Vanadate oligomers: in vivo effects in hepatic vanadium accumulation and stress markers

The formation of vanadate oligomeric species is often disregarded in studies on vanadate effects in biological systems, particularly in vivo, even though they may interact with high affinity with many proteins. We report the effects in fish hepatic tissue of an acute intravenous exposure (12, 24 h and 7 days) to two vanadium(V) solutions, metavanadate and decavanadate, containing different vanadate oligomers administered at sub-lethal concentration (5 mM; 1 mg/kg). Decavanadate solution promotes a 5-fold increase (0.135 +/- 0.053 microg V(-1) dry tissues) in the vanadium content of the mitochondrial fraction 7 days after exposition, whereas no effects were observed after metavanadate solution administration. Reduced glutathione (GSH) levels did not change and the overall reactive oxygen species (ROS) production was decreased by 30% 24 h after decavanadate administration, while for metavanadate, GSH levels increased 35%, the overall ROS production was depressed by 40% and mitochondrial superoxide anion production decreased 45%. Decavanadate intoxication did not induce changes in the rate of lipid peroxidation till 12 h, but later increased 80%, which is similar to the increase observed for metavanadate after 24 h. Decameric vanadate administration clearly induces different effects than the other vanadate oligomeric species, pointing out the importance of taking into account the different vanadate oligomers in the evaluation of vanadium(V) effects in biological systems.     

Decavanadate inhibits catalysis by ribonuclease A

Pentavalent organo-vanadates have been used extensively to mimic the transition state of phosphoryl group transfer reactions. Here, decavanadate (V(10)O(28)6-) is shown to be an inhibitor of catalysis by bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry shows that the Kd for the RNase A decavanadate complex is 1.4 microM. This value is consistent with kinetic measurements of the inhibition of enzymatic catalysis. The interaction between RNase A and decavanadate has a coulombic component, as the affinity for decavanadate is diminished by NaCl and binding is weaker to variant enzymes in which one (K41A RNase A) or three (K7A/R10A/K66A RNase A) of the cationic residues near the active site have been replaced with alanine. Decavanadate is thus the first oxometalate to be identified as an inhibitor of catalysis by a ribonuclease. Surprisingly, decavanadate binds to RNase A with an affinity similar to that of the pentavalent organo-vanadate, uridine 2',3'-cyclic vanadate.     

Recent advances into vanadyl, vanadate and decavanadate interactions with actin

Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 μM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 μM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 μM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance.     

Decavanadate effects in biological systems

Vanadium biological studies often disregarded the formation of decameric vanadate species known to interact, in vitro, with high-affinity with many proteins such as myosin and sarcoplasmic reticulum calcium pump and also to inhibit these biochemical systems involved in energy transduction. Moreover, very few in vivo animal studies involving vanadium consider the contribution of decavanadate to vanadium biological effects. Recently, it has been shown that an acute exposure to decavanadate but not to other vanadate oligomers induced oxidative stress and a different fate in vanadium intracellular accumulation. Several markers of oxidative stress analyzed on hepatic and cardiac tissue were monitored after in vivo effect of an acute exposure (12, 24 h and 7 days), to a sub-lethal concentration (5 mM; 1 mg/kg) of two vanadium solutions ("metavanadate" and "decavanadate"). It was observed that "decavanadate" promote different effects than other vanadate oligomers in catalase activity, glutathione content, lipid peroxidation, mitochondrial superoxide anion production and vanadium accumulation, whereas both solutions seem to equally depress reactive oxygen species (ROS) production as well as total intracellular reducing power. Vanadium is accumulated in mitochondria in particular when "decavanadate" is administered. These recent findings, that are now summarized, point out the decameric vanadate species contributions to in vivo and in vitro effects induced by vanadium in biological systems.     

