BACKGROUND: Endotoxin, the component of outermembrane of gram negative organism, plays an important role in the initiation and amplification of inflammatory reaction by its effects on inflammatory cells. Until recently, there have been continuing efforts to delinate the mechanisms of the signal trasduction pathway of endotoxin stimuli on inflammatory cells. By uncovering the mechanisms of signal transduction pathway of endotoxin stimuli, we can expect to have tools to control the excessive inflammatory responses which sometimes may be fatal to the involved host. It was generally accepted that endotoxin exerts its inflammatory effects through inflammatory cytokines that are produced by endotoxin-stimulated inflammatory cells and there were some reports on the importance of protein kinase C and protein tyrosine kinase activation in the production of inflammatory cytokines by endotoxin. So we evaluated the effect of pretreatment of protein kinase C inhibitors (H7, Staurosporin) and protein tyrosine kinase inhibitors(Herbimycin, Genistein) on the endotoxin-stimulated cytokines(IL-8 & TNF-alpha) mRNA expression. METHOD: Peripheral blood monocytes were isolated from healthy volunteers by Ficoll-Hypaque density gradient method and purified by adhesion to 60mm Petri dishes. Endotoxin(LPS 100ng/ml) was added to each dishes except one control dish, and each endotoxin-stimulated dishes was preincubated with H7, Staurosporin(protein kinase C inhibitor), Herbimycin or Genistein(protein tyrosine kinase inhibitor) respectively except one dish. Four hours later the endotoxin stimulation, total RNA was extracted and Northern blot analysis for IL-8 mRNA and TNF-alpha mRNA was done. RESULT: Endotoxin stimulation increased the expression of IL-8 mRNA and TNF-alpha mRNA expression in human peripheral blood monocyte as expected and the stimulatory effect of endotoxin on TNF-alpha mRNA expression was inhibited by protein kinase C inhibitors(H7, Staurosporin) and protein tyrosine kinase inhibitors (Herbimycin, Genistein). The inhibitory effect of each drugs was increased with increasing concentration. The stimulatory effect of endotoxin on IL-8 mRNA was also inhibited by H7 and protein tyrosine kinase inhibitors (Herbimycin, Genistein) dose-dependently but not by Staurosporin. CONCLUSION: Protein kinase C and protein tyrosine kinase are involved in the endotoxin induced signal transduction pathway in human peripheral blood monocyte.
Blotting, Northern ; Cytokines ; Healthy Volunteers ; Humans ; Interleukin-8 ; Monocytes* ; Phosphotransferases ; Protein Kinase C* ; Protein Kinases* ; Protein-Tyrosine Kinases* ; RNA ; RNA, Messenger ; Signal Transduction* ; Tumor Necrosis Factor-alpha

The regulatory role of cyclic nucleotides in the expression of neutrophil responses has been examined. fMLP-stimulated superoxide production in neutrophils was inhibited by dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), histamine, adenosine + theophylline, cAMP elevating agents, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and sodium nitroprusside, cGMP elevating agents. Staurosporine, a protein kinase C inhibitor, genistein, a protein tyrosine kinase inhibitor and chlorpromazine, a calmodulin inhibitor, inhibited superoxide production by fMLP, but they did not further affect the action of DBcAMP on the stimulatory action of fMLP. DBcAMP, histamine, adenosine + theophylline and genistein inhibited myeloperoxidease release evoked by fMLP, whereas BrcGMP, sodium nitroprusside and staurosporine did not affect it. The elevation of (Ca2+)-i evoked by fMLP was inhibited by genistein and chlorpromazine but was not affected by staurosporine. DBcAMP exerted little effect on the initial peak in (Ca2+)-i response to fMLP but effectively inhibited the sustained rise. On the other hand, BrcGMP significantly inhibited both phases. fMLP-induced Mn-2+ influx was inhibited by either DBcAMP or BrcGMP. These results suggest that fMLP-stimulated neutrophil responses may be regulated by cAMP more than cGMP. cAMP and cGMP appear not affect stimulated responses by direct protein kinase C activation. Their regulatory action on the stimulated neutrophil responses may be not influenced by other activation processes.
Adenosine ; Bucladesine ; Calmodulin ; Chlorpromazine ; Genistein ; Hand ; Histamine ; Neutrophils* ; Nitroprusside ; Nucleotides, Cyclic ; Protein Kinase C ; Protein-Tyrosine Kinases ; Staurosporine ; Superoxides ; Theophylline

