Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm, Bombyx mori.

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine-specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal-regulated kinase (ERK). The phosphorylation of another 120-kDa protein showed dose- and time-dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day-6 last-instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH-stimulated phosphorylation of the 120-kDa protein was markedly attenuated by genistein, a broad-spectrum tyrosine kinase inhibitor, but not by HNMPA-(AM)(3) , a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more-selective inhibitor of the Src-family tyrosine kinases, partially inhibited PTTH-stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120-kDa protein, which is not Src-family tyrosine kinase, is likely also involved in PTTH-stimulated ecdysteroidogenesis in B. mori.

The aryl hydrocarbon receptor (AhR) inhibits vanadate-induced vascular endothelial growth factor (VEGF) production in TRAMP prostates.

Hypoxia-inducible factor-1 alpha (HIF-1alpha) and aryl hydrocarbon receptor nuclear translocator (ARNT) are basic helix-loop-helix/per-arnt-sim (PAS) family transcription factors. During angiogenesis and tumor growth, HIF-1alpha dimerizes with ARNT, inducing expression of many genes, including vascular endothelial growth factor (VEGF). ARNT also dimerizes with the aryl hydrocarbon receptor (AhR). AhR-null (Ahr(-/-)) transgenic adenocarcinoma of the mouse prostate (TRAMP) mice develop prostate tumors with greater frequency than AhR wild-type (Ahr(+/+)) TRAMP mice, even though prevalence of prostate epithelial hyperplasia is not inhibited. This suggests that Ahr inhibits prostate carcinogenesis. In TRAMP mice, prostatic epithelial hyperplasia results in stabilized HIF-1alpha, inducing expression of VEGF, a prerequisite for tumor growth and angiogenesis. Since ARNT is a common dimerization partner of AhR and HIF-1alpha, we hypothesized that the AhR inhibits prostate tumor formation by competing with HIF-1alpha for ARNT, thereby limiting VEGF production. Prostates from Ahr(+/+), Ahr(+/-) and Ahr(-/-) C57BL/6J TRAMP mice were cultured in the presence of graded concentrations of vanadate, an inducer of VEGF through the HIF-1alpha-ARNT pathway. Vanadate induced VEGF protein in a dose-dependent fashion in Ahr(+/-) and Ahr(-/-) TRAMP cultures, but not in Ahr(+/+) cultures. However, vanadate induced upstream proteins in the phosphatidylinositol 3-kinase-signaling cascade to a similar extent in TRAMPs of each Ahr genotype, evidenced by v-akt murine thymoma viral oncogene homolog (Akt) phosphorylation. These findings suggest that AhR sequesters ARNT, decreasing interaction with HIF-1alpha reducing VEGF production. Since VEGF is required for tumor vascularization and growth, these studies further suggest that reduction in VEGF correlates with inhibited prostate carcinogenesis in Ahr(+/+) TRAMP mice.

Carnosol inhibits beta-catenin tyrosine phosphorylation and prevents adenoma formation in the C57BL/6J/Min/+ (Min/+) mouse.

Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated beta-catenin, dissociation of beta-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and beta-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc(+/+) wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and beta-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated beta-catenin. Thus, the Apc(Min) allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including beta-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress beta-catenin tyrosine phosphorylation.

Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

Combined treatment with benzylamine and low dosages of vanadate enhances glucose tolerance and reduces hyperglycemia in streptozotocin-induced diabetic rats.

Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO, such as benzylamine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes. Here we examined whether acute and chronic administration of benzylamine and vanadate in vivo enhances glucose tolerance and reduces hyperglycemia in diabetic rats. Acute intravenous administration of these drugs enhanced glucose tolerance in nondiabetic rats and in streptozotocin (STZ)-induced diabetic rats. This occurred in the absence of changes in plasma insulin concentrations. However, the administration of benzylamine or vanadate alone did not improve glucose tolerance. The improvement caused by benzylamine plus vanadate was abolished when rats were pretreated with the SSAO-inhibitor semicarbazide. Chronic administration of benzylamine and vanadate exerted potent antidiabetic effects in STZ-induced diabetic rats. Although daily administration of vanadate alone (50 and 25 micromol x kg(-1) x day(-1) i.p.) for 2 weeks had little or no effect on glycemia, vanadate plus benzylamine reduced hyperglycemia in diabetic rats, enhanced basal and insulin-stimulated glucose transport, and upregulated GLUT4 expression in isolated adipocytes. In all, our results substantiated that acute and chronic administration of benzylamine with low dosages of vanadate have potent antidiabetic effects in rats.

A structural model for the catalytic cycle of Ca(2+)-ATPase.

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.

Regulation of vascular and gastric smooth muscle contractility by pervanadate.

1. The contractile actions of vanadate (VO4) and pervanadate (PV, peroxide(s) of vanadate) were studied in rat gastric longitudinal muscle strips and in aortic rings. The roles of extracellular sodium and calcium were evaluated and the potential effects of nerve-released agonists were considered. The possibility that these responses were due to the potentiation of tyrosine kinase activity, as a result of PV-mediated tyrosine phosphatase inhibition was explored with the use of tyrosine kinase inhibitors (genistein, tyrphostin) and by Western blot analysis of phosphotyrosyl proteins in PV-treated tissues. The ability of PV to mimic the action of the tyrosine kinase receptor-associated agonist, epidermal growth factor-urogastrone (EGF-Uro), in the gastric preparation was also studied. 2. PV caused concentration-dependent contractions in both gastric and aorta-derived tissues, with a potency that was 1 to 2 orders of magnitude greater than that of VO4. 3. Although repeated exposure of gastric and aortic tissues to a fixed concentration of VO4 caused reproducible contractions in both tissues, repeated exposure of gastric tissue to PV caused an increased contractile response plateauing after 3 exposures. In contrast, a single exposure of aortic tissue to PV (20 microM) caused a prolonged desensitization of the tissue to the subsequent contractile actions of PV or other agonists. 4. The contractile responses to PV were unaffected in both preparations by tetrodotoxin, atropine, yohimbine and phenoxybenzamine; and in the aortic preparation, the responses to VO4 and PV were the same in the presence or absence of a functional endothelium. 5. PV-induced contractions in both tissues were observed in the absence of extracellular sodium but required extracellular calcium and were attenuated by 1 micro M nifedipine.6. In the gastric preparation, the characteristics of the contractile actions of PV paralleled those of EGF-Uro in terms of (1) inhibition by genistein, (2) inhibition by indomethacin and (3) a requirement for extracellular calcium. These response characteristics differed from those of other contractile agonists such as carbachol.7. In both the gastric and aortic preparations genistein was able to inhibit PV-induced contractions selectively without causing comparable inhibition of KCI-induced contractions. Tyrphostin (AG18) also selectively blocked PV-induced contractions in the gastric, but not in the aortic preparation.8. In both the gastric and aortic tissue, in step with an increased contractile response, PV caused increases in tissue phosphotyrosyl protein content, as detected by Western blot analysis using a monoclonal antiphosphotyrosine antibody; the increases in phosphotyrosyl protein content were reduced when tissues were treated with PV at the same time as a tyrosine kinase inhibitor.9 PV, at sub-contractile concentrations, potentiated the contractile action of angiotensin II in both the gastric and aorta tissue.10 We conclude that the growth factor-mimetic agent, PV, is a much more potent contractile agonist than V04 in both vascular and gastric smooth muscle tissue. PV can cause enhanced tissue phosphotyrosyl protein content most likely via the inhibition of tissue protein tyrosine phosphatases. The contractile actions of PV, which require extracelullar calcium and are independent of extracellular sodium, would appear not to be due either to Na+/Ca2" exchange, promoted by Na+/K+-ATPase inhibition or to the inhibition of Ca2+-ATPase and might be best explained by the ability of PV, via tyrosine phosphatase inhibition, to potentiate a tyrosine kinase pathway linked to calcium entry and to the contractile process.