Recent perspectives into biochemistry of decavanadate

The number of papers about decavanadate has doubled in the past decade. In the present review, new insights into decavanadate biochemistry, cell biology, and antidiabetic and antitumor activities are described. Decameric vanadate species (V10) clearly differs from monomeric vanadate (V1), and affects differently calcium pumps, and structure and function of myosin and actin. Only decavanadate inhibits calcium accumulation by calcium pump ATPase, and strongly inhibits actomyosin ATPase activity (IC50 = 1.4 μmol/L, V10), whereas no such ef- fects are detected with V1 up to 150 μmol/L; prevents actin polymerization (IC50 of 68 μmol/L, whereas no effects detected with up to 2 mmol/L V1); and interacts with actin in a way that induces cysteine oxidation and vanadate reduction to vanadyl. Moreover, in vivo decavanadate toxicity studies have revealed that acute exposure to polyoxovanadate induces different changes in antioxidant enzymes and oxidative stress parameters, in comparison with vanadate. In vitro studies have clearly demonstrated that mitochondrial oxygen consumption is strongly affected by decavanadate (IC50, 0.1 μmol/L); perhaps the most relevant biological effect. Finally, decavanadate (100 μmol/L) increases rat adipocyte glucose accumulation more potently than several vanadium complexes. Preliminary studies sug- gest that decavanadate does not have similar effects in human adipocytes. Although decavanadate can be a useful biochemical tool, further studies must be carried out before it can be conf irmed that decavanadate and its complexes can be used as anticancer or antidiabetic agents.     

Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell line

Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 μM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85 ± 0.40 versus 3.95 ± 0.10 μg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 μM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems. Daniel M. Tiago and Vincent Laizé1 contributed equally to this work.     

Vanadium and cadmium in vivo effects in teleost cardiac muscle: metal accumulation and oxidative stress markers

Several biological studies associate vanadium and cadmium with the production of reactive oxygen species (ROS), leading to lipid peroxidation and antioxidant enzymes alterations. The present study aims to analyse and compare the oxidative stress responses induced by an acute intravenous exposure (1 and 7 days) to a sub-lethal concentration (5 mM) of two vanadium solutions, containing different vanadate n-oligomers (n=1-5 or n=10), and a cadmium solution on the cardiac muscle of the marine teleost Halobatrachus didactylus (Lusitanian toadfish). It was observed that vanadium is mainly accumulated in mitochondria (1.33+/-0.26 microM), primarily when this element was administrated as decameric vanadate, than when administrated as metavanadate (432+/-294 nM), while the highest content of cadmium was found in cytosol (365+/-231 nM). Indeed, decavanadate solution promotes stronger increases in mitochondrial antioxidant enzymes activities (catalase: +120%; superoxide dismutase: +140%) than metavanadate solution. On contrary, cadmium increases cytosolic catalase (+111%) and glutathione peroxidases (+50%) activities. It is also observed that vanadate oligomers induce in vitro prooxidant effects in toadfish heart, with stronger effects induced by metavanadate solution. In summary, vanadate and cadmium are differently accumulated in blood and cardiac subcellular fractions and induced different responses in enzymatic antioxidant defence mechanisms. In the present study, it is described for the first time the effects of equal doses of two different metals intravenously injected in the same fish species and upon the same exposure period allowing to understand the mechanisms of vanadate and cadmium toxicity in fish cardiac muscle.     

Do vanadate polyanions inhibit phosphotransferase enzymes?

Decavanadate inhibits hexokinase, adenylate kinase and phosphofructokinase; neither mono-, tri nor tetrameric vanadate anion is an inhibitor. Decavanadate inhibits phosphofructokinase obtained from bacterial and protistic sources. No form of vanadium(V) anion inhibits galacto-, glycero-, pyruvate and creatine kinase, or inorganic pyrophosphatase. Decavanadate appears to be a non-competitive inhibitor of both hexokinase substrates.     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Decavanadate possesses α-adrenergic agonist activity and a structural motif common with trans-β form of noradrenaline

Decavanadate, an inorganic polymer of vanadate, produced contraction of rat aortic rings at a relatively high concentration compared to phenylephrine, an agonist of -adrenergic receptor. This effect was blocked by two known a-adrenergic receptor antagonists, prazosin and phenoxybenzamine. Decavanadate, formed by possible dimerization of V5 under acid conditions, possessed a structural feature of two pairs of unshared oxygen atoms at a distance of 3.12 Å, not found in its constituents of V4 or V5. A structural motif of O..O..O using such oxygen atoms is recognized in decavanadate. This matches with a similar motif of N..O..O that uses the essential amino and hydroxyl groups of the side-chain and the m-hydroxyl group in trans-b form of noradrenaline. The interaction of such a structural motif with the membrane receptor is likely to be the basis of the unusual noradrenaline-mimic action of decavanadate.     

