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At least 19 recordsLinked to original sources
PubMed1992

Vanadate stimulates system A amino acid transport activity in skeletal muscle. Evidence for the involvement of intracellular pH as a mediator of vanadate action.

Sodium orthovanadate caused a 2-fold stimulation of system A transport activity in soleus muscle, as assessed by the uptake of the nonmetabolizable analog 2-(methylamino)isobutyric acid (MeAIB). The effect of vanadate on system A was rapid, concentration-dependent and was characterized by an increased Vmax without modification of Km for MeAIB. Under these conditions, vanadate also activated 3-O-methylglucose uptake and lactate production. The effects of vanadate on muscle metabolism showed a complex interaction with the effects of insulin. Thus, the stimulatory effects of vanadate and insulin on MeAIB and 3-O-methylglucose uptake were not additive; however, the effects of insulin and vanadate on lactate production were additive. In spite of the lack of additivity, insulin- and vanadate-induced stimulation of system A differed in their sensitivity to gramicidin D, being the vanadate effect more susceptible to inhibition by gramicidin D than the insulin effect. System A transport activity shows a dependence on pH, and recent results suggest the presence of critical histidine residues on the A carrier that may be responsible for its pH dependence (Bertran, J., Roca, A., Pola, E., Testar, X., Zorzano, A. & Palacín, M. (1991) J. Biol. Chem. 266, 798-802). In this regard, a rise in extracellular pH led to a substantial activation of system A. Furthermore, lowering of muscle intracellular pH induced by ethylisopropylamiloride (EIPA), a specific inhibitor of sodium/proton exchange activity, led to inhibition of system A. This suggests that critical histidine residues are present in an intracellular localization on the A carrier. Furthermore, the rate of muscle glycolysis was also altered in response to a rise in extracellular pH or to EIPA treatment. Regarding the mechanisms involved in vanadate action, vanadate treatment in the incubated soleus muscle did not cause any significant stimulation of tyrosine kinase activity after partial purification of muscle insulin receptors. On the other hand, vanadate but not insulin caused a substantial increase in muscle intracellular pH as assessed by 5,5'-dimethyloxazolidine-2,4-dione equilibrium. This effect of vanadate on intracellular pH was not due to activation of the sodium/proton exchanger, since it was not blocked by EIPA. Based on these findings, we suggest that alkalinization of muscle intracellular pH might mediate the effects of vanadate on system A and on glycolysis.

Amiloride↗
PubMed1990

Plasma membrane-stimulated vanadate-dependent NADH oxidation is not the primary mediator of vanadate toxicity in Saccharomyces cerevisiae.

Interactions of oxyvanadium compounds with cellular metabolism have recently been demonstrated. Membrane-stimulated vanadate-dependent NADH oxidation has been hypothesized to involve the cellular accumulation of H2O2, which may cause the vanadate sensitivity of animals and microbes. This report shows that the vanadate-dependent NADH oxidation activity of the yeast plasma membrane requires oxygen and is present in vanadate-resistant mutants of Saccharomyces cerevisiae. In addition, the vanadate sensitivity of growth in S. cerevisiae is the same during aerobic and anaerobic growth. These results imply that neither plasma membrane-mediated vanadate-stimulated NADH oxidation, nor any other oxidative process, is the primary cause of vanadate sensitivity in yeast cells.

Aerobiosis↗
PubMed1986

Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation.

The interaction of vanadate with phenol and N-acetyltyrosine ethyl ester in aqueous solution has been studied by using 51V nuclear magnetic resonance spectroscopy. On the basis of these studies, it has been concluded that vanadate rapidly esterifies the hydroxyl group of the aromatic ring to yield a phenyl vanadate. For phenol, the equilibrium constant for this reaction in terms of the convention that the activity of liquid water is 1.0 is K1 = [phenyl vanadate]/[phenol][vanadate] = 0.97 +/- 0.02. This value is well over 4 orders of magnitude larger than estimates from the literature for the corresponding equilibrium constant for the esterification of phenol by phosphate. The equilibrium constant for esterification of the phenol moiety of N-acetyltyrosine ethyl ester is similar to that for esterification of phenol. The relevance of these observations to processes that are regulated by reversible phosphorylation/dephosphorylation of tyrosine residues is discussed, in particular the insulin-like effect of vanadate.

Hydrogen-Ion Concentration↗
PubMed1985

Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase. Nonenzymic formation of glucose 6-vanadate.