Pervanadate stimulation of wortmannin-sensitive and -resistant 2-deoxyglucose transport in adipocytes.

Pervanadate mimics several distinct insulin effects, including stimulation of hexose uptake in the in vitro system, and reduces the blood glucose level in streptozotocin-treated diabetic rats. It has been proposed that pervanadate induces insulin-like effects mediated through autophosphorylation and activation of insulin receptor (IR) even in the absence of insulin by inhibiting protein tyrosine phosphatases. This study focused on the mechanism of pervanadate action on hexose uptake. Both insulin (100 nM) and pervanadate (100 microM), a protein tyrosine phosphatase inhibitor, induced a marked increase in the phosphorylation at tyrosine residues of IR and insulin receptor substrate 1 (IRS-1) and in 2-deoxyglucose uptake in 3T3-L1 adipocytes. Wortmannin (1 microM), a specific phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, inhibited the increased 2-deoxyglucose uptake by insulin completely but that by pervanadate only partially. On the other hand, both insulin- and pervanadate-stimulated PI 3-kinase activities were inhibited completely by wortmannin (100 nM), suggesting that the pervanadate-induced wortmannin-resistant effect on hexose uptake may be mediated through a PI 3-kinase-independent pathway. This pervanadate-induced wortmannin-resistant effect was abolished by ST-638, a specific tyrosine kinase inhibitor. These data suggest that at least two distinct tyrosine phosphorylation pathways may be involved in the insulin-like effect of pervanadate.

Ca(2+)-independent contraction of uterine smooth muscle induced by vanadate and its inhibition by Ca2+.

Vanadate, 30 microM, contracts uterine smooth muscle of estrogen-dominated non-pregnant rats in Ca(2+)-free medium after preincubation with 3 mM EGTA. In spite of the phosphorylation of the myosin light chain during this contraction, studies with fura-2 suggested that this contraction was not accompanied by an increase in the cytosolic Ca2+ level. Inhibitors of the myosin light chain kinase and protein kinase C partly inhibited this contraction. Vanadate seems to enter the cell through anion channels to inhibit phosphatases, resulting in phosphorylation via basal activities of the myosin light chain kinase and protein kinase C. An increase in the cytosolic free Ca2+ level resulted in relaxation of the contracting muscle in the same manner as in the oxytocin-induced Ca(2+)-free contraction.

Effect of antioxidants on vanadate-induced toxicity towards isolated perfused rat livers.

The effect of trolox C, a water soluble vitamin E analogue, propyl gallate and ascorbate on vanadate hepatotoxicity was investigated in vitro. In isolated perfused livers from fasted rats, sodium orthovanadate (2 mmol/l) led to toxic responses including reduction of oxygen consumption, release of cytosolic (glutamate-pyruvate-transaminase (GPT) and lactate dehydrogenase (LDH)) and mitochondrial (glutamate-dehydrogenase (GLDH)) enzymes, intracellular accumulation of calcium, a marked depletion of glutathione (GSH) and an enhanced formation and release of thiobarbituric acid- (TBA) reactive material. Trolox C and propyl gallate inhibited the release of GPT and LDH partially and that of GLDH totally, but had no influence on vanadate-induced calcium accumulation or on the reduction of oxygen consumption. Both agents suppressed vanadate-induced lipid peroxidation (LPO) and partially prevented GSH depletion. Ascorbate failed to provide any protection probably due to the interference of its pro-oxidant potential with its antioxidant activity. The protection, mainly of mitochondria, afforded by those agents which also inhibited LPO substantiates our previous findings that the pro-oxidant activity of vanadate is mainly responsible for its direct hepatotoxic actions [2]. Besides, reduction of organ perfusion rate due to vasoconstriction also contributes to vanadate toxicity, but oxidative stress is not involved in this indirect toxic activity.

Tiron administration minimizes the toxicity of vanadate but not its insulin mimetic properties in diabetic rats.

