Inhibitory Effect Of Vanadate Research Articles

Effect of sodium orthovanadate on ovulation isolated ovarian follicles in Siberian Sturgeon (Acipenser baerii) In Vitro

Effects of sodium orthovanadate on oocyte ovulation were examined during in vitro culture of Siberian sturgeon ovarian follicles from hibernating fish. It was shown that sodium orthovanadate stimulates ovulation of Siberian sturgeon oocytes in a dose-dependent manner. The stimulating or inhibitory effect of vanadate depends on the time of addition to the incubation medium. It was also shown that the stimulating effects of orthovanadate depend on the physiological status of hibernating females whose oocytes were isolated

Sodium orthovanadate suppresses DNA damage-induced caspase activation and apoptosis by inactivating p53

We previously reported that p42/SETbeta is a substrate for caspase-7 in irradiated MOLT-4 cells, and that treating the cells with sodium orthovanadate (vanadate) inhibits p42/SETbeta's caspase-mediated cleavage. Here, we initially found that the inhibitory effect of vanadate was due to the suppression of caspase activation but not of caspase activity. Further investigations revealed that vanadate suppressed upstream of apoptotic events, such as the loss of mitochondrial membrane potential, the conformational change of Bax, and p53 transactivation, although the accumulation, total phosphorylation, and phosphorylation of six individual sites of p53 were not affected. Importantly, vanadate suppressed p53-dependent apoptosis, but not p53-independent apoptosis. Finally, gel-shift and chromatin immunoprecipitation assays conclusively demonstrated that vanadate inhibits the DNA-binding activity of p53. Vanadate is conventionally used as an inhibitor of protein tyrosine phosphatases (PTPs); however, we recommend that the influence of vanadate not only on PTPs but also on p53 be considered before using it.

Phosphorus deficiency enhances plasma membrane H+-ATPase activity and citrate exudation in greater purple lupin (Lupinus pilosus).

The response of greater purple lupin (Lupinus pilosus L.) to a combination of phosphorus (P) deficiency and aluminium (Al) toxicity is unknown, and the mechanisms involved in the exudation of organic anions from greater purple lupin have not been reported. Therefore, plants grown with (+P) or without (-P) 250 µm P were exposed to 0 or 50 µm AlCl3 and the amount of organic anions exuded and the activities of plasma membrane H+-ATPase (E.C 3.6.3.6) and H+-pumps were investigated. Twenty days of P deficiency resulted in significantly reduced shoot growth and increased proteoid root formation. Exposure to 50 µm AlCl3 did not induce citrate exudation but did induce some malate exudation in -P plants. In contrast, P deficiency did induce exudation of citrate. Enhanced citrate exudation was attributed to the large increase in the activity of plasma membrane H+-ATPase and associated H+ transport. This was shown by the inhibitory effect of vanadate on plasma membrane H+-ATPase activity in vitro and on citrate exudation in vivo. However, vanadate did not suppress the exudation of malate. During 9 h of Al exposure, exudation of citrate showed a continuing increase for both -P and +P plants, while malate exudation increased only during the first 3 h, after which it rapidly declined. The total amount of organic anion exudation was significantly higher for -P plants. In the presence of 50 µm anion channel blockers [anthracene-9-carboxylic acid (A-9-C), niflumic acid (NIF) and phenylglyoxal (PG)], the exudation of citrate and malate was reduced by 25-40%. It was concluded that P deficiency induces citrate exudation by enhancing the activity of plasma membrane H+-ATPase and H+ export. In L. pilosus, exudation of organic anions occurs primarily in response to P deficiency but not Al toxicity. This contrasts with previous results obtained in Brassica napus L.

