Recent studies showed that Eph/Ephrin tyrosine kinase family plays an important role in the development and functional maintenance of the nervous system, but its function in the sympathetic nervous system is still obscure. In the present study, we examined the effect of Eph/Ephrin-B1 signaling on the whole-cell currents mediated by either alpha7 or alpha3-nicotinic acetylcholine receptors (nAChRs) in acutly dissociated ciliary ganglion (CG) neurons. Firstly, we detected the effect of Ephrin-B1 on nAChRs currents. The neurons were randomly divided into control group, Ephrin-B1Fc-treated group that was stimulated by recombinant Ephrin-B1Fc, IgG-treated group, and Ephrin-B1-treated group. Secondly, we studied the regulatory mechanism of Ephrin-B1Fc on nAChRs currents. The neurons were randomly divided into control group, Ephrin-B1Fc-treated group, PP2 (inhibitor of Src tyrosine kinase) or PD98095 (antagonist of mitogen-activated protein kinase)-treated group, Ephrin-B1Fc + PP2 or PD98095-treated group. The results showed that there was no significant difference between the currents in control group, IgG-treated group and Ephrin-B1-treated group, but Ephrin-B1Fc significantly suppressed both alpha3-nAChRs and alpha7-nAChRs-mediated currents (P=0.002, P=0.003). Pretreatment with PP2 or PD98095 could partially rescue the Ephrin-B1Fc-induced suppression of currents mediated by alpha3-nAChRs or alpha7-nAChRs respectively. These results suggest that the Eph/Ephrin-B1 signaling may inhibit alpha3-nAChRs and alpha7-nAChRs-mediated currents on CG neurons, involving Src tyrosine kinase and mitogen-activated protein kinase signaling in the regulation of sympathetic nervous system.
Ephrin-B1 ; metabolism ; Ganglia, Parasympathetic ; enzymology ; Mitogen-Activated Protein Kinases ; metabolism ; Neurons ; enzymology ; Receptors, Nicotinic ; metabolism ; Signal Transduction ; alpha7 Nicotinic Acetylcholine Receptor ; metabolism ; src-Family Kinases ; metabolism

OBJECTIVETo investigate the effect of Src kinase inhibitor PP2 on intercellular communication of gap junction in breast cancer cells.

METHODSCultured breast cancer Hs578T cells were treated with various concentrations of pp2 (0,1,2,4,8,16,32 μmol/L) for 24h. Cell growth was determined by MTT assay; dye spread in Hs578T cells was measured by Parachute assay; and the expression of Src kinase in Hs578T cells was detected by Western blot.

RESULTSMTT assay showed that the survive rate of Hs578T cells treated with PP2 (1 ≊ 8 μmol/L) was 98% ± 3% ≊ 94 % ± 4%. Parachute assay showed that compared to control group the standard normalized dye spread rates of Hs578T cells treated with 1,2,4 and 8 μmol/L PP2 were 1.60 ± 0.08,2.00 ± 0.05,2.20 ± 0.05 and 2.70 ± 0.09,respectively (all P<0.01). Moreover,compared to control group at the same time points,the standard normalized dye spread of Hs578T cells treated with 8 μmol/L PP2 for 6,12 and 24 h were 1.4 ± 0.05,1.7 ± 0.06,and 2.2 ± 0.07,respectively (all P<0.01). Western blot showed that the expression ratios of Src kinase/β-actin of Hs578T cells treated with 1,2,4 and 8 μmol/L PP2 for 24 h were 0.93 ± 0.02,0.70 ± 0.09,0.66 ± 0.09 and 0.36 ± 0.10,which were significantly inhibited compared with control group (P<0.05 or 0.01). And the expression ratio of Src kinase/β-actin of Hs578T cells treated with 8 μmol/L PP2 for 6,12 and 24h was 0.82 ± 0.03,0.66 ± 0.08 and 0.59 ±0.09, which were all inhibited significantly compared to control group (P<0.01).

CONCLUSIONPP2 enhances the gap junction function in breast cancer Hs578T cells, which is probably related to the inhibition of Src kinase.