Although it has been reported that vanadate is effective in diminishing the expression of diabetes in the rat, the severe toxic side effects noted in the vanadate-treated animals suggest that chronic oral administration of vanadate argues against its use in human diabetes. The present study was conducted to evaluate the effects of the chelator Tiron on the mobilization of vanadium after administration of sodium metavanadate in the drinking water (0.20 mg/ml) of streptozotocin-induced diabetic rats for 35 days. Intraperitoneal treatment with Tiron (300 or 600 mg/kg) was initiated after three weeks of vanadate administration and continued for two weeks. The ameliorative effects of vanadium with respect to diabetes were not diminished by the administration of Tiron, but the accumulation of vanadium in kidney and bone was significantly decreased in the Tiron-treated groups and diabetes associated increases in serum GOT, GPT and cholesterol were diminished with Tiron treatment. It is concluded that the coadministration of metavanadate and Tiron may be of potential value for treatment of diabetes mellitus.

Bruton's tyrosine kinase prevents activation of the anti-apoptotic transcription factor STAT3 and promotes apoptosis in neoplastic B-cells and B-cell precursors exposed to oxidative stress.

Bruton's tyrosine kinase (BTK) was previously demonstrated to be a mediator of oxidative stress-induced apoptosis in irradiated neoplastic B-cells and B-cell precursors. Defective BTK expression in leukaemic B-cell precursors from infants with t(4;11) acute lymphoblastic leukaemia has been associated with radiation resistance. The present study examined whether BTK mediates apoptosis during oxidative stress by interfering with the anti-apoptotic function of signal transducer and activator of transcription 3 (STAT3). BTK physically associated with and tyrosine phosphorylated STAT3; this association was promoted by pervanadate (PV)-induced oxidative stress. The BTK/STAT3 interaction appeared to prevent STAT3 response to oxidative stress, because PV-induced STAT3 activation was markedly enhanced in DT40 chicken lymphoma B-cells that were rendered BTK-deficient by targeted disruption of the btk gene as well as in BTK-deficient RAMOS-1 human lymphoma B-cells. These BTK-deficient cells were highly resistant to oxidative stress-induced apoptosis triggered by PV treatment. Reconstitution of BTK-deficient DT40 cells with wild-type human BTK gene eliminated the amplification of the STAT3 response and restored the PV-induced apoptotic signal. Similarly, while the BTK-positive NALM-6 human leukaemic B-cell precursor cell line showed no STAT3 activation after PV treatment and was exquisitely sensitive to PV-induced apoptosis, PV failed to induce apoptosis in BTK-deficient RAMOS-1 human lymphoma B-cells that showed a robust STAT3 response. These results provide unprecedented biochemical and genetic evidence for a unique mode of cross-talk that occurs between BTK and STAT3 pathways during oxidative stress, whereby BTK may trigger apoptosis via negative regulation of the anti-apoptotic STAT3 activity.

Prevention by Tiron (sodium 4,5-dihydroxybenzene-1,3-disulfonate) of vanadate-induced developmental toxicity in mice.

Vanadate is embryotoxic and fetotoxic in golden hamsters, mice and rats. Tiron (sodium 4,5-dihydroxybenzene-1,3-disulfonate), a chelating agent widely used in analytical chemistry, is an effective antidote in the treatment of oral or parenteral vanadate poisoning. The present study evaluated the effect of administration of Tiron on sodium metavanadate (NaVO3)-induced developmental toxicity in mice. NaVO3 (25 mg/kg, i.p.) was injected on day 12 of gestation, whereas Tiron was injected subcutaneously at 0, 24, 48, and 72 hr after NaVO3 administration. Tiron effectiveness was assessed at dosage levels of 0, 250, 500, and 1,000 mg/kg. Cesarean sections were performed on gestation day 18. All live fetuses were examined for external, internal, and skeletal malformations and variations. Amelioration by Tiron of NaVO3 developmental toxicity was evidenced by a significant decrease in the number of resorbed fetuses, an increase in the mean fetal weight, and a reduction in the incidence of the skeletal variations caused by NaVO3. According to these results, Tiron offers encouragement with regard to its therapeutic potential for pregnant women exposed to vanadate. However, further investigations, including the effect of increasing the time interval between acute vanadate exposure and initiation of Tiron therapy, are required.

