To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 microL was established. Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP). The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78). A total of 24,240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening. Finally, six compounds with high inhibitory activity were identified, whose IC50 values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 microg x mL(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.
Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; analysis ; pharmacology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Humans ; Inhibitory Concentration 50 ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; metabolism ; Sensitivity and Specificity ; Vanadates ; pharmacology

OBJECTIVE: Triptolide (TP) has been reported to suppress the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), of which main function is to inactivate the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2), and to exert antiproliferative and pro-apoptotic activities. However, the mechanisms underlying antiproliferative and pro-apoptotic activities of TP are not fully understood. The purpose of this study was to examine whether the down-regulation of MKP-1 expression by TP would account for antiproliferative activity of TP in immortalized HT22 hippocampal cells. METHODS: MKP-1 expression and MAPK phosphorylation were analyzed by Western blot. Cell proliferation was assessed by 3H-thymidine incorporation. Small interfering RNA (siRNA) against MKP-1, vanadate (a phosphatase inhibitor), U0126 (a specific inhibitor for ERK-1/2), SB203580 (a specific inhibitor for p38 MAPK), and SP600125 (a specific inhibitor for JNK-1/2) were employed to evaluate a possible mechanism of antiproliferative action of TP. RESULTS: At its non-cytotoxic dose, TP suppressed MKP-1 expression, reduced cell growth, and induced persistent ERK-1/2 activation. Similar growth inhibition and ERK-1/2 activation were observed when MKP-1 expression was blocked by MKP-1 siRNA and its activity was inhibited by vanadate. The antiproliferative effects of TP, MKP-1 siRNA, and vanadate were significantly abolished by U0126, but not by SB203580 or SP600125. CONCLUSION: Our findings suggest that TP inhibits the growth of immortalized HT22 hippocampal cells via persistent ERK-1/2 activation by suppressing MKP-1 expression. Additionally, this study provides evidence supporting that MKP-1 may play an important role in regulation of neuronal cell growth.
Anthracenes ; Blotting, Western ; Butadienes ; Cell Proliferation ; Diterpenes ; Down-Regulation ; Epoxy Compounds ; Imidazoles ; Neurons ; Nitriles ; p38 Mitogen-Activated Protein Kinases ; Phenanthrenes ; Phosphorylation ; Protein Kinases ; Pyridines ; RNA, Small Interfering ; Vanadates

This study is to evaluate the effect of resveratrol on carotid baroreceptor activity (CBA). The functional curve of carotid baroreceptor (FCCB) was constructed and the functional parameters of carotid baroreceptor were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. Resveratrol (30, 60 and 120 micromol x L(-1)) inhibited CBA, which shifted FCCB to the right and downward. There was a marked decrease in peak slope (PS) and peak integral value (PIV) of carotid sinus nerve charge in a concentration-dependent manner. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol x L(-1)), an inhibitor of nitric oxide synthase (NOS), eliminated the inhibitory effect of resveratrol. Pretreatment with Bay K8644 (an agonist of L-type calcium channel, 500 nmol x L(-1)) abolished the effect of resveratrol on CBA. A potent inhibitor of tyrosine phosphatase (sodium orthovanadate, 1 mmol x L(-1)) did not influence the effect of resveratrol on CBA. Resveratrol inhibits carotid baroreceptor activity, which may be mediated by the locally released NO and decreased calcium influx. Several studies have showed a cardioprotective effect of resveratrol, with the penetrating study of resveratrol, it may show a potential value in the clinical treatment of cardiovascular disease as an alternative medicine.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Anesthesia ; Animals ; Carotid Sinus ; drug effects ; physiology ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Pressoreceptors ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology ; Vanadates ; pharmacology

Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.
Vanadates/pharmacology ; Tetradecanoylphorbol Acetate/pharmacology ; Protein-Tyrosine-Phosphatase/*metabolism ; Phosphotyrosine/metabolism ; Phosphorylation/drug effects ; Nitric Oxide/metabolism ; Humans ; Fibroblasts ; Epidermal Growth Factor/pharmacology ; Enzyme Inhibitors/pharmacology ; Econazole/pharmacology ; Cytochrome P-450 Enzyme System/antagonists & inhibitors/*metabolism ; Cyclic GMP/analogs & derivatives/metabolism ; Cells, Cultured ; Calcium Channels/*metabolism ; Calcium/metabolism ; Bradykinin/pharmacology

