OBJECTIVETo investigate the roles of cytochrome c (Cyt-c), caspase-9, and caspase-3 in pentavalent vanadium-induced neuronal apoptosis and to provide a basis for mechanism research.

METHODSNeurons from rats aged 1-3 days were cultured and treated with vanadium pentoxide (V2O5) at 5, 10, or 20 mmol/L. Neuronal apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL). The protein levels of Cyt-c, caspase-9, and caspase-3 were determined by Western blot.

RESULTSApoptosis bodies were detected in the nuclei of neurons by TUNEL. The number of neurons with apoptosis bodies increased with increasing dose of V2O5 The apoptosis index (AI) was significantly higher in the 10 and 20 mm/L exposure groups than in the control group (P < 0.05 or P < 0.01). Western blot showed that the protein expression levels of Cyt-c and caspase-3 significantly increased in the 5 mmol/L exposure group as compared with the control group (P < 0.05). In the 10 and 20 mmol/L exposure groups, the protein expression of Cyt-c, caspase-9, and caspase-3 all increased as compared with the control group (P < 0.01). Neuronal AI was positively correlated with Cyt-c, caspase-9, and caspase-3 (r = 0.954, P < 0.01; r = 0.938, P < 0.01; r = 0.943, P < 0.01).

CONCLUSIONPentavalent vanadium may induce neuronal apoptosis. The protein expression of Cyt-c, caspase-9, and caspase-3 may play an important role in neuronal apoptosis induced by pentavalent vanadium.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cytochromes c ; metabolism ; Neurons ; drug effects ; metabolism ; Rats ; Vanadium ; toxicity ; Vanadium Compounds ; toxicity

AIMTo study the hypoglycemic effect of bis (alpha-furancarboxylato) oxovanadium (IV) (VO-FA) in normal rats and streptozotocin (STZ)-diabetic rats.

METHODSRats were injected intraperitoneally STZ 50 mg.kg(-1) to induce diabetes. Blood glucose, glycohemoglobin, glycogen and serum insulin were observed after administering intragastrically VO-FA for four weeks.

RESULTSAfter 2 weeks administration, VO-FA reduced the blood glucose in STZ-rats (P < 0. 01) dose-dependently, and up to 4 weeks the blood glucose was normalized (below 11.1 mmol.L(-1)) in some of STZ-rats, whereas did not decrease in normal rats. After administration of VO-FA at the dosage of 56.8 and 113.6 mg.kg(-1), the serum insulin levels were lowered in normal rats and STZ-rats, respectively. Moreover, VO-FA reduced glycohemoglobin, improved the glucose tolerance, and increased the liver glycogen and muscle glycogen contents in STZ-rats in a dose-dependent manner (P < 0. 05, P < 0. 01), but not in normal rats.

CONCLUSIONVO-FA could improve the glycometabolism in STZ-rats, but not in normal rats.

Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; blood ; metabolism ; Dose-Response Relationship, Drug ; Glucose Tolerance Test ; Glycated Hemoglobin A ; metabolism ; Hypoglycemic Agents ; administration & dosage ; pharmacology ; Insulin ; blood ; Liver Glycogen ; metabolism ; Male ; Organometallic Compounds ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Vanadium ; administration & dosage ; pharmacology