Inorganic vanadate (Vi) activates catalysis by glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+. As the concentration of Glu-6-P dehydrogenase is increased, the rate of the vanadate-activated glucose oxidation becomes less sensitive to increases in enzyme concentration. The rate of glucose oxidation in the absence of Vi increases linearly with Glu-6-P dehydrogenase concentration. These results are interpreted in terms of nonenzymic formation of glucose 6-vanadate. At high enzyme concentration, vanadate ester formation becomes partially rate-limiting, and extrapolation to infinite Glu-6-P dehydrogenase concentration allows determination of the second order rate constant for formation of the ester. In separate experiments designed to test the proposed mechanism, it was found that Vi, at concentrations at which it strongly activates catalysis by Glu-6-P dehydrogenase of glucose oxidation, has no effect on the rates of oxidation of glucose 6-phosphate or 6-deoxyglucose catalyzed by Glu-6-P dehydrogenase. Sulfate, which is known to activate glucose oxidation and to inhibit glucose 6-phosphate oxidation, strongly activates 6-deoxyglucose oxidation. These experiments show that the 6-hydroxyl group of glucose is essential for the observed activation by Vi and are also consistent with the formation of glucose 6-vanadate. Also, the rate of the sulfate-activated glucose oxidation increases linearly with Glu-6-P dehydrogenase concentration. These results are consistent with the proposed mechanism for sulfate activation which involves sulfate binding to the enzyme (Anderson, W. B., Horne, R. N., and Nordlie, R. C. (1968) Biochemistry 7, 3997-4004). The second order rate constant calculated for formation of glucose 6-vanadate at pH 7.0 is 2.4 M-1 s-1. The corresponding values for glucose 6-phosphate and glucose 6-arsenate formation are approximately 9 X 10(-11) M-1 s-1 and 6.3 X 10(-6) M-1 s-1 (Lagunas, R. (1980) Arch. Biochem. Biophys. 205, 67-75).

Glucose↗
PubMed1990

Role of glutathione in vanadate reduction in young and mature rats: evidence for direct participation of glutathione in vanadate inactivation.

The influence of renal glutathione content, modulated by the glutathione synthesis inhibitor buthionine sulphoximine or by glutathione infusion, on the polyuric and natriuretic effects of i.v. administered vanadate was investigated in 20- and 55-day-old rats. The modulation of renal glutathione content led to significant changes in urine volume and sodium excretion, independently of age. A decrease in the renal glutathione level led to intensification and prolongation of the diuretic effects of vanadate in 55-day-old animals. Treatment with glutathione abolished and treatment with buthionine sulphoximine increased the polyuric effect of vanadate. These observations indicate a role for renal glutathione in vanadate inactivation. Age-dependent differences in the polyuric and natriuretic effectiveness of vanadate are caused by differences in renal glutathione content during maturation of the kidney.

Aging↗
PubMed1992

Superoxide-independent reduction of vanadate by rat liver microsomes/NAD(P)H: vanadate reductase activity.

It has been reported that vanadate-stimulated oxidation of NAD(P)H by microsomal systems can proceed anaerobically, in contrast to the general notion that the oxidation proceeds exclusively by an O(2-)-dependent free radical chain mechanism. The current study indicates that microsomal systems are endowed with a vanadate-reductase property, involving a NAD(P)H-dependent electron transport cytochrome P450 system. Our ESR measurements demonstrated the formation of a vanadium(IV) species in a mixture containing vanadate, rat liver microsomes, and NAD(P)H. This vanadium(IV) species was identified as the vanadyl ion (VO2+) by comparison with the ESR spectrum of VOSO4. The initial rate of vanadium(IV) formation depends linearly on the concentration of microsomes. The Michaelis-Menten constants were found to be: km = 1.25 mM and Vmax = 0.066 mumol (min)-1 (mg microsomes)-1, respectively. Pretreatment of the microsomes with carbon monoxide or K3Fe(CN)6 reduced vanadium(IV) generation, suggesting that the NAD(P)H-dependent electron transport cytochrome P450 system plays a significant role in the microsomal reduction of vanadate. Measurements under argon or in the presence of superoxide dismutase caused only minor (less than 10%) reductions in vanadium(IV) generation. The VO2+ species was also detected in NAD(P)H oxidation by fructose plus vanadate, a reaction known to proceed via an O(2-)-mediated chain mechanism. However, the amount of vanadium(IV) generated by this reaction was an order of magnitude smaller than that by the microsomal system and was inhibitable by superoxide dismutase, affirming the conclusion that the microsomal/NAD(P)H system is endowed with the (O(2-)-independent) vanadium(V) reductase property.