Kinetics of vanadate dissociation: estimation of the rate by inhibitor inactivation.

Vanadate (+5) is a potent inhibitor of a variety of ATPases including dynein ATPase. We describe a method useful for estimating the functional dissociation rate of vanadate from the active site which does not rely on classical physical separation techniques. The method involves spectrophotometrically monitoring the enzymatic activity as the inhibitor dissociates from the enzyme and is inactivated by norepinephrine. Norepinephrine effectively reverses vanadate inhibition by reducing vanadate (+5) to oxovanadium (+4). This reduction by norepinephrine is sufficiently fast for these purposes--addition of vanadate after norepinephrine shows no inhibition of ATPase activity. The mathematical estimation procedure is generally useful for estimation of dissociation rates of other reversible inhibitors which can be quickly inactivated after dissociation from the enzyme. The rate of dissociation of vanadate from dynein with ATP and 2-N3ATP as substrates using this method was estimated to be in the ranges 0.0023-0.0042 and 0.0057-0.0075 s-1, respectively. These rates permit estimation of the rates of vanadate association with dynein by using the reported dissociation constant for vanadate. The results are consistent with the very fast and potent inhibition of dynein ATPase activity observed.

Antagonistic action of reductants against vanadate-induced EP decrease.

The antagonistic action of ascorbic acid or glutathione against vanadate was studied by observing changes in EP in guinea pig cochlea. After intravenous injection of ascorbic acid or glutathione, the EP decrease induced by perfusion of the perilymphatic space with vanadate solution was suppressed and the EP showed a remarkable recovery in some animals. Intravenous preadministration of either of the two compounds inhibited or prevented the vanadate-induced EP decrease. When ascorbic acid or glutathione was added to the vanadate solution, the EP decrease was also inhibited. The chemical action of both reductants against vanadate is discussed. From the results obtained, it is speculated that a chemical balance between some oxidants and reductants in the stria vascularis, must be kept constant in order to maintain the EP at a constant potential level.

The role of estradiol receptor in the proliferative activity of vanadate on MCF-7 cells.

Vanadate stimulates growth of the estradiol-responsive MCF-7 cells in the absence of estrogens through a mechanism requiring tyrosine kinase activity. The proliferative effect of vanadate is mediated by estradiol receptor, and is inhibited by three antiestrogens, hydroxytamoxifen, ICI 164,384, and ICI 182,780. Estradiol abolishes the inhibitory effect of ICI 164,384 or ICI 182,780. Before stimulating cell proliferation, vanadate induces accumulation of tyrosine phosphorylation in several proteins including estradiol receptor and epidermal growth factor receptor. In addition, vanadate increases the binding activity of the estradiol receptor for its ligand. This is the first evidence of in vivo association between estradiol receptor tyrosine phosphorylation and its hormone-binding activation. Antiestrogens abolish the vanadate effect on estradiol receptor and epidermal growth factor receptor phosphorylation and reduce it on general protein tyrosine phosphorylation. These findings show that vanadate, apparently through estradiol receptor tyrosine phosphorylation, triggers activity of this receptor, which in turn stimulates protein tyrosine phosphorylation and induces cell proliferation.

Secophalloidin as a novel activator of skinned cardiac muscle.