Bone formation by osteoblast may be closely related to the increase in intracellular Ca2+ activity of osteoblast. In order to study the effects of changes in Ca2+ activity of osteoblast-like cell on fracture healing, we changed intracellular Ca2+ activity of osteoblast-like cells by vanadate and verapamil. And the process of fracture healing was observed after injection of the treatment osteoblast-like cells into the fracture site by hematoxylin-eosin (H-E) stain and bromodeoxyuridine (BrdU) stain. The results were as follow: 1) The most effective range of concentration which could facilitate bone formation was 10-6 to 10-5 M. 2) H-E stain showed more abundant osteoblast and osteoid tissues, more active mitotic division of osteoblast, and earlier appearance of chondroblast and chondroid tissue, making the maturation of woven bone faster in the vanadate-treated group than in the control group. The opposite was true in the verapamil-treated group compared with the control group. 3) BrdU labeling index showed more active osteoblastic proliferation in the vanadate-treated than in the control group. The opposite was observed in the verapamil-treated group compared with the control group. From these results, the fracture healing appears to be facilitated and decelerated by vanadate which apparently increase intracellular Ca2+ activity of osteoblast and verapamil which decreases it, repectively. Therefore the change of intracellular Ca2+ activity of osteoblast can be considered to be one of fracture healing mechanisms and expected to be applied for clinical purpose.
Bromodeoxyuridine ; Chondrocytes ; Fracture Healing ; Osteoblasts ; Osteogenesis ; Vanadates ; Verapamil

Intracellular ions which have a major role in cellular function have been reported to affect repair of radiation damage. Recently it has been reported that ouabain sensitizes A549 tumor cells hut not CCL-120 normal cells to radiation. Ouabain inhibits the Na+-K+-pump rapidly thus it increases intracellular Na concentration. Vanadate which is distributed extensively in almost all living organisms in known to be a Na+-K+-ATPase inhibitors. This study was performed to see any change in radiosensitivity of tumor cell by vanadate and any role of Na+-K+-ATPase in radiosensitization. Experiments have been carried out by pretreatment with vanadate in human cell line(A549, JMG) and mouse cell line(L1210, spleen). For the cell survival MTT assay was performed for A549 and JMG cell and trypan blue dye exclusion test for L120, and spleen cells. Measurements of Na+-K+-ATPase activity in control, vanadate treated cell, radiation treated cell (9 Gy for A549 and JMG, 2 Gy for L1201, spleen), and combined 10-6 M vanadate and radiation treated cells were done. The results were summarized as follows. 1. L1210 cell was most radiosensitive, and spleen cell and JMG cell were intermediate, and A549 cell was least radiosensitive. 2. Minimum or cytotoxicity was seen with vanadate below concentration of 10-6 M. 3. In A549 cells there was a little change in radiosensitivity with treatment of vanadate. However radiation sensitization was shown in low dose level of radiation i. E. 2-Gy. In JMG cells no change in radiosensitivity was noted. Both L1210 and spleen cell had radiosensitization but change was greater in tumor cell. 4. Na+-K+-ATPase activity was inhibited significantly in tumor cell by treatment of vanadate. 5. Radiation itself inhibited Na+-K+-ATPase activity of tumor cell with high Na+- K+-ATPase concention. Increase in radiosensitivity by vanadate was closely associated with original Na+-K+-ATPase contents. From the above results vanadate had little cytotoxicity and it sensitized tumor cells to radiation. Inhibitory effect of vanadate on Na+-K+-ATPase activity might be one of the contributing factors for radiosensitization to tumor cells which has greater enzyme activity than that of normal cell. It was suggested vanadate could be used as a potential radiosensitizer for tumor cells.
Animals ; Cell Survival ; Humans ; Ions ; Mice ; Ouabain ; Radiation Tolerance* ; Spleen ; Trypan Blue ; Vanadates*

