Stimulatory Effects Of Vanadate Research Articles

Stimulatory effects of vanadate on amylase release from isolated rat pancreatic acini

The effects of vanadate on exocrine pancreatic function were examined in isolated rat pancreatic acini. Vanadate caused a concentration-dependent stimulation of amylase release above a concentration of 1 mM. Co-incubation of vanadate with vasoactive intestinal polypeptide, 8-bromoadenosine 3′:5′-cyclic monophosphate, and the Ca 2+ ionophore A23187 produced a synergistic pattern of amylase release, whereas co-incubation with cholecystokinin octapeptide (CCK-8), carbamylcholine, and 12-O-tetradecanoylphorbol 13-acetate produced an additive effect. Vanadate alone had no influence on acinar cyclic AMP content, Ca 2+ efflux, or intracellular Ca 2+ concentration. However, preincubation with vanadate prevented the plateau phase of CCK-8-induced Ca 2+ transient increase from returning to baseline. Moreover, depletion of the intracellular Ca 2+ pool by pretreatment of acini with CCK-8 in Ca 2+-free medium (plus ethyleneglycol bis[β-aminoethylether]-N ,N′-tetraacetic acid) had no effect on subsequent stimulation by vanadate, although it abolished the response to both CCK-8 and carbamylcholine stimulation. The protein kinase C (PKC) inhibitors staurosporine and calphostin C significantly inhibited vanadate-stimulated amylase release, whereas the protein tyrosine kinase inhibitor genistein had no inhibitory effect. Moreover, vanadate caused a significant translocation of PKC from cytosol to membrane fraction in pancreatic acinar cells. This translocation was inhibited significantly by staurosporine and calphostin C but not by genistein. These results suggest that vanadate acts directly on pancreatic acini and stimulates amylase release by activating PKC without an effect on Ca 2+ mobilization, cyclic AMP, or protein tyrosine kinase.

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate- inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads.

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.

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.

Mechanism of vanadate-induced activation of tyrosine phosphorylation and of the respiratory burst in HL60 cells. Role of reduced oxygen metabolites

Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+)-OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover.

Vanadate stimulates the pumped movements of Na in skeletal muscle.

We have measured the effects of concentrations of vanadate ranging between 0.01 and 10 mM on the 22Na efflux of frog sartorius muscles. The addition of vanadate had no effects when concentrations lower than 0.5 mM were used; higher concentrations increased Na efflux. The increase was abolished by the addition of ouabain (10(-5)M). In muscles pretreated with ouabain vanadate did not modify Na efflux. The stimulatory effects of vanadate on Na efflux were also observed in Na-free solutions indicating that the efflux of vanadate was not caused mainly either by an increase in the exchange of Na for Na or by an increase in Na entry into the muscle. We also examined the effects of vanadate on muscles immersed in solutions containing 20 mM K+; both vanadate and increased K+ produced stimulations of Na efflux that were additive. Similarly when the effects of vanadate and insulin were measured on the Na efflux of the same muscle, additive effects were found. As the ouabain-sensitive Na efflux in frog muscle is generally agreed to be due to the activity of the Na-K ATPase, our findings suggest that the net effect of vanadate in intact muscle cells is an increase in the activity of the Na pump. Since vanadate affects many enzymes it is quite possible that the stimulatory action is not due to a direct effect on the Na-K ATPase but may be mediated through an intermediary step.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory (insulin-mimetic) and inhibitory (ouabain-like) action of vanadate on potassium uptake and cellular sodium and potassium in heart cells in culture

(1) The influence of vanadate (Na 3VO 4) on sodium and potassium uptake as well as on cellular ion contents of sodium and potassium has been studied in heart muscle and non-muscle cells obtained from various species. An ouabain-like inhibition of potassium uptake (up to 50%), combined with a decrease of cellular potassium (up to 20%) has been observed by vanadate (10 −4−10 −3 M) in heart non-muscle cells obtained from neonatal guinea pigs and chick embryos. In heart muscle and non-muscle cells prepared from neonatal rats, as well as in Girardi human heart cells, a vanadate-induced stimulation of potassium uptake (up to 100%), combined with a rise in cellular potassium (up to 20%) and without significant alteration of cellular sodium, has been found. A slight increase of 22Na + influx can be measured in rat heart muscle cells and in Girardi human heart cells in the presence of vanadate (10 −4−10 −3 M). (2) In beating rat heart muscle cells in culture, detrimental effects of serum deprivation—concerning beating properties, potassium uptake and cellular potassium—can at least in part be overcome by addition of vanadate. Furthermore, this compound prevents ouabain-induced signs of toxicity (contractures) in these cells. (3) The stimulatory effects of vanadate on potassium can be mimicked by insulin (1–10 mU/ml). Furthermore, vanadate produces an insulin-like stimulation of 2-deoxy- d-glucose uptake in rat heart muscle and non-muscle cells as well as in Girardi human heart cells. (4) The experimental data demonstrate an ouabain-like inhibition as well as an insulin-mimetic stimulation of potassium-uptake in various heart cells. The reason for this antagonistic mode of action may be due to the different capabilities of the heart cell types to reduce vanadium in the V-valence state to vanadium in the IV-valence state, thereby favouring either ouabain-like inhibition (vanadium V) or insulin-mimetic stimulation (vanadium IV) of potassium transport.

Stimulatory effect of vanadate on cyclic AMP levels in cat papillary muscle

Stimulatory effect of vanadate on cyclic AMP levels in cat papillary muscle

Stimulatory effect of vanadate on the adenylate cyclase of cardiac tissue

Stimulatory effect of vanadate on the adenylate cyclase of cardiac tissue

Stimulatory effect of vanadate on (Na+ + K+)-ATPase activity and on 3H-ouabain-binding in a cat heart cell membrane preparation

VANADATE has been reported to inhibit (Na+ + K+)-ATPase activity in membrane preparations from dog kidney1 and in red cells2. This inhibition, which occurs in the nanomolar concentration range in the presence of high Mg2+ concentrations and in the micromolar range at physiological Mg2+ concentrations2,3 is specific as other ATPase enzymes are not affected in these conditions1. Therefore vanadate has been suggested to be ‘an ideal specific regulator’ of (Na+ + K+)-ATPase1. We have shown4 that vanadate has a distinct positive inotropic effect in cat papillary muscle. It has been claimed that the positive inotropic action of cardiac glycosides, which are specific inhibitors of (Na+ + K+)-ATPase activity5,6, is associated with the inhibition of that enzyme. If that is so, vanadate would be expected to inhibit (Na+ + K+)-ATPase, in a concentration-dependent manner, in parallel with its effect on force of contraction. To test this hypothesis, we measured the effect of vanadate on (Na+ + K+)-ATPase activities and on 3H-ouabain-binding in identical conditions in a membrane preparation taken from cat hearts that were also used for experiments on contraction. We report here, however, that vanadate may stimulate (Na+ + K+)-ATPase activity transiently and 3H-ouabain-binding permanently in these conditions.