Anaerobiosis↗
PubMed1991

Effects of valinomycin on vanadate-sensitive and vanadate-resistant H+ transport in vesicles from turtle bladder epithelium: evidence for a K+/H+ exchanger.

The vanadate-sensitive component of the ATP-dependent H+ gradient formed in isolated vesicles from a urinary epithelium was abolished by valinomycin omission. This suggests that vanadate-sensitive H+ transport has an absolute requirement for intravesicular K+ and that the transport may be due to a K+/H+ exchanger. Sensitivity to the inhibitor SCH28080 supports this conclusion. On the other hand, valinomycin affects the initial velocity of vanadate-resistant transport without altering its maximum gradient. This is consistent with the development of a membrane potential consequent to electrogenic uniport H+ transport.

Adenosine Triphosphate↗
PubMed1988

Vanadate-stimulated NADH oxidation requires polymeric vanadate, phosphate and superoxide.

NADH oxidation, catalyzed by the microsomal enzyme system is stimulated on addition of polymeric vanadate. Maximum stimulation by polymeric vanadate was obtained in the presence of phosphate buffer. The small stimulation obtained by metavanadate (500 microM) increased on acidification followed by neutralization, or on adding a trace amount of polymeric vanadate (1 microM).

Animals↗
PubMed1990

Vanadate regulates glucose transporter (Glut-1) expression in NIH3T3 mouse fibroblasts.

Vanadate, the major oxidized form of the essential trace element vanadium, has rapid effects on glucose transport in vitro and more delayed effects on glucose transport in vivo. We addressed the question that one potential mechanism for the delayed effects of vanadate on glucose homeostasis could be altered expression of one or more of the genes encoding glucose transporters. To do this we studied vanadate regulation of Glut-1 and Glut-4 in NIH3T3 mouse fibroblasts. Vanadate (5-40 microM) induced cells to proliferate to higher cell densities, and in addition, 40 microM vanadate caused the cells to exhibit a transformed morphology. Glut-1 mRNA was maximally induced 4- to 5-fold over the control value after 6-h exposure to 30 microM vanadate. Unlike the response to serum and growth factors, the vanadate-induced increase in Glut-1 mRNA remained elevated over the control value in the presence of vanadate for 5 days. The vanadate effect was serum dependent and was fully reversible when vanadate was removed from the medium. In the absence of vanadate, the half-life of Glut-1 mRNA was 0.5-1 h, whereas after treatment for 5 h with 30 microM vanadate the half-life was increased to 1.5-2 h. Thus, mRNA stabilization accounts for at least a part of the increase in glucose transporter mRNA levels after vanadate treatment. Glut-4 mRNA was not detected in these cells in either the absence or presence of vanadate. While the importance of this increased Glut-1 gene expression for the vanadate effect on normalization of blood glucose in vivo remains to be determined, an association between vanadate-induced cell proliferation and transformed phenotype, and vanadate-induced Glut-1 mRNA in vitro has been made. Possible potential therapeutic use of vanadate for treatment of diabetes must, therefore, be viewed with caution.

Animals↗
PubMed1990

Vanadate dimer and tetramer both inhibit glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

Vanadate dimer and tetramer inhibit glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. The inhibition by a vanadate mixture containing vanadate monomer, dimer, tetramer, and pentamer was determined by measuring the rates of glucose 6-phosphate oxidation and reduction of NAD (or NADP) catalyzed by glucose-6-phosphate dehydrogenase. The inhibition by vanadate is competitive with respect to NAD or NADP and noncompetitive (a mixed type) with respect to glucose 6-phosphate (G6P) when NAD or NADP are cofactors. This inhibition pattern varies from that observed with phosphate and thus suggests vanadate interacts differently than a phosphate analogue with the enzyme. 51V NMR spectroscopy was used to directly correlate the inhibition of vanadate solutions to the vanadate dimer and/or tetramer, respectively. The activity of the vanadate oligomer varied depending on the cofactor and which substrate was being varied. The vanadate dimer was the major inhibiting species with respect to NADP. This is in contrast to the vanadate tetramer, which was the major inhibiting species with respect to G6P and with respect to NAD. The inhibition by vanadate when G6P was varied was weak. The competitive inhibition pattern with respect to NAD and NADP suggests the possibility that vanadate oligomers may also inhibit catalysis of other NAD- or NADP-requiring dehydrogenases. Significant concentrations of vanadate dimer and tetramer are only found at fairly high vanadate concentrations, so these species are not likely to represent vanadium species present under normal physiological conditions. It is however possible the vanadate dimer and/or tetramer represent toxic vanadate species.