Vanadium is an essential trace element but has not been identified with a specific biogical role. To study the direct effects of vanadium on osteoblast, we incubated murin osteoblast-like (MC3T3-E1) cells with various concentration of vanadium oxide & sodium orthovanadate. This study was designed to investigate the effect of vanadium on DNA synthesis, alkaline phosphatase (ALP) activity, cAMP formation responsive to parathormone(PTH) and type I alpha 2 collagen ribonucleic acid (mRNA) level in murin osteoblast-like (MC3T3-E1) cells. The cells were cultured in a -minimal essential medium(alpha -MEM) supplemented with 10% fetal bovine serum (FBS) and then changed to 0.1% FBS with various concentration of vanadium oxide & sodium orthovanadate. Quiescent cultured MC3T3-E1 cells incubated for 24 hours with 2,5,10,15,20 micrometer vanadium oxide incorporated [3H]Thymidine; every concentration showed increases in [3H]Thymidine incorporations dose dependant manner, the greatest response occurred at 20micrometer. Quiescent cultured MC3T3- E1 cells incubated for 3days with 2,5,10,15,20 micrometer vanadium oxide, for 2 days with sodium orthovanadate and alkaline phosphatase was assayed with disodium phenyl phosphate as substrate. Vanadium oxide increased the alkaline phosphatase content in MC3T3- E1 cells at 2 micrometer & 6micrometer; the greatest response occurred at 2micrometer. But decreased at other content. sodium orthovanadate increased alkaline phosphatase content in MC3T3-E1 cells at all concentration ; the greatest response occurred at 4micrometer. Quiescent cultured MC3T3- E1 cells incubated for 3days with 5,10micrometer vanadium oxide, with 5,8micrometer sodium orthovanadate and cAMP formation was measured by Radioimmunoassay(RIA). Vanadium oxide & sodium orthovanadate showed the tendency of inhibitory effects on cAMP responsiveness to PTH in MC3T3-E1 cells. Quiescent cultured MC3T3-E1 cells incubated for 24hours with 10,20micrometer vanadium oxide, with 5,10micrometer sodium orthovanadate and Type I alpha2 collagen ribonucleic acid (mRNA) expression was studied by Northern blot analysis. Northern blot analysis of vanadium oxide treated cells showed decreasing effects 0& sodium orthovanadate revealed increasing effects in type I &2 collagen ribonucleic acid (mRNA) level.
Alkaline Phosphatase ; Blotting, Northern ; Collagen ; DNA ; Osteoblasts ; RNA ; Sodium* ; Vanadates* ; Vanadium*

It is the aim of this study to investigate the effects of sodium fluoride and sodium orthovanadate upon the proliferation and activity of the osteoblast (MC3T3-E1 cells). MC3T3-E1 cells were cultured in alpha-MEM containing 10% FBS and various concentration of sodium fluoride and sodium orthovanadate was appended to serum free media. DNA synthesis was examined through the [3H] thymidine incorporation into DNA. Collagen synthesis was examined through the [3H] proline incorporation into collagenase digestible protein and noncollagen protein. The following results were drawn; 1. Sodium fluoride stimulated the DNA synthesis of osteoblast significantly in dose-dependent manner within the concentration from 2micrometer to 10micrometer (P<0.005). 2. Sodium orthovanadate stimulated the DNA synthesis of osteoblast significantly in dosedependent manner within the concentration from 2micrometer to 8micrometer, however showed diminution at 10micrometer (P<0.001). 3. Sodium fluoride and sodium orthovanadate stimulated the percent collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from 5micrometer to 10micrometer (P<0.001). 4. Sodium fluoride and sodium orthovanadate stimulated the non collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from 5micrometer to 10micrometer (P<0.001). In conclusion, sodium fluoride and sodium orthovanadate stimulate the proliferation and activity of osteoblast by stimulation of DNA synthesis and collagen and noncollagen synthesis in osteoblast.
Cell Line* ; Collagen ; Collagenases ; Culture Media, Serum-Free ; DNA ; Osteoblasts* ; Proline ; Sodium Fluoride* ; Sodium* ; Thymidine ; Vanadates*