Adenosine is a naturally occurring breakdown product of adenosine triphosphate and plays an important role in different physiological and pathological conditions. Adenosine also serves as an important trigger in ischemic and remote preconditioning and its release may impart cardioprotection. Exogenous administration of adenosine in the form of adenosine preconditioning may also protect heart from ischemia-reperfusion injury. Endogenous release of adenosine during ischemic/remote preconditioning or exogenous adenosine during pharmacological preconditioning activates adenosine receptors to activate plethora of mechanisms, which either independently or in association with one another may confer cardioprotection during ischemia-reperfusion injury. These mechanisms include activation of K(ATP) channels, an increase in the levels of antioxidant enzymes, functional interaction with opioid receptors; increase in nitric oxide production; decrease in inflammation; activation of transient receptor potential vanilloid (TRPV) channels; activation of kinases such as protein kinase B (Akt), protein kinase C, tyrosine kinase, mitogen activated protein (MAP) kinases such as ERK 1/2, p38 MAP kinases and MAP kinase kinase (MEK 1) MMP. The present review discusses the role and mechanisms involved in adenosine preconditioning-induced cardioprotection.
Adenosine Triphosphate ; Adenosine* ; Heart ; Inflammation ; Mitogen-Activated Protein Kinase Kinases ; Nitric Oxide ; Phosphotransferases ; Protein Kinase C ; Protein-Tyrosine Kinases ; Proto-Oncogene Proteins c-akt ; Receptors, Opioid ; Receptors, Purinergic P1 ; Reperfusion Injury

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 micrometer) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.
Adenosine Triphosphate ; Animals ; Calcium ; Constriction ; Extracellular Space ; Gallic Acid ; Genistein ; HEPES ; Indoles ; Maleimides ; Nimodipine ; PC12 Cells ; Phenol ; Protein Kinase C ; Protein-Tyrosine Kinases ; Thapsigargin

Nuclear factor kappaB (NF- kappaB) activation is modulated by various protein kinases. Activation of NF- kappaB is known to be important in the regulation of cell viability. The present study investigated the effect of inhibitors of protein tyrosine kinase (PTK), protein kinase C (PKC) and protein kinase A (PKA) on NF- kappaB activity and the viability of bovine cerebral endothelial cells (BCECs). In serum-deprivation-induced BCEC death, low doses of TNF alpha showed a protective effect. TNF alpha induced NF- kappaB activation within 4 h in serum-deprivation. PTK inhibitors (herbimycin A and genistein) and PKC inhibitor (calphostin C) prevented NF- kappaB activation stimulated by TNF alpha. Likewise, these inhibitors prevented the protective effect of TNF alpha. In contrast to TNF alpha-stimulated NF- kappaB activity, basal NF- kappaB activity of BCECs in media containing serum was suppressed only by calphostin C, but not by herbimycin A. As well BCEC death was also induced only by calphostin C in serum-condition. H 89, a PKA inhibitor, did not affect the basal and TNF alpha-stimulated NF- kappaB activities and the protective effect of TNF alpha on cell death. These data suggest that modulation of NF- kappaB activation could be a possible mechanism for regulating cell viability by protein kinases in BCECs.
Cell Death* ; Cell Survival ; Cyclic AMP-Dependent Protein Kinases ; Endothelial Cells* ; Protein Kinase C ; Protein Kinases* ; Protein-Tyrosine Kinases