Effect of oxyanions of the early transition metals on rabbit skeletal muscle phosphorylase

The differential effects of the oxyanions of the early transition metals ions V(V), W(VI), and Mo(VI) on the catalytic activity and coenzyme binding of rabbit skeletal muscle phosphorylase are studied. The oligoanions of V(V), W(VI), and Mo(VI) are potent inhibitors of phosphorylase. Kinetic studies revealed that oligovanadates inhibit pyridoxal-reconstituted phosphorylase b by competing with both the substrate, glucose 1-phosphate, and the activator, phosphite, with K1 values of 4 microM and 6 microM, respectively. Oligovanadates in the millimolar concentration range inhibit phosphorylases a and b by competing with glucose 1-phosphate binding. The polymeric decavanadate and paratungstates caused time-dependent inactivation of phosphorylase. Spectral studies with tungstate and phosphorylase b revealed that the inactivation is due to deformation of the coenzyme site. Kinetic studies and the protective effects of substrate and effectors on inactivation and deformation by tungstate or vanadate suggest that deformation and inactivation is caused by a primary binding of the oligoanions at the glucose 1-phosphate site. Nuclear magnetic resonance (NMR) studies of vanadate-phosphorylase complexes and vanadate solutions under different conditions were carried out to ascertain the nature of vanadate ions interacting with functional groups in phosphorylase. The results suggest that decavanadate is the major protein-bound species. NMR studies also showed that guanidino groups react with decavanadate and suggest that arginine residues in phosphorylase are potential functional groups that can interact with decavanadate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.     

Vanadium distribution, lipid peroxidation and oxidative stress markers upon decavanadate in vivo administration

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.     

Oxidative stress in toadfish (Halobactrachus didactylus) cardiac muscle. Acute exposure to vanadate oligomers

Vanadate solutions as ‘metavanadate’ (containing ortho and metavanadate species) and ‘decavanadate’ (containing manly decameric species) (5 mM; 1 mg/kg) were injected intraperitoneously in Halobatrachus didactylus (toadfish), in order to evaluate the contribution of decameric vanadate species to vanadium (V) intoxication on the cardiac tissue. Following short-term exposure (1 and 7 days), different changes on antioxidant enzyme activities—superoxide dismutase (SOD), catalase (CAT), selenium-glutathione peroxidase (Se-GPx), total glutathione peroxidase (GPx), lipid peroxidation and subcellular vanadium distribution were observed in mitochondrial and cytosolic fractions of heart ventricle toadfish. After 1 day of vanadium intoxication, SOD, CAT and Se-GPx activities were decreased up to 25%, by both vanadate solutions, except mitochondrial CAT activity that increased (+23%) upon decavanadate administration. After 7 days of exposure, decavanadate versus metavanadate solutions promoted different effects mainly on cytosolic CAT activity (−56% versus −5%), mitochondrial CAT activity (−10% versus +10%) and total GPx activity (+1% versus −35%), whereas lipid peroxidation products were significantly increased (+82%) upon 500 μM decavanadate intoxication. Accumulation of vanadium in total (0.137±0.011 μg/g) and mitochondrial (0.022±0.001 μg/g) fractions was observed upon 7 days of metavanadate exposure, whereas for decavanadate, the concentration of vanadium increased in cytosolic (0.020±0.005 μg/g) and mitochondrial (0.021±0.009 μg/g) fractions. It is concluded that decameric vanadate species are responsible for a strong increase on lipid peroxidation and a decrease in cytosolic catalase activity thus contributing to oxidative stress responses upon vanadate intoxication, in the toadfish heart.     