The present study was intended to examine the effect of sodium vanadate on contractility of vascular smooth muscle. Aortic ring preparations were made from the rabbit thoracic aorta and endothelial cells were removed from the ring. The contractility of the aortic ring was measured under various conditions. The results were summarized as follows; 1) Sodium vanadate induced contraction of vascular smooth muscle in a dose-dependent fashion. 2) The contractile effects were not blocked by treatments with adrenergic blocking agent(phentolamine) and indomethacin, indicating the direct action of the drug on vascular smooth muscle. 3) In the presence of ouabain, Na(+)-K(+)-ATPase inhibitor, sodium vanadate still increased the contractility of vascular smooth muscle. 4) Treatment with 4.4'-diisothiocyanostilbene-2.2'-disulfonic acid(DIDS) blocked completely the contractile effects of sodium vanadate. 5) In the presence of verapamil, lanthanum and ryanodine, the contractility of the vascular smooth muscle by sodium vanadate was decreased. From the above results. it was suggested that sodium vanadate acts directly on vascular smooth muscle and causes contraction. It was probably due to inhibition of Ca(++)-ATPase in plasma membrane as well as increasing the release of Ca(++) from sarcoplasmic reticulum and Ca(++) influx across the plasma membrane, but not inhibition of Na(+)-K(+)-ATPase.
Aorta, Thoracic ; Cell Membrane ; Endothelial Cells ; Indomethacin ; Lanthanum ; Muscle, Smooth, Vascular* ; Ouabain ; Ryanodine ; Sarcoplasmic Reticulum ; Sodium* ; Vanadates* ; Verapamil

PURPOSE: The purpose: of this study was to know the effect of inhibition of protein dephosphorylation on the synthesis of collagen and fibronectin (FN), alkaline phosphatase (ALP) activity, and the formation of bone nodule in MC3T3-E1 osteoblasts using orthovanadate (OVA) which is a potent protein tyrosine phosphatase (PTPases) inhibitor. MATERIALS AND METHODS: The synthesis of collagen, noncollagenous protein (NCP), and percent collagen in MC3T3-E1osteoblasts with or without OVA treatment according to concentration and time sequence was determined by incorporation of [3 H]-proline, synthesis of FN by [35 S] methionine incorproated immunoprecipitation after treatment with 100 M OVA for 24 hours, mRNA expression of collagen and FN by Northern blotting, activity of ALP by spectrophotometric method, and formation of bone nodule by staining method. RESULTS: OVA increased collagen and NCP synthesis concentration dependently, until 12 hours in short-time culture, and time dependently through the differentiation until 29 days, however, there was no significant effect on the percent collagen production. OVA increased percent collagen synthesis significantly at 6 hours, and decreased in a long time culture. Total FN synthesis and FN synthesis in cell layer were increased by OVA, however, FN synthesis in medium was not changed. OVA decreased collagen mRNA level dose-dependently and increased the steady-state level of FN mRNA. OVA inhibited activity of ALP in both short and long-time culture. OVA inhibited bone nodule formation in MC3T3-E1 osteoblasts. CONCLUSION: These results indicate that the inhibition of PTPase by OVA increased the synthesis of collagen, FN, and decreased ALP activity and it resulted in the inhibition of bone formation in MC3T3-E1 osteoblast cells.
Alkaline Phosphatase* ; Blotting, Northern ; Collagen* ; Fibronectins* ; Immunoprecipitation ; Methionine ; Osteoblasts* ; Osteogenesis ; Ovum ; Protein Tyrosine Phosphatases ; RNA, Messenger ; Vanadates*

The effects of vanadate on cellular Ca2+ movements across the sarcolemma of cardiac muscle cells were investigated by measuring the intracellular and extracellular Ca2+ activities of guinea pig papillary muscle with Ca2+-selective electrodes. During the rest period following a steady-state of 2 contractions per second the extracellular Ca2+ concentration was increased over the basal level within a minute. During the rest period Ca2+ was transported across the sarcolemma into the extracellular space. Vanadate decreased the change in extracellular Ca2+ concentration during the rest period implying that the Ca2+ efflux across the sarcolemma was decreased by vanadate. Vanadate increased intracellular Ca2+ activities significantly (from 1.9 X 10(-7) M to 10(-6)M) resulting in an increase in resting tension. These results suggest that vanadate decreases Ca2+ efflux from the cells into the extracellular space by blocking Ca2+ transport across the sarcolemma, possibly blocking the Na+-Ca2+ exchange transport.
Animal ; Calcium/metabolism* ; Female ; Guinea Pigs ; Ion Channels/drug effects* ; Male ; Membrane Potentials/drug effects ; Papillary Muscles/drug effects* ; Vanadates ; Vanadium/pharmacology*

