corn mitochondria, cauliflower mitochondria show poor initial respiratory control ratios and phosphate uptake, but these are normalized after the first ADP addition. Sonication or high pH treatment releases a high rate of oligomycin-sensitive ATPase, indicating ATP transport into cauliflower mitochondria is the limiting factor. A brief period of respiration will activate, or “prime,” the 2,4-dinitrophenol-stimulated ATPase of cauliflower mitochondria, and the activity is inhibited by atractyloside, mersalyl, and oligomycin. Influx pumping of phosphate or arsenate extends the time the priming period lasts after respiration ceases to 1–2 min unless the 2,4-dinitrophenol is added before the ATP, in which case the priming is collapsed. Respiratory priming seems to consist of creating a transmembrane potential, possibly in the form of a phosphate gradient, for driving the ATP^4?-ADP^3? transporter.