Modulation of mitochondrial complex I activity by reversible Ca2+ and NADH mediated superoxide anion dependent inhibition

Complex I, a key component of the mitochondrial respiratory chain, exhibits diminished activity as a result of cardiac ischemia/reperfusion. Cardiac ischemia/reperfusion is associated with increases in the levels of mitochondrial Ca(2+) and pro-oxidants. In the current in vitro study, we sought evidence for a mechanistic link between Ca(2+), pro-oxidants, and inhibition of complex I utilizing mitochondria isolated from rat heart. Our results indicate that addition of Ca(2+) to solubilized mitochondria results in loss in complex I activity. Ca(2+) induced a maximum decrease in complex I activity of approximately 35% at low micromolar concentrations over a narrow physiologically relevant pH range. Loss in activity required reducing equivalents in the form of NADH and was not reversed upon addition of EGTA. The antioxidants N-acetylcysteine and superoxide dismutase, but not catalase, prevented inhibition, indicating the involvement of superoxide anion (O2(*-)) in the inactivation process. Importantly, the sulfhydryl reducing agent DTT was capable of fully restoring complex I activity implicating the formation of sulfenic acid and/or disulfide derivatives of cysteine in the inactivation process. Finally, complex I can reactivate endogenously upon Ca(2+) removal if NADH is present and the enzyme is allowed to turnover catalytically. Thus, the present study provides a mechanistic link between three alterations known to occur during cardiac ischemia/reperfusion, mitochondrial Ca(2+) accumulation, free radical production, and complex I inhibition. The reversibility of these processes suggests redox regulation of Ca(2+) handling.     

The effect of hyperoxia on superoxide production by lung submitochondrial particles

Terbium ion (Tb³⁺), like other rare earth lanthanides, has traditionally been viewed as binding nucleic acids at or near their ionized phosphate groups only. Here evidence is presented from ¹H NMR studies that confirms this mode of binding in Tb³⁺-mono-nucleotide complexes. However, in polynucleotides, we find that Tb³⁺ coordinately binds at two distinct sites, the phosphate moiety and electron donor groups on purine and pyrimidine bases. This two-site binding is best illustrated by complexes of Tb³⁺-polyuridylic acid, where the relative sensitivities of the uracil protons H5 and H6 to induced chemical shift and nuclear spin relaxation are the inverse of that seen in Tb³⁺-uridine monophosphate complexes. These data substantiate recently reported results derived from ultraviolet absorption and fluorescence spectroscopy (D. S. Gross and H. Simpkins, 1981, J. Biol. Chem.256, 9593–9598) that two-site binding is characteristic of the terbium(III)-polynucleotide interaction.     

Inhibition of superoxide anion production by extracellular acidification in neutrophils

Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O₂⁻) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E₁, also inhibited the formyl peptide-induced O₂⁻ production. The inhibitory action on the O₂⁻ production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O₂⁻ production.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Inhibition of mitochondrial Na+-Ca2+ exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.     

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2- generation in mitochondria respiring on the complex I substrates pyruvate+malate, an effect fully inhibited by rotenone. Antimycin A increased O2- production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2- production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2- formation driven with the complex II substrate succinate. At 0.6 microM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2- formation; however, at 40 microM myxothiazol (which completely inhibits both complexes I and III) eliminated O2- production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2- from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Inhibition of brain mitochondrial Ca2+ transport by amiloride analogues

Rat brain mitochondrial Ca2+ uptake and release were examined in the presence of amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)-pyrazinecarboxamide) and nineteen amiloride analogues. Amiloride, an inhibitor of Na+-Ca2+ exchange in plasmalemma membranes, did not affect energy-dependent Ca2+ uptake, whereas several other analogues were inhibitors. Similarly, amiloride did not alter Ca2+ release in the presence or absence of Na+. However, some analogues were found that stimulated and others that inhibited Ca2+ release. While many of these analogues reduced mitochondrial respiratory control ratios, two analogues were identified which inhibited Ca2+ uptake but did not alter mitochondrial respiratory control. Similarly two analogues were identified which inhibited Ca2+ efflux without affecting respiratory control.     

Opening mitoKATP increases superoxide generation from complex I of the electron transport chain

Opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of mitoK(ATP)-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the K(ATP) channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K(+) influx as K(ATP) channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K(+) influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoK(ATP)-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

Inhibition of the veratridine-induced increase in cytosolic Ca2+ and respiration by Ca2+ antagonists in isolated cardiac myocytes.