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

Actin as a potential target for decavanadate

ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1 × 10^− 3 s^− 1), and an apparent dissociation constant (k_dapp) of 227.4 ± 25.7 μM and 112.3 ± 8.7 μM was obtained in absence or presence of 20 μM V₁₀, respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V₁₀) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V₁) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V₁ (0-200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: phosphate dependence and effects of other oxyanions

The effects of various oxyanions on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) were examined. No evidence could be found for an absolute dependence of the kinase activity on inorganic phosphate as was recently reported by M. Laloux, E. Van Schaftingen, and H.-G. Hers ((1985) Eur. J. Biochem. 148, 155-159). Rather, phosphate and arsenate activated the kinase by decreasing the enzyme's Km for fructose 6-phosphate without affecting its Km for ATP or Vmax. The Km of the kinase for fructose 6-phosphate in the presence of inorganic phosphate was found to be significantly lower (6 microM) than previously reported (30 microM) when the hydrolysis of fructose 2,6-bisphosphate by the concomitant bisphosphatase activity at low Fru 6-P concentrations was taken into account. The KA's for phosphate and arsenate activation of the kinase were 0.2 and 0.3 mM, respectively. A number of other oxyanions, including pyrophosphate, sulfate, tungstate, selenate, and molybdate all inhibited the kinase by increasing the Km for fructose 6-phosphate. The apparent Ki's for inhibition of the kinase were in the 0.5-1 mM range. In contrast, all of these oxyanions activated the bisphosphatase, with half-maximal effects requiring millimolar concentrations. Inorganic phosphate was the most potent activator with a KA of 1 mM. In contrast to the other oxyanions, vanadate and meta-periodate inhibited the kinase but had no effect on the bisphosphatase. Vanadate appeared to be a noncompetitive inhibitor since its effects were not overcome by Pi, ATP, or fructose 6-phosphate, and the species responsible was shown to be decavanadate. Like vanadate, meta-periodate had no effect on the bisphosphatase, though it was a potent inhibitor (I0.5 = 30 microM) of the kinase. Its effects were shown to be time-dependent and reversed by dithiothreitol, suggesting that it acted by an oxidative mechanism. These results augment the mounting body of evidence that the enzyme's two reactions are catalyzed at discrete active sites.     

Oxidative stress in toadfish (Halobactrachus didactylus) cardiac muscle: Acute exposure to vanadate oligomers

Vanadate solutions as ‘metavanadate’ (containing ortho and metavanadate species) and ‘decavanadate’ (containing manly decameric species) (5 mM; 1 mg/kg) were injected intraperitoneously in Halobatrachus didactylus (toadfish), in order to evaluate the contribution of decameric vanadate species to vanadium (V) intoxication on the cardiac tissue. Following short-term exposure (1 and 7 days), different changes on antioxidant enzyme activities—superoxide dismutase (SOD), catalase (CAT), selenium-glutathione peroxidase (Se-GPx), total glutathione peroxidase (GPx), lipid peroxidation and subcellular vanadium distribution were observed in mitochondrial and cytosolic fractions of heart ventricle toadfish. After 1 day of vanadium intoxication, SOD, CAT and Se-GPx activities were decreased up to 25%, by both vanadate solutions, except mitochondrial CAT activity that increased (+23%) upon decavanadate administration. After 7 days of exposure, decavanadate versus metavanadate solutions promoted different effects mainly on cytosolic CAT activity (−56% versus −5%), mitochondrial CAT activity (−10% versus +10%) and total GPx activity (+1% versus −35%), whereas lipid peroxidation products were significantly increased (+82%) upon 500 μM decavanadate intoxication. Accumulation of vanadium in total (0.137±0.011 μg/g) and mitochondrial (0.022±0.001 μg/g) fractions was observed upon 7 days of metavanadate exposure, whereas for decavanadate, the concentration of vanadium increased in cytosolic (0.020±0.005 μg/g) and mitochondrial (0.021±0.009 μg/g) fractions. It is concluded that decameric vanadate species are responsible for a strong increase on lipid peroxidation and a decrease in cytosolic catalase activity thus contributing to oxidative stress responses upon vanadate intoxication, in the toadfish heart.