Breast Neoplasms ; pathology ; Cell Line, Tumor ; Female ; Gap Junctions ; drug effects ; Humans ; Pyrimidines ; administration & dosage ; pharmacology ; src-Family Kinases ; metabolism

Intracellular signal transduction plays an important role in the process of cellular metabolism, segmentation, differentiation, biological behaviour and cell death. Overactive signal transduction relates to tumor development and progression. Signaling pathways operated by protein tyrosine kinases (PTKs) will be illuminated here briefly. The Ras/Raf/MAPK and PI-3K/Akt pathways through receptor protein tyrosine kinases (RTKs), the Src, Bcr-Abl and JAK/STAT pathways by non-receptor protein tyrosine kinases (nrPTKs) are shown separately. Antitumor agents targeting the key proteins involved in the above five signalling routes are also summarized in this review.
Animals ; Antineoplastic Agents ; pharmacology ; Humans ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Receptor Protein-Tyrosine Kinases ; metabolism ; STAT Transcription Factors ; metabolism ; Signal Transduction ; drug effects ; ras Proteins ; metabolism ; src-Family Kinases ; metabolism

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria. METHODS: A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg•d) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells. RESULTS: In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group (<0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group (<0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group (<0.01). CONCLUSION Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.
Acupuncture Points ; Animals ; Connective Tissue ; metabolism ; Electroacupuncture ; Mast Cells ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Syk Kinase ; metabolism ; Urticaria ; therapy ; src-Family Kinases ; metabolism

Pervanadate, a complex of vanadate and H2O2, has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen- activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.
Animals ; Cell Line ; Enzyme Activation/drug effects ; Fibroblasts ; Mitogen-Activated Protein Kinases/metabolism ; Phospholipase D/*metabolism ; Rats ; Receptor, Epidermal Growth Factor/*agonists/*metabolism ; Vanadates/*pharmacology ; src-Family Kinases/metabolism

Tyrosine kinases are one of the most important regulators for intracellular signal transduction related to inflammatory responses. However, there are no reports describing the effects of tyrosine kinases on neutrophil apoptosis induced by Entamoeba histolytica. In this study, isolated human neutrophils from peripheral blood were incubated with live trophozoites in the presence or absence of tyrosine kinase inhibitors. Entamoeba-induced receptor shedding of CD16 and PS externalization in neutrophils were inhibited by pre-incubation of neutrophils with the broad-spectrum tyrosine kinase inhibitor genistein or the Src family kinase inhibitor PP2. Entamoeba-induced ROS production was also inhibited by genistein or PP2. Moreover, genistein and PP2 blocked the phosphorylation of ERK and p38 MAPK in neutrophils induced by E. histolytica. These results suggest that Src tyrosine kinases may participate in the signaling event for ROS-dependent activation of MAPKs during neutrophil apoptosis induced by E. histolytica.
*Apoptosis ; Cells, Cultured ; Entamoeba histolytica/*immunology/*pathogenicity ; GPI-Linked Proteins/metabolism ; Genistein/metabolism ; Humans ; Neutrophils/*immunology ; Protein Kinase Inhibitors/metabolism ; Pyrimidines/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, IgG/metabolism ; src-Family Kinases/antagonists & inhibitors/*metabolism

Annexin A1 (ANXA1) is a kind of endogenous scaffold protein. Previous research showed that ANXA1 could increase markedly with multiple increase of drug resistance in K562/imatinib cell lines in vitro. Here the stable transfection cell strains K562-pEGFP-N1 which was the native control and K562-pEGFP-N1-ANXA1 which can stably express ANXA1 were established using the Lipofectamine 2000 in order to find whether ANXA1 involved in the drug resistance. Cell growth inhibition experiment via MTT and cell proliferation experiment via MTS showed that K562-pEGFP-N1-ANXA1 cell strain was more sensitive to imatinib than the K562-pEGFP-N1 cell strain, and however the ability of proliferation of K562-pEGFP-N1-ANXA1 cell strain did not change compared with the negative control. Western blotting results showed that the expression of proteins in Annexin family did not change; drug resistance proteins, Bcr-Abl/p-Bcr-Abl (Tyr245), Src family kinase for example, did not change; proteins related with cell proliferation and cell cycle, such as ERK1/2MAPK, p-38MAPK, CDK1 and Wee 1, did not change either in the K562-pEGFP-N1-ANXA1 cell strain compared with the negative control. The co-immunoprecipitation result showed that the interaction between ANXA1 and beta-actin in the K562-pEGFP-N1-ANXA1 cell strain increased markedly. The deduction was that ANXA1 may make the K562-pEGFP-N1-ANXA1 cell strain more sensitive to imatinib due to the increased uptake of imatinib via the increase of ANXA1 and the interaction between ANXA1 and beta-actin in the K562-pEGFP-N1-ANXA1 cell strain in vitro.
Actins ; metabolism ; Annexin A1 ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; pharmacology ; Immunoprecipitation ; K562 Cells ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Transfection ; src-Family Kinases ; metabolism

OBJECTIVETo investigate the expression of Csk-binding protein (CBP) in esophageal carcinoma and its association with the tumorigenesis and progression of esophageal cancer.