The hypoglycemic effects after oral administration of vanadium have been studied previously in many species such as rats, mice and even humans. However, there has been no prior report on the glucose lowering effect of vanadium on diabetic dogs. Therefore, the purpose of this study was to evaluate the hypoglycemic effects of oral vanadium on diabetic dogs. Diabetes mellitus in the dogs studied was induced by alloxan monohydrate intravenous injection. The dogs were divided into two groups, one was the diabetic control (DC) group (n = 4) and the other was the vanadium treated (DV) group (n = 6). Fresh water was supplied to the dogs in the DC group, but sodium metavanadate solution (0.1~0.2 mg/ml) was given to the dogs in DV group from one week after the alloxan injection. The fasting glucose levels, fructosamine and serum chemistry profiles were compared between the two groups weekly for three weeks. The fasting blood glucose levels in DV group were significantly lower than those in the DC group (p < 0.01). Fructosamine levels in the DV group were also lower than those in the DC group (p < 0.05). The serum chemistry profiles were not significantly different in comparisons between the two groups. However, the cholesterol levels were significantly lower in the DV group compared to the DC group (p < 0.05). Our findings showed that oral vanadium administration had a hypoglycemic effect on chemically induced diabetic dogs.
Alanine Transaminase/blood ; Alkaline Phosphatase/blood ; Animals ; Aspartate Aminotransferases/blood ; Blood Glucose/metabolism ; Blood Urea Nitrogen ; Chlorides/blood ; Cholesterol/blood ; Creatinine/blood ; Diabetes Mellitus, Experimental/blood/*drug therapy ; Dog Diseases/blood/*drug therapy ; Dogs ; Female ; Fructosamine/blood ; Hypoglycemic Agents/*pharmacology ; Male ; Pancreas/drug effects/pathology ; Potassium/blood ; Random Allocation ; Sodium/blood ; Triglycerides/blood ; Vanadates/*pharmacology

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.
Animals ; Cell Movement/drug effects/*physiology ; Cells, Cultured ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Genistein/pharmacology ; Hydrogen Peroxide/pharmacology ; Muscle, Smooth, Vascular/cytology/*physiology ; *Oxidative Stress/drug effects ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Protein Kinase C/*metabolism ; Protein-Tyrosine Kinase/antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/drug effects ; Vanadates/pharmacology ; Vascular Diseases/metabolism

The effects of genistein (GST) on intracellular calcium concentration ([Ca(2+)](i)) were investigated in guinea pig ventricular myocytes. [Ca(2+)](i) was detected by confocal microscopy and represented by relative fluorescent intensity (FI-F(0)) /FI(0), %). The results showed that GST (10-40 micromol/L) reduced [Ca(2+)](i) in normal Tyrode's solution, Ca(2+)-free Tyrode's solution and normal Tyrode's solution containing 3 mmol/L EGTA in a concentration-dependent manner. The effects of GST on [Ca(2+)](i) in normal Tyrode's solution were partially inhibited by pretreatment with sodium orthovanadate, a potent inhibitor of protein tyrosine phosphatase, or L-type Ca(2+) channel agonist Bay K8644. GST also markedly inhibited the ryanodine-induced [Ca(2+)](i) responses in Ca(2+)-free Tyrode's solution. When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, GST (40 micromol/L) could block the propagating waves of elevated [Ca(2+)](i), and reduce the velocity and duration of propagating waves. These results suggest that GST may reduce the [Ca(2+)](i) in isolated guinea pig ventricular myocytes. The inhibition of voltage-dependent Ca(2+) channel, tyrosine kinase inhibition, and alleviation of Ca(2+) release from SR are possibly involved in the GST effect.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; Genistein ; pharmacology ; Guinea Pigs ; Heart Ventricles ; Male ; Microscopy, Confocal ; Myocytes, Cardiac ; cytology ; metabolism ; ultrastructure ; Protein Tyrosine Phosphatases ; antagonists & inhibitors ; Sarcoplasmic Reticulum ; metabolism ; Vanadates ; pharmacology