Bacterial Proteins↗
PubMed1985

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae.

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.

Biological Transport↗
PubMed1990

51V NMR study of vanadate binding to myosin and its subfragment 1.

The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predominant vanadate form was monomeric, while at high concentration, it was tetrameric. The presence of myosin or S-1 in the solution produced a significant broadening of the signal of each form of vanadate, indicating that all of them bind to the protein. Addition of ATP, which does not affect the 51V NMR spectra in the absence of proteins, causes their significant alteration in the presence of myosin or S-1. The changes, which include the broadening of the signal of the monomeric and the narrowing of the signal of the oligomeric vanadate forms, indicate that more monomeric and less oligomeric vanadate binds to the proteins in the presence than in the absence of ATP. Irradiation by near-UV light in the presence of vanadate cleaves S-1 at three specific sites--at 23, 31, and 74 kDa from the N-terminus. The cleavages at 23 and 31 kDa are specifically inhibited by the addition of ATP. The vanadate-associated photocleavage of S-1 also depends on the total concentration of vanadate; it is observed only when the concentration of vanadate is at least 0.2 mM. This was also the lowest concentration at which oligomeric vanadate was detected in the 51V NMR spectra. From the parallel concentration dependence of the photocleavage and the appearance of the tetrameric vanadate, it is concluded that photocleavage occurs only when tetrameric vanadate binds to S-1.

Adenosine Triphosphate↗
PubMed1987

Down-regulation of cell surface insulin receptors in primary cultured rat adipocytes by sodium vanadate.

Sodium vanadate, a potent phosphotyrosine phosphatase inhibitor, was found to down-regulate the number of cell surface receptors in primary cultured adipocytes (50% receptor loss by 24 h; 65% loss by 48 h; ED50, 17 and 9 microM, respectively). The characteristics of vanadate-induced down-regulation were distinguished by three salient features. First, time-course studies revealed a lag period of 4-6 h preceding vanadate-induced receptor loss. This lag period was not due to slow diffusion of vanadate into the cell, since it could not be shortened using a very high dose of vanadate (1 mM). Second, vanadate was found to trigger receptor loss, such that down-regulation continued after vanadate removal. Thus, no immediate down-regulation was observed in adipocytes exposed to 80 microM vanadate for 6 h. However, when cells were washed and reincubated in vanadate-free medium for an additional 18 h (24 h total), a 50% receptor loss was seen. Lastly, we found that vanadate-induced down-regulation was not readily reversible, since receptor recovery was not observed when 24-h down-regulated cells were reincubated in vanadate-free medium for an additional 24 h. These three features of vanadate-induced receptor loss are remarkably similar to the characteristics of insulin-induced down-regulation. In studies examining the relationship between protein synthesis and vanadate-induced down-regulation, we found that vanadate markedly stimulated the overall rate of protein synthesis in 24-h treated adipocytes (a 42% increase; ED50, 3 microM), and that cycloheximide treatment markedly blunted vanadate-induced loss of cell surface receptors. Overall, these studies are consistent with the hypothesis that tyrosine phosphorylation plays a role in regulating the number of cell surface insulin receptors.

Adipose Tissue↗
PubMed1988

Insulin-like effects of vanadate in isolated rat adipocytes.

Vanadate has been shown to have a number of insulin-like effects in various cells, including isolated rat adipocytes. In the present study we compared the activities of vanadate and insulin in isolated fat cells using a number of different assays of insulin-like activity. Both insulin and vanadate stimulated [2-3H]glucose incorporation into fat cell lipid in a dose-dependent manner, but the maximal effect of vanadate was markedly greater than that of insulin. At 10(-2) M vanadate the effect was 3-4 times as great as the maximal effect of insulin. This effect was dependent on specific glucose transport. Combinations of insulin and vanadate were not more effective than vanadate alone. Vanadate also produced antilipolysis with an effect somewhat greater than that of insulin. Using [U-14C]glucose both vanadate and insulin stimulated 14CO2 production and [14C]glucose incorporation into lipid, and again the effect of vanadate was greater than that of insulin. Vanadate had a greater effect on 14CO2 production than on [14C]glucose incorporation into lipid. When [1-14C]glucose was used vanadate again had a significantly greater effect on 14CO2 production than did insulin, but when [6-14C]glucose was used the effects of vanadate and insulin were equal. These results demonstrate that vanadate has insulin-like effects in isolated fat cells, but it selectively stimulates certain pathways to a greater extent than does insulin. The greater effect of vanadate than insulin appears to be primarily on the pentose phosphate shunt, suggesting that this agent may be useful for examination of this intracellular pathway in fat cells.