THE INVOLVEMENT OF A PLASMA MEMBRANE H+‐ATPase IN THE BLUE‐LIGHT ENHANCEMENT OF PHOTOSYNTHESIS IN LAMINARIA DIGITATA (PHAEOPHYTA)1

Many brown algae, including the kelp Laminaria digitata (Huds.) Lamour., exhibit enhanced photosynthesis when they are given a small amount of blue‐light in addition to a background of saturating red light. This blue light effect is correlated with an increased uptake of carbon. In the present study, we tested the hypothesis that blue light acts by increasing the activity of a plasma membrane H+‐ATPase, thereby promoting an active carbon uptake across the plasma membrane. Photosynthetic carbon uptake was studied in pH‐drift experiments under illumination with red and blue light and using different inhibitors. Vanadate, an inhibitor of plasma membrane H+‐ATPases, had a minor inhibitory effect on carbon uptake rates under saturating red light conditions, but inhibited the blue‐light enhancement by approximately 60%. An inhibitor of external carbonic anhydrase, acetazolamide, decreased the carbon uptake in both red light and in red plus blue light by 48% and 68%, respectively. These results suggest that photosynthetic carbon uptake depends on an external carbonic anhydrase under both red and red plus blue light conditions, and that blue light induces an increased activity of a P‐type H+‐ATPase in the plasma membrane. The proton buffer Tris, which has a buffering capacity similar to vanadate in seawater, had no inhibitory effect on carbon uptake rates neither in red light nor in red plus blue light, showing that the inhibitory effect of vanadate is not caused by its effect as a buffer. The blue‐light enhancement was also abolished by a protein kinase inhibitor (H‐7), suggesting that the transduction of the blue‐light signal involves a protein kinase, which activates the plasma membrane H+‐ATPase by phosphorylation.

Vanadate disrupts mammary gland development in whole organ culture.

Protein tyrosine kinases and phosphatases are signaling molecules involved in all aspects of development, including proliferation, differentiation, and apoptosis. How disruption of protein tyrosine phosphatase affects mammary gland development is not entirely clear. We examined the effects of sodium vanadate, which is known to primarily inhibit tyrosine phosphatases, in mouse mammary gland development in whole organ culture. Mammary epithelial differentiation was effectively inhibited by vanadate in a dose-dependent manner as indicated by lack of epithelial alveoli compared to the contralateral non-treated gland controls. Mammary glands in the differentiation medium after four days in the presence of vanadate did not differentiate into alveoli. Instead, they exhibited prominent terminal end buds and lost the distinctive epithelial structures. The inhibitory effect of vanadate on mammary epithelial cell differentiation was irreversible after one day of treatment. Immunohistochemical staining for PCNA (Proliferating Cell Nuclear Antigen) showed that vanadate-treated glands exhibited elevated proliferation signals in the differentiation medium. Expression of beta-casein protein in the vanadate-treated glands decreased dramatically and progressively. Short-term exposure (up to 72 hours) of mammary glands to vanadate resulted in an increase in mammary epithelial cell density and loss of organization of the mammary structures. TUNEL assay of mammary glands with prolonged exposure to vanadate revealed widespread apoptosis. Furthermore, some cells were still proliferating or expressing beta-casein after prolonged exposure to vanadate. Taken together, these data indicate that vanadate treatment blocks mammary epithelial cell differentiation and promotes abnormal proliferation and apoptosis, likely through the inhibition of protein tyrosine phosphatase-mediated signaling.

Inhibitory effect of vanadate on protein phosphatase, glycogen phosphorylase and protein kinase in extracts from several rat tissues

The purpose of this study was to determine the in vitro effect of increasing vanadate concentrations (1 n m-I m m) on protein phosphatase (PP) and glycogen phosphorylase (GP) of extracts from several rat tissues. PP activity was inhibited in various brain structures by moderate concentrations of vanadate; however, in the spinal cord, comparatively low concentrations (10 n m) of vanadate caused a steep increase in enzyme activity. Only at a high concentration (1 m m) did vanadate produce moderate PP inhibition. Reciprocal correlation was obtained between vanadate effects on PP and GP in various parts of the nervous system. In the last series of experiments the effect of vanadate upon protein kinase (PK) activity in various regions of the CNS was determined; low and intermediate vanadate concentrations produced a moderate activation; higher vanadate concentrations suppressed the enzyme activity. Vanadate at a concentration of 0.1 m m produced activation of PK in the medulla oblongata, whereas at 1 m m vanadate there was significant inhibition of PK. The spinal cord enzyme was more sensitive to vanadate, being inhibited at concentrations of 10 n m-1 m m.

Increase of (Ca(2+)-Mg2+)-ATPase activity in hepatic plasma membranes of rats administered orally calcium: the endogenous role of regucalcin.