BACKGROUND: The p38 mitogen-activated protein kinases (p38Map kinases) are a family of prolinedirected serine/threonine kinases. At least four isoforms of p38Map kinases have been identified; however, their physiological significances remain to be understood. Recently, the role of p38Map kinase in insulin-stimulated glucose uptake has been suggested. The present study aimed to investigate which isoform(s) were responsive to insulin stimulation. In addition, the activities of p38 Map kinase isoforms that may participate in the insulin's antiapoptotic function in CHO-IR cells were also determined. METHODS: Chinese hamster ovary cells, expressing wild- or mutated human insulin receptors (CHO-IR cells), were used to investigate whether insulin can stimulate any of the isoform(s) of the p38Map kinases. The p38Map kinase activity was determined by measuring the degree of 32P-labelling of ATF-2 protein, a specific substrate of p38Map kinase. A DNA laddering assay was performed to examine the degree of apoptosis and a RT-PCR analysis to determine which isoform(s) of the p38Map kinases were expressed in response to insulin. RESULTS: p38Map kinase activation by insulin was sharply suppressed in only the CHO-IR/A1018K cells, which lack the intrinsic tyrosine kinase activity of insulin receptors. Insulin stimulation of p38Map kinase was insensitive to SB203580, an inhibitor of the alpha(alpha)-and beta(beta)-isoforms of p38Map kinases. Moreover, orthovanadate, known as a specific stimulator of the gamma(gamma)-and delta(delta-) isoforms, stimulated the p38Map kinase activity in CHO-IR cells. Insulin increased the degree of mRNA expression of the delta-isoform, but not that of the alpha-isoform p38Map kinase. Interestingly, PD98059, an inhibitor of ERK, suppressed p38Map kinase stimulation, as well as the antiapoptotic protection of cells by insulin. As insulin was found to still protect ERK-lacking cells (CHO-IR/ SOS) from apoptosis, any substantial role(s) of ERK might be excluded. CONCLUSION: Our data suggest that insulin may stimulate the activity and expression of the-isoform of p38Map kinase in a MEK1/2-dependent manner. The involvement of the delta-isoform of p38Map kinase in insulin's antiapoptotic protection was also suggested, but remains to be investigated further to clarify the nature of its mechanism of action
Animals ; Apoptosis ; Cricetinae ; Cricetulus ; DNA ; Female ; Glucose ; Humans ; Insulin ; Ovary ; p38 Mitogen-Activated Protein Kinases ; Phosphotransferases* ; Protein Isoforms ; Protein-Tyrosine Kinases ; Receptor, Insulin ; RNA, Messenger ; Vanadates

BACKGROUND: The p38 mitogen-activated protein kinases (p38Map kinases) are a family of prolinedirected serine/threonine kinases. At least four isoforms of p38Map kinases have been identified; however, their physiological significances remain to be understood. Recently, the role of p38Map kinase in insulin-stimulated glucose uptake has been suggested. The present study aimed to investigate which isoform(s) were responsive to insulin stimulation. In addition, the activities of p38 Map kinase isoforms that may participate in the insulin's antiapoptotic function in CHO-IR cells were also determined. METHODS: Chinese hamster ovary cells, expressing wild- or mutated human insulin receptors (CHO-IR cells), were used to investigate whether insulin can stimulate any of the isoform(s) of the p38Map kinases. The p38Map kinase activity was determined by measuring the degree of 32P-labelling of ATF-2 protein, a specific substrate of p38Map kinase. A DNA laddering assay was performed to examine the degree of apoptosis and a RT-PCR analysis to determine which isoform(s) of the p38Map kinases were expressed in response to insulin. RESULTS: p38Map kinase activation by insulin was sharply suppressed in only the CHO-IR/A1018K cells, which lack the intrinsic tyrosine kinase activity of insulin receptors. Insulin stimulation of p38Map kinase was insensitive to SB203580, an inhibitor of the alpha(alpha)-and beta(beta)-isoforms of p38Map kinases. Moreover, orthovanadate, known as a specific stimulator of the gamma(gamma)-and delta(delta-) isoforms, stimulated the p38Map kinase activity in CHO-IR cells. Insulin increased the degree of mRNA expression of the delta-isoform, but not that of the alpha-isoform p38Map kinase. Interestingly, PD98059, an inhibitor of ERK, suppressed p38Map kinase stimulation, as well as the antiapoptotic protection of cells by insulin. As insulin was found to still protect ERK-lacking cells (CHO-IR/ SOS) from apoptosis, any substantial role(s) of ERK might be excluded. CONCLUSION: Our data suggest that insulin may stimulate the activity and expression of the-isoform of p38Map kinase in a MEK1/2-dependent manner. The involvement of the delta-isoform of p38Map kinase in insulin's antiapoptotic protection was also suggested, but remains to be investigated further to clarify the nature of its mechanism of action
Animals ; Apoptosis ; Cricetinae ; Cricetulus ; DNA ; Female ; Glucose ; Humans ; Insulin ; Ovary ; p38 Mitogen-Activated Protein Kinases ; Phosphotransferases* ; Protein Isoforms ; Protein-Tyrosine Kinases ; Receptor, Insulin ; RNA, Messenger ; Vanadates

OBJECTIVETo evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level.

METHODSImmature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level.

RESULTSIn dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 micromol/L SOV and control groups (P < 0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 micromol/L. In incontinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 micromol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P < 0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed.

CONCLUSIONDynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or M I to anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.

Animals ; Cells, Cultured ; Cyclin B ; genetics ; metabolism ; Cyclin B1 ; Dyneins ; antagonists & inhibitors ; Female ; Gene Expression Regulation ; Meiosis ; drug effects ; Mice ; Mice, Inbred BALB C ; Oocytes ; drug effects ; growth & development ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vanadates ; pharmacology