Secophalloidin (SPH) is known to activate skinned cardiac muscle in the absence of Ca(2+). We hypothesized that SPH-induced changes in cross-bridge properties underlie muscle activation. We found that force responsiveness to orthovanadate was drastically reduced in SPH activated muscles compared to Ca(2+)-activated contraction. Moreover, SPH caused approximately 30% increase in Ca(2+)-independent force in muscles where Ca(2+) sensitivity was totally destroyed by troponin I extraction with 10mM vanadate. Thus, SPH and Ca(2+) activation differ in both properties of the cross-bridge cycle and protein requirements for thin filament regulation. In addition, we tested the relationship between the activating effects SPH and EMD 57033, a Ca(2+) sensitizer that increases resting force in cardiac muscle. After maximal activation by either SPH or EMD 57033, the other compound was found to further increase force, indicating that SPH activates muscle via a novel mechanism.

Vanadate inhibition of hepatocytic autophagy. Calcium-modulated and osmolality-modulated antagonism by asparagine.

The phosphate analogue vanadate, at 10 mM, strongly (approximately 90%) inhibited the autophagic sequestration of endogenous lactate dehydrogenase in isolated rat hepatocytes. The effect of vanadate was markedly (approximately 80%) antagonized by asparagine (20 mM), and to a lesser extent by glutamine, glycine, and alanine. The antagonism was only observed in the presence of Ca2+ when an isotonic standard incubation medium was used, but by increasing the medium osmolality this Ca2+ requirement could be eliminated. Asparagine induced a cell swelling (17% at 20 mM) that might account for at least part of its vanadate antagonism, since hypotonic cell swelling by itself stimulated autophagy (with a maximal effect at approximately 200 mosM). Conversely, hypertonic media inhibited autophagy and were additive to vanadate. In a strongly hypotonic medium (less than 200 mosM), both asparagine and vanadate were inhibitory. However, since vanadate alone had no effect on cell volume, the vanadate-asparagine antagonism could not be exerted exclusively at the level of cell volume regulation. An additional mechanism might be a partial deamination of asparagine, generating ammonia, which was found to oppose the vanadate inhibition of autophagy while having no effect on cell volume. Other metabolizable amino acids, like alanine and glycine, were moderately vanadate-antagonistic while failing to induce cell swelling. These results are compatible with a vanadate-antagonistic effect of asparagine mediated partly through an unknown mechanism (possibly pH change) by its deamination product, ammonia, partly through cell swelling and a secondary Ca2+ influx that could compensate for a vanadate-induced depletion of intracellular calcium stores.

Vanadyl causes hydroxyl radical mediated degradation of deoxyribose.

Vanadyl caused a time- and dose-dependent degradation of deoxyribose to carbonyl products detectable with thiobarbituric acid. This process was inhibited by catalase, ethanol or HEPES; whereas superoxide dismutase was without effect. Vanadate did not substitute for vanadyl even in the presence of a source of O2- plus H2O2; but it did so in the presence of reductants such as thiols or NADH. It appears that hydrogen peroxide, generated by the autoxidation of vanadyl, is reduced by vanadyl to the hydroxyl radical; which, in turn, was responsible for the degradation of deoxyribose. A similar process might contribute to the toxic and pharmacological effects of vanadium salts.

Glucose transport by osmotic shock and vanadate is impaired by glucosamine.

In 3T3-L1 adipocytes, we previously reported that glucosamine impairs insulin stimulation of glucose transport, which is accompanied by impaired insulin stimulation of serine/threonine kinase Akt. To examine the role of Akt in glucosamine-induced insulin resistance, we investigated time course for insulin stimulation of Akt activity and glucose transport during recovery from glucosamine-induced insulin resistance. After induction of insulin resistance by glucosamine, we washed cells to remove glucosamine and incubated them for various times. After one hour, insulin stimulated-glucose transport was significantly increased and continued to increase up to 6-24 h. Insulin stimulation of Akt, however, did not increase after 1-3 h and began to slightly increase after 6 h. Next, we investigated effects of osmotic shock and vanadate on glucose transport in glucosamine-treated cells and found that glucosamine completely inhibited their actions in these cells. These data suggest that an Akt-independent mechanism is operative in glucosamine-induced insulin resistance and glucosamine impairs glucose transport stimulated by various stimuli involving and not involving Akt activation.