The alteration of the plasma membrane (Ca(2+)-Mg2+)-ATPase activity in the liver of rats administered orally calcium chloride solution was investigated. The plasma membrane (Ca(2+)-Mg2+)-ATPase activity was significantly increased by a single oral administration of calcium (10, 25 and 50 mg/100 g body weight) in rats. This increase was seen between 10 and 60 min after the administration. The presence of anti-regucalcin IgG (1.0-5.0 micrograms/ml) in the enzyme reaction mixture caused a complete inhibition for the elevation of the plasma membrane (Ca(2+)-Mg2+)-ATPase activity by the addition of regucalcin (0.25 microM). Also, the calcium administration-induced increase in hepatic plasma membrane (Ca(2+)-Mg2+)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0 and 2.5 micrograms/ml). Moreover, the calcium administration-induced increase in hepatic plasma membrane (Ca(2+)-Mg2+)-ATPase activity was not inhibited by vanadate (0.1 and 0.2 mM) addition into the enzyme reaction mixture, although the inhibitory effect of vanadate was seen in the plasma membranes from normal rat liver. Now, the activating effect of regucalcin (0.25 microM) on hepatic plasma membrane (Ca(2+)-Mg2+)-ATPase was not inhibited by vanadate addition. The endogenous regucalcin may play a role in the calcium administration-induced increase of (Ca(2+)-Mg2+)-ATPase activity in the liver plasma membranes of rats.

Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells.

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.

The nonspecific nature of the vanadate inhibition of rat ileal (Na, K)-ATPase

Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis we examined the stimulatory and inhibitory effects of vanadate on 86∧Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal stimulation of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme.

Effect of vanadate on water transport by the toad bladder

Vanadate increases renal Na and water excretion. The mechanism whereby vanadate impairs water transport was examined in the toad bladder. Vanadate did not alter baseline water transport but caused a significant inhibition of water transport elicited by high doses of AVP. The inhibition of AVP stimulated water flow by vanadate was dose dependent with inhibition present with concentration as low as 10 −7 and maximal inhibition occurring at 10 −5M. Vanadate also inhibited water transport stimulated by cyclic AMP or by phosphodiesterase inhibition indicating that vanadate has an effect beyond cyclic AMP step, in addition to whatever effect it might have on adenylate cyclase. The inhibitory effect of vanadate on AVP stimulated water flow was not altered by prior Na-K-ATPase or prostaglandin inhibition. Since vanadate has been shown to stimulate adenylate cyclase in other tissues we examined whether addition of vanadate 10 minutes after addition of AVP would enhance water transport. Vanadate caused a transient enhancement of AVP stimulated water flow. These data demonstrate that vanadate can inhibit or stimulate water flow in the toad bladder.

Effect of noradrenaline and vanadate on sodium, potassium-activated ATPase of human brain cortical homogenate

Human brain cortical homogenate derived from surgical operations exhibited Na(+), K(+)-ATPase and K-p-nitrophenylphosphatase activity values of 2.12 +/- 0.08 ?mol P(i)/mg protein/15 min and 0.38 +/- 0.01 ?mol p-nitrophenol/mg protein/15 min, respectively which is in the range of those observed in rat brain cortical homogenates. Vanadate concentration dependently inhibited the activity of both enzymes. Noradrenaline, dopamine and isoprenaline reversed the inhibitory effect of vanadate in the presence of EDTA (0.2 mM). When Mg(2+) concentration was enhanced from 4 to 24 mM, the inhibitory effect of vanadate (1 ?M) was significantly potentiated. Evidence has been obtained that the effect of catecholamines is not a receptor mediated process; antagnoists such as phentolamine, phenoxybenzamine, propranolol, haloperidol failed to prevent the effect of adrenoceptor agonists. It is concluded that there is an interaction between vanadate and noradrenaline on human brain Na, K-ATPase.