Adipose Tissue↗
PubMed1988

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes.

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.

Binding, Competitive↗
PubMed1987

Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. Characterization and mode of action.

The effect of oral administration of vanadate, in normalizing blood glucose levels of streptozotocin-treated rats (ST-rats), is further characterized and its mode of action is determined. We have examined the effects of two orally administered doses of sodium metavanadate. High concentrations of orally administered vanadate (0.8 mg/ml in drinking water) reduced blood glucose levels within 2-4 days of application and led to the appearance of hypoglycemia in test animals. Lower concentrations of vanadate (0.2 mg/ml in drinking water) also lowered blood glucose levels within 4 days, but did not lead to hypoglycemia for at least 3 weeks. These effects of vanadate were found to be reversible; hyperglycemia recurred within 2 days after removal of vanadate from the drinking water. In streptozotocin-treated rats receiving low vanadate treatment, circulating levels of vanadate were about 0.8 microgram/ml after 3 weeks of treatment. These rats became anabolic, while rats receiving high vanadate treatment remained catabolic. Subsequent to vanadate treatment, adipocytes derived from ST-rats responded to lower insulin concentrations. In addition, vanadate treatment lowered the increased insulin binding capacity of liver plasma membranes derived from ST-rats. Insulin binding capacity under these conditions approached that of control non-ST-rats. Basal rates of hexose uptake in muscle and liver tissues were doubled in vanadate-treated ST-rats. It is concluded that the oral administration of vanadate leads to normoglycemia by stimulating glucose uptake. Treatment with "low vanadate" leads to the formation of a stable anabolic and normoglycemic state in ST-rats and appears to restore insulin responsiveness of target tissues, without apparent signs of toxicity. Vanadate treatment did not impair either kidney or liver function, as assayed by the measurement of serum urea, creatinine, and glutamic-oxaloacetic transaminase.

Adipose Tissue↗
PubMed1991

Existence of high- and low-affinity vanadate-binding sites on Ca(2+)-ATPase of the sarcoplasmic reticulum.

The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca(2+)-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1 microM, and the low-affinity sites one of 36 microM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Airways hyperreactivity and bronchoconstriction induced by vanadate in the guinea-pig.

1 The characteristics of vanadate-induced bronchoconstriction and airways hyperreactivity were observed in spontaneously breathing anaesthetized guinea-pigs by measurement of airways resistance (Raw) and dynamic lung compliance (Cdyn). Vanadate (0.3-3 mg kg-1 i.v. over 25 min) increased Raw and decreased Cdyn in a reversible, dose-related manner. This action (1 mg kg-1 vanadate) was not inhibited by atropine (1 mg kg-1 i.v.), propranolol (1 mg kg-1 i.v.) or bilateral vagotomy, suggesting a direct effect on the airways smooth muscle. 2 An aerosol of vanadate (10% w/v in H2O) for 3 min decreased Cdyn by 19.5% (P less than 0.05, n = 6) but caused no change in Raw. 3 Histamine (3 micrograms kg-1 i.v.) caused a bronchoconstriction which was enhanced by vanadate in a dose-related manner. This hyperreactivity (after 1 mg kg-1 i.v. vanadate) was unchanged after propranolol or bilateral vagotomy, but was partly blocked by atropine (enhancement by vanadate of the Cdyn change to histamine was diminished, P less than 0.02, n = 3). 4 Bronchoconstrictor responses to acetylcholine (6 micrograms kg-1 i.v.) and 5-hydroxytryptamine (6 micrograms kg-1 i.v.) were also enhanced by vanadate (1 mg kg-1 i.v.) Hyperreactivity after vanadate to the three bronchoconstrictors tested continued during vanadate infusion and was reversed 45 min after cessation of infusion. 5 Histamine (3 ;Lgkg-' i.v.) caused a transient tachypnoea which was also enhanced by vanadate (0.3-3mgkg-'i.v.), in a dose-related manner, in association with the increased reactivity of the airways (r = 0.66, n = 11). 6 It is concluded that vanadate-induced airways hyperreactivity is non-vagal (efferent) and largely non-cholinergic in origin and appears to involve an action of vanadate within the lung itself.

Acetylcholine↗