BACKGROUND: The proinflammatory cytokine, IFN-y has been shown to exert pleiotropic effects in a variety of pathophysiologic conditions in autoimmune thyroid disease. The thyrocyte response to IFN-y is mediated two distinct classes of proteins, Janus kinases(Jakl and Jak2) and Signal Transducers and Activation of Transcription(STATl). The activation of STAT 1 is involved in the regulation of many interferon stimulated genes, such as MHC class II, intercellular adhesion molecules-1(ICAM-1) and MHC class II transactivator(CIITA) after the binding to the GASgFN- pactivated site) of the gene promoters. Recently we found TSH/forskolin inhibits IFN-y stimulated maximal expression of ICAM-1 in FRTL-5 cell. IFN-y action is localized between -175 bp and -97 bp from the start of translation of ICAM-1 gene which contains regulatory elements known to be involved in IFN-y action in other eukaryotic cells, palindromic IFN-y activated site(GAS)(5-TTTCCGGGAAA-3) which could bind STAT1, STAT3, STAT5, STAT6. Furthermore, the addition of TSH and forskolin causes a decrease in ICAM-1 promoter activity and its action was localized in GAS. These findings suggested TSH/cAMP signaling pathways downregulate IFN-y activated Janus kinase-STAT signaling path. We wanted to explore the possible involvement of elevated cAMP in the negative regulation of IFN-y induced STAT1 activation in thyroid cells. METHOD: We made several 5-deletion constructs of rat ICAM-1 promoter and analyzed the promoter activities by measuring the luciferase activity after tranfection into FRTL-5 cells. The protein/DNA complex was measured by electrophoretic mobility shift analysis using labeled oligonucleotide. We checked the level of total and phosphorylated STATl protein by immunoblot analysis using specific antibodies. RESULTS: Stimulation of IFN-y in FRTL-5 cells resulted in rapid activation of STATl/DNA binding activity, which was apparent after several minute of stimulation, maintains its activity until 48 h. Incubation of cells with TSH result in suppression of IFN-p mediated STAT1/DNA binding activity throughout the time course of activation by IFN-y. Addition of TSH into 5H maintained FRTL-5 cells did not change the total amount of latent STAT1 amount and also not affect IFN-y mediated production of total STAT1 until 4 h. IFN-y(100 U/mL) rapidly induced phosphorylation of STAT1 within 30 min. and maintained its level without significant change until 48 hours. Cells treated with TSH dramatically lowered the level of IFN-y induced production and phosphorylation of STAT1 after 12 h, 24 h, 36 h, and 48 h but TSH had no effect on the level of phosphorylated STATl within 4 h after IFN-y stimulation. The proteasome inhibitor, MG132 and phosphatase inhibitor, sodium orthovanadate did not block the TSH or forskolin mediated downregulation of phosphorylated STAT1. CONCLUSION: These results indicate a regulatory mechanism which TSH signaling can modulate the prolonged activation of Jak/Stat by IFN-y. We identified one of mechanisms related to TSH mediated negative suppression of the ICAM-1 gene; TSH/cAMP signaling pathways downregulate the cytokine activated Janus kinase-STAT signaling path.
Animals ; Antibodies ; Colforsin ; Down-Regulation ; Eukaryotic Cells ; Gene Expression* ; Intercellular Adhesion Molecule-1 ; Interferons ; Luciferases ; Phosphorylation ; Proteasome Inhibitors ; Rats ; Sodium ; Thyroid Diseases ; Thyroid Gland ; Thyrotropin* ; Transducers* ; Vanadates