Inhibitory effects of vanadate on the contractile responses in vascular smooth muscle

The effects of vanadate (Na 3VO 4) on the contractions induced by high concentrations of K, histamine and norepinephrine in isolated guinea-pig aorta were investigated. Vanadate ( −3M) relaxed the high K contraction only when the external K concentration was above 85.4 mM. Vanadate showed no inhibitory action on the contraction induced by either Na-containing 45.4 mM K solution or Na-poor 45.4 mM K solution. Ouabain (2 × 10 −5 M) had no inhibitory effects on the contractions induced by these high K solutions. Vanadate failed to relax the contraction induced by histamine or norepinephrine in normal (5.4 mM) medium, while it inhibited the contraction in the presence of 142.2 mM K. Ouabain also did not inhibit the histamine- or norepinephrine-induced contraction in either normal or high K solution. These results suggest that the inhibitory actions of vanadate are not due to an inhibition of Na pump and that these effects depend on the presence of a high concentration of external K ions.

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, 45Ca-exchange and (Na +, K +)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[ 14C]glucose as well as the efflux of 3-O-[ 14 C] methylglucose . (3) Within the same concentration range, vanadate induced an early increase in 45Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of 3-O-[ 14 C] methylglucose and 45Ca were significantly correlated ( P < 0.001, r = 0.98). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of 3-O-[ 14 C] methylglucose and this effect was preceded by a rise in the washout of 45Ca. The maximum increases in the fractional losses of 3-O-[ 14 C] methyglucose and 45Ca were significantly correlated ( P < 0.005, r = 0.98). (5) In extensor digitorum longus and soleus muscles, vanadate increased K +-contents and decreased Na + contents. (6) The stimulation of 45Ca-washout presumably reflects an increase in the cytoplasmic Ca 2+ level, brought about by an inhibitory effect of vanadate on the Ca 2+-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca 2+ level.

Vanadate inhibition of auxin-enhanced H + secretion and elongation in pea epicotyls and oat coleoptiles

In experiments carried out to investigate the acid secretion theory of auxin action, we utilized sodium orthovanadate, an agent found to be a selective inhibitor of a plasma membrane-associated H(+)-pumping ATPase in Neurospora [Bowman, B. J. & Slayman, C. W. (1979) J. Biol. Chem. 245, 2928-2934]. At 1 mM, vanadate inhibited auxin-enhanced medium acidification by pea epicotyl segments within 5 min, whether added 0.5 or 2.5 hr after auxin. Inhibition of acidification was total after 10-15 min but could be reversed within 10 min after vanadate removal. When given as a 40-min pretreatment, vanadate completely prevented any auxin-enhanced acidification. Vanadate inhibition of medium acidification by oat coleoptile segments was also total and reversible, but both inhibition and reversal occurred after longer lag times than in pea. Inhibitory effects of vanadate on elongation in pea and oat tissue closely paralleled its effects on acidification, and the inhibitory effect of vanadate on elongation could be reversed by an acidic buffer. Vanadate did not inhibit respiration or protein synthesis in pea epicotyl segments, although it strongly inhibited L-[(14)C]leucine uptake. These results indicate the importance of cell wall acidification for short- and long-term auxin-enhanced growth and suggest the participation in wall acidification of a plasma membrane-associated ATPase acting as an H(+) pump.

Inhibition of progesterone receptor activation by vanadate

The cell-free activation of progesterone receptor from the avian oviduct was determined by measuring the binding of activated receptor to ATP-Sepharose. When transformation of the receptor-hormone complex to the activated form was initiated by temperature elevation, this process could be blocked by sodium vanadate. However, vanadate has no effect on receptor that is already in the activated state; therefore, the inhibitor acts in preventing the activation process. The inhibitory effect of vanadate appears to be on the rate of receptor activation rather than on the maximum extent of activation. Inhibition by vanadate resembles that previously shown for molybdate. However, vanadate is a more potent inhibitor and, unlike molybdate, its action can be reversed by addition of the chelator, EDTA, and by the reducing agent, sodium borohydride. Whether receptor activation involves phosphate metabolism, as is the case with several vanadate-sensitive enzymes, remains unknown.

Influence of Ca ++ and Mg ++ on the vanadate inhibition of the Ca ++-ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca ++-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10 −5 M. Mg ++ promotes this inhibition by vanadate whereas increasing Ca ++-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio Mg ++ ATP constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10 −3 M ATP. Whenever the ratio Mg ++ ATP was higher than 1:1 the inhibitory effect of vanadate on the Ca ++-ATPase was increased.