OBJECTIVETo investigate the effect of the non-PLC-dependent protein kinase C (PKC) pathway of parathyroid hormone (PTH) on the apoptosis and proliferation of osteoblast MC-3T3E1 cells.

METHODSMC-3T3E1 cells were seeded in 96-well plates at the density of 1.5×10(4) cells/mL and incubated for 3 day. The cells were then exposed to 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-28), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34)+1 µmol/L Go6983, 1 µmol/L Go6983, or deionized water (control) for 1, 24 or 48 h. After the treatments, cell counting kit-8 (CCK-8) and Caspase-Glo® 3/7 Assay (Caspase-3) were used to examine the proliferation and apoptosis of MC3T3-E1 cells.

RESULTSCCK-8 results showed that hPTH(1-34) increased the number of MC3T3-E1 cells compared with hPTH(1-34)+Go6983 at 1 h and 24 h, but this difference was not statistically different. At 48 h, treatment with hPTH(1-34), as compared with hPTH(1-28), significantly increased the number of MC3T3-E1 cells (P<0.05), and this effect was blocked by the PKC inhibitor Go6983 (P<0.05). hPTH(1-34) did not result in significant inhibition of MC3T3-E1 cell apoptosis at 1 h and 24 h as compared with hPTH(1-34)+Go6983, but significantly inhibited the cell apoptosis as compared with hPTH(1-28) (P<0.05); this inhibitory effect was blocked by Go6983 (P<0.05).

CONCLUSIONs A relatively long time (for 48 h) of exposure to PTH can inhibit apoptosis and promote the proliferation of MC3T3-E1cells through a non-PLC-dependent PKC pathway.

3T3 Cells ; Animals ; Apoptosis ; Cell Proliferation ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Mice ; Osteoblasts ; Parathyroid Hormone ; pharmacology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Signal Transduction

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Female ; Guanine Nucleotide Exchange Factors ; antagonists & inhibitors ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; MCF-7 Cells ; Maleimides ; pharmacology ; Protein Isoforms ; genetics ; metabolism ; Protein Kinase C ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; toxicity

OBJECTIVETo explore the roles of PKCβ/P66Shc oxidative stress signal pathway in mediating hyperoxia-induced reactive oxgen species (ROS) production in alveolar epithelial cells (A549) and the protective effects of PKCβ inhibitor on hyperoxia-induced injuries of alveolar epithelial cells.

METHODSA549 cells were cultured in vitro and randomly divided into three groups: control, hyperoxia and PKCβ inhibitor LY333531 treatment. The hyperoxia group was exposed to a mixture of O2 (950 mL/L) and CO2 (50 mL/L) for 10 minutes and then cultured in a closed environment. The LY333531 group was treated with PKCβ inhibitor LY333531 of 10 µmol/L for 24 hours before hyperoxia induction. Cells were collected 24 hours after culture and the levels of PKCβ, Pin1, P66Shc and P66Shc-Ser36 were detected by Western blot. The intracellular translocation of P66Shc, the production of ROS and cellular mitochondria membrane potential were measured using the confocal microscopy.

RESULTSCompared with the control group, the levels of PKCβ, Pin1, P66Shc and P-P66Shc-Ser36 in A549 cells 24 hours after culture increased significantly in the hyperoxia group. These changes in the hyperoxia group were accompanied with an increased translocation rate of P66Shc from cytoplasm into mitochondria, an increased production of mitochondrial ROS, and a reduced mitochondrial membrane potential. Compared with the hyperoxia group, the levels of Pin1, P66Shc and P66Shc-Ser36 in A549 cells, the translocation rate of P66Shc from cytoplasm into mitochondria and the production of mitochondrial ROS decreased significantly, while the mitochondrial membrane potential increased significantly in the LY333531 treatment group. However, there were significant differences in the above mentioned measurements between the LY333531 treatment and control groups.

CONCLUSIONSHyperoxia can increase the expression of PKCβ in alveolar epithelial cells and production of mitochondrial ROS and decrease mitochondrial membrane potential. PKCβ inhibitor LY333531 can partially disrupt these changes and thus alleviate the hyperoxia-induced alveolar epithelial cell injury.

Cell Hypoxia ; Cells, Cultured ; Epithelial Cells ; metabolism ; Humans ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Oxidative Stress ; Protein Kinase C beta ; physiology ; Pulmonary Alveoli ; cytology ; metabolism ; Reactive Oxygen Species ; metabolism ; Shc Signaling Adaptor Proteins ; physiology ; Signal Transduction ; physiology ; Src Homology 2 Domain-Containing, Transforming Protein 1

A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹⁰-1 × 10⁻⁵ mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻⁶ mol/L). Also it could be removed by intracellular dialysis of either GDP-β-S (5 × 10⁻⁴mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻⁶ mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.
Animals ; Arginine Vasopressin ; pharmacology ; Ganglia, Spinal ; cytology ; Guanosine Diphosphate ; analogs & derivatives ; pharmacology ; Indoles ; Maleimides ; Membrane Potentials ; Neurons ; drug effects ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; metabolism ; Signal Transduction ; Thionucleotides ; pharmacology ; gamma-Aminobutyric Acid ; pharmacology

Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3beta, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3beta inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3+), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3beta. Furthermore, SIC was abrogated by wortmannin and Gd3+. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3+ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.
Androstadienes/pharmacology ; Animals ; Gadolinium/pharmacology ; Glycogen Synthase Kinase 3/*metabolism ; Indoles/pharmacology ; *Ischemic Preconditioning, Myocardial ; Lithium/pharmacology ; Male ; Maleimides/pharmacology ; Myocardial Reperfusion Injury/enzymology/physiopathology/*prevention & control ; Phosphatidylinositol 3-Kinase/*antagonists & inhibitors/metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt/*metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Specific Pathogen-Free Organisms

OBJECTIVETo investigate the effect of different concentrations of glucose on the permeability of human umbilical vein endothelial cell monolayer and the protective effect of protein kinase C (PKC) inhibitor Ro-31-8425 against high-glucose exposure.

METHODSCultured human umbilical vein endothelial cell line EA.hy926 cells were exposed to 5.5 mmol/L glucose (control) and high-concentration glucose (10, 15, 20, 25.5, and 30 mmol/L), and the endothelial monolayer permeability was assessed by measuring the flux of FITC-labeled dextran (FITC-DΧ) across the endothelial cells. The cultured EA.hy926 cells were treated with 5.5 mmol/L glucose +saline, high glucose (25.5 mmol/L) +saline, or high glucose (25.5 mmol/L) +Ro-31-8425(10 µmol/L), and the level of PKC phosphorylation and endothelial monolayer permeability were evaluated.

RESULTSHigh glucose dose-dependently increased the permeability of the endothelial cell monolayer (P<0.01), and glucose at 25.5 mmol/L significantly increased the phosphorylation level of PKCα and PKCβ II in the cells (P<0.01). Treatment with 10 µmol/L Ro-31-8425 obviously attenuated high-glucose-induced PKCα and PKCβ II phosphorylation (P<0.01) as well as the increase of the cell monolayer permeability (P<0.01).

CONCLUSIONSHigh glucose increases the hyperpermeability of human umbilical vein endothelial cell monolayer mediated by the phosphorylation of PKC, and the PKC inhibitor Ro-31-8425 can reverse such effects.

Capillary Permeability ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; drug effects ; metabolism ; Glucose ; adverse effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Protein Kinase C ; antagonists & inhibitors

OBJECTIVETo research the effects of glycogen synthase kinase (GSK3β) overexpression and GSK3β inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway.

METHODSThe hepatic oval cells WBF-344 were divided into the blank control group, GSK3β over-expression group, DMSO control group and GSK3β inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3β over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3β, β-catenin and cyclin D1 were detected by Western blot.

RESULTSThe cells of GSK3β over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3β over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3β over-expression group. Western blot showed that the expression of GSK3β was increased, while the expression of β-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3β had no significant difference among the control group and inhibitor groups. However, the expression of β-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing.

CONCLUSIONSThe overexpression of GSK3β can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.

Animals ; Cell Line ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Glycogen Synthase Kinases ; metabolism ; Hepatocytes ; drug effects ; Indoles ; pharmacology ; Male ; Maleimides ; pharmacology ; Rats ; Transfection ; Wnt Signaling Pathway ; beta Catenin ; metabolism

The Wnt/β-catenin and bone morphogenetic proteins (BMPs) pathways play important roles in controlling osteogenesis. Using a cell-based kinase inhibitor screening assay, we identified the compound bisindoylmaleimide I (BIM) as a potent agonist of the cytosolic β-catenin accumulation in preosteoblast cells. Through suppressing glycogen synthase kinase 3β enzyme activities, BIM upregulated β-catenin responsive transcription and extended duration of BMP initiated signal. Functional analysis revealed that BIM promoted osteoblast differentiation and bone formation. The treatment of human mesenchymal stem cells with BIM promoted osteoblastogenesis. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by integration of the cellular signaling pathways.
Animals ; Bone Morphogenetic Proteins ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Indoles ; chemistry ; pharmacology ; Maleimides ; chemistry ; pharmacology ; Mesenchymal Stem Cells ; cytology ; metabolism ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Signal Transduction ; drug effects ; Wnt Proteins ; metabolism ; beta Catenin ; antagonists & inhibitors ; genetics ; metabolism

Myocardial fibrosis is the common results of the development of a variety of heart diseases which leads to extracellular matrix protein metabolic disorders and causes cardiac remodeling owing to cardiac fibroblasts proliferation, eventually results in malignant arrhythmia, heart failure, and even the occurrence of sudden cardiac death. Effective inhibition of myocardial remodeling could prevent the occurrence of sudden death. To know the protein kinase C (PKC) effective mechanism of regulation on myocardial fibrosis, a new therapeutic target for reversing myocardial remodeling might be provided.
Animals ; Cardiomegaly ; metabolism ; Fibrosis ; Humans ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Matrix Metalloproteinase 9 ; metabolism ; Myocardium ; pathology ; Protein Kinase C ; antagonists & inhibitors ; classification ; metabolism ; Ventricular Remodeling