1. We studied the effect of verapamil, nitrendipine, 3',4'-dichlorobenzamil (DCB) and Cd2+ on the increase in cytosolic free Ca2+ ([Ca2+]c) and the rate of O2-uptake induced by depolarization of isolated rat cardiac myocytes with veratridine. 2. The degree of inhibition by the several drugs tested on the increase in [Ca2+]c and respiration was dependent on extracellular Ca2+, pH and Na+. 3. Low verapamil and nitrendipine concentrations (2.5 microM) were fully effective in Ca2+ channel blockade, as indicated from experiments with isoproterenol and in a low-Na+ medium. 4. A complete inhibition of veratridine-induced increase in [Ca2+]c and O2-uptake was attained with higher Ca2+ blocker concentrations (25-30 microM), implying that these processes depend to a major extent on some other Ca2+ transport system, probably Na+/Ca2+ exchange.     

Inhibition by reactive aldehydes of superoxide anion radical production in stimulated human neutrophils

alpha,beta-Unsaturated aldehydes were investigated in vitro for their ability to inhibit superoxide anion radical (O2-.) production in stimulated human polymorphonuclear leukocytes (PMN). The aldehydes investigated were (i) trans-4-hydroxynonenal and malonaldehyde (MDA), two toxic lipid peroxidation products; (ii) acrolein and crotonaldehyde, two air pollutants derived from fossil fuel combustion; (iii) trans,trans-muconaldehyde, a putative hematotoxic benzene metabolite. Preincubation of PMN with reactive aldehydes followed by stimulation with the oxygen burst initiator phorbol myristate acetate (PMA) resulted in a dose-dependent inhibition of O2-. production. The concentration at which 50% inhibition (IC50) was observed was 21 microM for acrolein, 23 microM for trans,trans-muconaldehyde, 27 microM for trans-4-hydroxynonenal and 330 microM for crotonaldehyde. A similar inhibitory effect by these aldehydes was observed in digitonin- and concanavalin A-stimulated PMN. MDA inhibited O2-. production in PMA-stimulated PMN by 100% at 10(-2) M but gave no inhibition at 10(-3) M. The standard aldehyde propionaldehyde did not inhibit O2-. production at 10(-3)-10(-6) M. Preincubation of PMN with acrolein in the presence of cysteine completely protected against the inhibitory effect of this reactive aldehyde. The results indicate that the ability of toxic aldehydes to inhibit O2-. production in stimulated PMN correlates directly with their alkylation potential which is a function of the electrophilicity of the beta carbon.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

Inhibition and inactivation of NADH-cytochrome c reductase activity of bovine heart submitochondrial particles by the iron(III)-adriamycin complex.

The NADH-cytochrome c reductase activity of bovine heart submitochondrial particles was found to be slowly (half-time of 16 min) and progressively lost upon incubation with the Fe2(+)-adriamycin complex. In addition to this slow progressive inactivation seen on incubation, a reversible fast phase of inhibition was also seen. However, if EDTA was added to the incubation mixture within 15 s, the slow progressive loss in activity was largely preventable. Separate experiments indicated that EDTA removed about one-half of the iron from the Fe2(+)-adriamycin complex in about 40 s. These results indicated the requirement for iron for the inactivation process. Since the Vmax. for the fast phase of inhibition was decreased by the inhibitor, the inhibition pattern was similar to that seen for uncompetitive or mixed-type inhibition. The direct binding of both Fe3(+)-adriamycin and adriamycin to submitochondrial particles was also demonstrated, with the Fe3(+)-adriamycin complex binding 8 times more strongly than adriamycin. Thus binding of Fe3(+)-adriamycin to the enzyme or to the inner mitochondrial membrane with subsequent generation of oxy radicals in situ is a possible mechanism for the Fe3(+)-adriamycin-induced inactivation of respiratory enzyme activity.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Inhibition of the Na+/Ca2+ exchange in cardiac sarcolemmal vesicles by amiloride

The pyrazine diuretic amiloride inhibits the Na+/Ca2+ exchange activity of cardiac sarcolemmal vesicles in a concentration-dependent way. A good relationship between the uptake of amiloride by the vesicles and the inhibition of the exchanger has been found. Kinetic analyses indicate that the inhibition of Na+/Ca2+ exchange activity by amiloride is non-competitively removed by Ca2+ and competitively overcome by an outwardly directed Na+ gradient.