PURPOSE: Adenosine triphosphate (ATP) evokes several cellular responses in microglia including propagation. However, the role of the purinoceptor on ROS generation in microglia is unclear. In order to determine the action of the purinoceptor in microglia, the effects of ATP on ROS generation and cellular proliferation in BV-2 murine microglial cells were evaluated. An additional aim of this study was to investigate signal transduction pathways using several inhibitors. METHODS: The [Ca2+] was measured using Ca2+ sensitive indicator, Fura-2/AM. ROS production was observed by fluorescence-confocal microscope and cell proliferation was evaluated by counting cell number. RESULTS: ATP increased the intracellular calcium levels ([Ca2+]i) in BV-2 cells in a dose-dependent manner. This increase was attenuated by pretreatment with a calcium chelator (EGTA) and a phospholipase C (PLC) inhibitor (U-73122) while the protein tyrosine kinase (PTK) inhibitor (genistein) had no inhibitory effects. To identify the effects of the nucleotides, ROS generation was observed in the nucleotide-stimulated BV-2 cells. The treatment with 100 M ATP induced ROS generation, but 100 M adenosine and 100 M UTP did not. To investigate the signal transduction pathway in ATP-induced ROS generation, several inhibitors were pretreated before adding ATP. ATP- induced ROS production was blocked by pretreatment with either 0.5 mM EGTA or 10 M U73122 while 40 M genistein had an inhibitory effect on ATP action. Correspondingly, 40 M KN62 (CaM kinase II inhibitor), 1 M sphingosine (protein kinase C inhibitor), 1 nM DPI (NADPH oxidase inhibitor) and 50 M mepacrine (phospholipase A2 inhibitor) could suppress ATP-induced ROS generation. The effects of ATP on cell proliferation was observed 3 days after ATP treatment and its peak velocity after 4 days. NF-kB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-kB activation was obtained with 0.3 mM ATP while higher concentrations were less effective. CONCLUSION: Overall, we conclude that ATP in BV-2 cells induces ROS generation and cell propagation. The signal transduction pathways including calcium, CaM kinase II, PLC, protein kinase C, phospholipase A2 and NADPH oxidase are involved in ATP-induced ROS generation.
Adenosine ; Adenosine Triphosphate* ; Calcium ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Cell Count ; Cell Proliferation* ; Egtazic Acid ; Genistein ; Microglia ; NADPH Oxidase ; NF-kappa B ; Nucleotides ; Oxidoreductases ; Phospholipases A2 ; Phosphotransferases ; Protein Kinase C ; Protein-Tyrosine Kinases ; Quinacrine ; Reactive Oxygen Species* ; Receptors, Purinergic ; Signal Transduction ; Sphingosine ; Type C Phospholipases ; Uridine Triphosphate

ABBREVIATIONS: Akt/PKB protein kinase B, ALI acute lung injury, ARDS acute respiratory distress syndrome, CREB C-AMP response element binding protein, ERK extracelluar signal-related kinase, fMLP fMet-Leu-Phe, G-CSF granulocyte colony-stimulating factor, IL interleukin, ILK integrin-linked kinase, JNK Jun N-terminal kinase, LPS lipopolysaccharide, MAP mitogen-activated protein, MEK MAP/ERK kinase, MIP-2 macrophage inflammatory protein-2, MMP matrix metalloproteinase, MPO myeloperoxidase, NADPH nicotinamide adenine dinucleotide phosphate, NE neutrophil elastase, NF-kappaB nuclear factor-kappa B, NOS nitric oxide synthase, p38 MAPK p38 mitogen activated protein kinase, PAF platelet activating factor, PAKs p21-activated kinases, PMN polymorphonuclear leukocytes, PI3-K phosphatidylinositol 3-kinase, PyK proline-rich tyrosine kinase, ROS reactive oxygen species, TNF-alpha tumor necrosis factor-alpha.
Abbreviations ; Acute Lung Injury* ; Carrier Proteins ; Granulocyte Colony-Stimulating Factor ; Interleukins ; Leukocyte Elastase ; Macrophages ; N-Formylmethionine Leucyl-Phenylalanine ; NADP ; Neutrophil Activation* ; Neutrophils* ; NF-kappa B ; Nitric Oxide Synthase ; p21-Activated Kinases ; p38 Mitogen-Activated Protein Kinases ; Peroxidase ; Phosphatidylinositol 3-Kinase ; Phosphotransferases ; Platelet Activating Factor ; Protein Kinases ; Protein-Tyrosine Kinases ; Proto-Oncogene Proteins c-akt ; Reactive Oxygen Species ; Respiratory Distress Syndrome, Adult ; Response Elements ; Signal Transduction ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the role of extracellular signal-regulated kinase (ERK) in apoptosis of human colon cancer (HCT116) cells.