METHODSRT-PCR and Western blotting were employed to determine the expressions of CBP at the mRNA and protein levels in 50 pairs of fresh esophageal carcinoma tissue and the adjacent normal tissues.

RESULTSCBP mRNA and protein expressions in normal tissues were 1.43- and 1.28-fold higher than those in the cancer tissues, respectively (P<0.05). The expressions of CBP mRNA and protein were positively correlated (P=0.015). The decreased expressions of CBP were significantly correlated to lymph node metastasis of esophageal cancer (P<0.05).

CONCLUSIONThe expression of CBP gene is decreased in esophageal carcinoma, which might contribute to the tumorigenesis and progression of this malignancy.

Adenocarcinoma ; metabolism ; pathology ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Down-Regulation ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; src-Family Kinases ; genetics ; metabolism

OBJECTIVETo investigate the effect of hypoxia on the human pulmonary artery smooth muscle cells two pore domain potassium channels TASK-1 and the regulation of non-receptor tyrosine kinase c-Src in this process.

METHODSThe cultured human pulmonary artery smooth muscle cells (hPASMCs) were divided into: normal group, hypoxia 30 minute group, hypoxia 6 hours group and hypoxia 48 hour group, and hypoxia 48 hour + PP2 group, hypoxia 48 hour + PP3 group, hypoxia 48 hour + bpV group. Flow cytometry was used to analyze the cell cycle, RT-PCR and Western blot technique were carried out to detect the expression changes of TASK-1 mRNA and protein in different groups.

RESULTS(1) Cell Cycle Show: Compared with normal control group, with prolonged hypoxia, the percentages of hPASMCs in S phases of cell cycle were increased. While compared with hypoxia 48 hour group, the percentages of hypoxia 48 hour + PP2 group hPASMCs in S phases of cell cycle were decreased. The expression of TASK-1 mRNA on hPASMCs in acute hypoxia 6 hour group was increased, while the expression of TASK-1 protein on hPASMCs in the acute and chronic hypoxia group was decreased, and the expression of TASK-1 mRNA on hPASMCs in the chronic hypoxia group was decreased; After pre-incubation of a potent and selective inhibitor of the Src family of protein tyrosine kinases PP2, the expression of TASK-1 mRNA and protein in hypoxia 48 hour group was increased, however after pre-incubation of the inhibitor of the Src family of protein tyrosine phosphatase bpV, the expression of TASK-1 protein in hypoxia 48 hour group was decreased.

CONCLUSIONHypoxia promotes human pulmonary artery smooth muscle cell proliferation, and non-receptor tyrosine kinase c-Src may participate in the expression of two pore domain potassium channels TASK-1 regulated by hypoxia. Therefore, we hypothesized that TASK-1 channels and c-Src participatein the acute and chronic hypoxic human pulmonary vasoconstriction.

Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Humans ; Myocytes, Smooth Muscle ; cytology ; Nerve Tissue Proteins ; metabolism ; Potassium Channels, Tandem Pore Domain ; metabolism ; Pulmonary Artery ; cytology ; RNA, Messenger ; Vasoconstriction ; src-Family Kinases ; metabolism

OBJECTIVE: To investigated the effects of Src family kinases on the expression of mRNA and protein of Nav1.1 in spiral ganglion neurons. METHOD: RT-PCR and Western blot techniques respectively explored the level of expression of mRNA and protein of Nav1.1 in spiral ganglion neurons by Src family kinases inhibitor. RESULT: An application of the inhibitor of Src family kinases which was PP2 (10 micromol/L) and SU6656 (2 micromol/L) gived rise to the mRNA decreasing to 26% +/- 0.8% and 36% +/- 1.5% respectively (P < 0.05), and protein reducing to 39% +/- 12.5% and 53% +/- 1.7% severally (P < 0.05). CONCLUSION Administration of the inhibitor of Src family kinases could decrease the expression of mRNA and protein of Nav1.1 in spiral ganglion neurons.
Animals ; Indoles ; pharmacology ; Male ; NAV1.1 Voltage-Gated Sodium Channel ; metabolism ; Neurons ; metabolism ; Pyrimidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology ; Sulfonamides ; pharmacology ; src-Family Kinases ; antagonists & inhibitors ; metabolism