METHODSAfter the HCT116 cells were pretreated with specific ERK inhibitor (U0126) or specific siRNA and exposed to 10 mmol/L sodium butyrate (NaBT) for 24 h, their apoptosis was detected by flow cytometry, levels of SphK2 and ERK protein were measured by Western blot, and translocation of SphK2 was assayed by immunofluorescence microscopy.

RESULTSThe U0126 and siRNAs specific for SphK2 blocked the export of SphK2 from nuclei to cytoplasm and increased the apoptosis of HCT116 cells following NaBT exposure. Over-expression of PKD decreased NaBT-induced apoptosis of HCT116 cells, which was reversed by U0126. Furthermore, transfection of HCT116 cells with constitutively activated PKD plasmids recovered the U0126-blocked export of SphK2.

CONCLUSIONERK regulates the export of SphK2 and apoptosis of HCT116 cells by modulating PKD. Modulation of these molecules may help increase the sensitivity of colon cancer cells to the physiologic anti-colon cancer agent, NaBT.

Apoptosis ; drug effects ; physiology ; Butyric Acid ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; HCT116 Cells ; drug effects ; Humans ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Protein Kinase C ; genetics ; metabolism ; RNA, Small Interfering ; Signal Transduction ; drug effects

Rat aorta media, adventitia and cultured vascular smooth muscle cells (VSMCs) were used in this study to identify the source of nitric oxide (NO) generation from various cell types of vascular tissues and to elucidate the mechanisms involved in NO formation. Treatment of vascular media and VSMCs with lipopolysaccharide (LPS) or cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta)] resulted in a dose-dependent increase of NO release. Inducible nitric oxide synthase (iNOS) in the stimulated VSMCs was significantly upregulated as shown by Western blot analysis. Protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) prevented LPS-, TNF-alpha- and IL-1beta-induced NO production, whereas N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide (HA1004), an H7 analogue with little activity towards PKC, had no inhibition effect. The role of PKC in LPS- and cytokine-induced changes on NO formation was confirmed by using another structurally distinct PKC inhibitor chelerythrine. Treatment of VSMCs with protein tyrosine kinase (PTK) inhibitor genistein or tyrphostin AG18 also reduced the NO production evoked by LPS, TNF-alpha or IL-1beta, which was associated with inhibition of iNOS protein expression. In contrast, PKC inhibitor chelerythrine did not affect iNOS expression. These results suggest that PTK mediates LPS- and cytokine-induced NO formation by upregulation of iNOS expression. PKC may be involved in the post-translational modification of iNOS or the regulation of the availability of iNOS substrates and cofactors.
Animals ; Aorta, Thoracic ; cytology ; Cells, Cultured ; Cytokines ; pharmacology ; Interleukin-1beta ; pharmacology ; Lipopolysaccharides ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Protein Kinase C ; physiology ; Protein-Tyrosine Kinases ; physiology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUND/OBJECTIVES: Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism. MATERIALS/METHODS: Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement. RESULTS: CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose. CONCLUSION: CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment.
Acarbose ; alpha-Glucosidases* ; AMP-Activated Protein Kinases ; Blotting, Western ; Cordyceps* ; Food Habits ; Glucokinase ; Glucose Transport Proteins, Facilitative ; Glucose* ; Glycogen ; Glycogen Synthase Kinase 3 ; Glycolysis ; Hep G2 Cells* ; Hepatocyte Nuclear Factor 1-alpha ; Hypoglycemic Agents ; Liver ; Metabolic Diseases ; Metabolism* ; Motor Activity ; Obesity ; Oxidoreductases ; Phosphatidylinositol 3-Kinase ; Phosphoenolpyruvate ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; Pyruvic Acid ; Social Conditions ; Water*