OBJECTIVE: To observe the effect of chronic unpredicted sequence of mild stress on the expression of cAMP-dependent protein kinase A(PKA) and phosphorylated cAMP-responsive element binding protein (P-CREB) in hippocampus of rats and the antagonism of antidepressors (fluoxetine). METHODS: Thirty-six male Sprague Dawley rats were randomly and equally allocated to 3 groups: A normal control group, a model group, and a fluoxetine group. All rats except the control group were singly housed and exposed to an unpredicted sequence of mild stressors. The different distribution and expression of PKA and P-CREB in the hippocampus of rats in different groups were investigated with immunohistochemistry and Westernblot technique. RESULTS: The positive PKA and P-CREB cells in the hippocampus of normal controls were the pyramidal cells and the granule cells. The PKA and P-CREB protein expression levels in the hippocampus of model rats were significantly lower than those of the normal controls (P<0.05). The PKA and P-CREB protein expression levels in the hippocampus of the fluoxetine group were significantly higher than those of the model group (P<0.05). CONCLUSION Chronic unpredicted mild stress can affect the PKA and P-CREB expression in hippocampus of rats and fluoxetine has antagonism against it.
Animals ; Antidepressive Agents, Second-Generation ; antagonists & inhibitors ; Cyclic AMP Response Element-Binding Protein ; biosynthesis ; genetics ; Cyclic AMP-Dependent Protein Kinases ; biosynthesis ; genetics ; Depression ; etiology ; metabolism ; Fluoxetine ; antagonists & inhibitors ; Hippocampus ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological ; metabolism

BACKGROUNDAdipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms.

METHODSFully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0-50 μg/ml) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber.

RESULTSOxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 μg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPβ protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPβ protein level in oxLDL-induced 3T3-L1 adipocytes.

CONCLUSIONSOxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

3T3-L1 Cells ; Animals ; CCAAT-Enhancer-Binding Protein-beta ; analysis ; physiology ; Chemokine CCL2 ; genetics ; secretion ; Cyclic AMP ; physiology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; Humans ; Lipoproteins, LDL ; antagonists & inhibitors ; pharmacology ; Mice ; Peptides ; pharmacology ; Signal Transduction ; physiology

OBJECTIVETo observe the changes in glucose 6-phosphate dehydrogenase (G6PD) activity, cAMP and respiratory burst function in THP-1 cells exposed to high glucose and identify the possible signaling pathways to mediate these changes.

METHODSTHP-1 cells were treated with high glucose, high glucose plus the PKA inhibitor (PKI), or normal glucose plus Forskolin. The changes in the G6PD activity and cAMP in the exposed cells were assayed using the spectrophotometric method, and the reactive oxygen species (ROS) content in the cell culture was determined using the fluorescent probe DCFH-DA. Western blotting was employed to examine the expression of phosphorylated p47(phox) in the cells.

RESULTSCompared with the normal control cells, the cells exposed to high glucose and to normal glucose and Forskolin showed a significantly lowered G6PD activity, ROS content and expression of phosphorylated p47(phox), but with a increased cAMP content (P<0.01). High glucose exposure in the presence of PKI caused no significant changes in G6PD activity, ROS level, phosphorylated p47(phox) or cAMP compared to those in the normal control cells (P>0.01).

CONCLUSIONHigh glucose causes inhibition of G6PD activity in THP-1 cells via activation of PKA and thus leads to respiratory burst dysfunction, which is the probable mechanism underlying the lowered leucocyte function and susceptibility to infections in diabetic patients.

Cell Line ; Cyclic AMP ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; antagonists & inhibitors ; metabolism ; Glucose ; adverse effects ; Glucosephosphate Dehydrogenase ; metabolism ; Humans ; Phosphorylation ; Respiratory Burst ; drug effects ; Signal Transduction ; drug effects

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^-1), whereas higher concentrations (>5 μmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca²⁺-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
8-Bromo Cyclic Adenosine Monophosphate/pharmacology* ; Acrosome/metabolism* ; Acrosome Reaction/drug effects* ; Calcimycin/pharmacology* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors* ; Exocytosis/drug effects* ; Guanine Nucleotide Exchange Factors/metabolism* ; Humans ; Male ; Protein Kinase Inhibitors/pharmacology* ; Signal Transduction/drug effects* ; Spermatozoa/metabolism* ; Thapsigargin/pharmacology*

OBJECTIVETo explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.

METHODSWashed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer.

RESULTSAfter PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor.

CONCLUSIONPKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.

Blood Platelets ; drug effects ; Calpain ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; antagonists & inhibitors ; Flow Cytometry ; Humans ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Platelet Glycoprotein GPIb-IX Complex ; biosynthesis

Phosphodiesterase (PDE) 4 inhibitors have been shown to induce the cAMP-mediated signaling pathway by inhibiting cAMP hydrolysis. This study investigated the effect of a PDE4 inhibitor on the expression of the inducible cAMP early repressor (ICER), which is an endogenous inhibitor of CRE- mediated transcription, in osteoblastic cells. RT-PCR analysis revealed that rolipram, a PDE4 inhibitor, stimulates the ICER mRNA in a dose dependent manner. The induction of ICER mRNA expression by rolipram was suppressed by the inhibitors of protein kinase A (PKA) and p38 MAPK, suggesting the involvement of PKA and p38 MAPK activation in ICER expression by rolipram. It was previously shown that rolipram induced the expression of TNF-related activation-induced cytokine (TRANCE, also known as RANKL, ODF, or OPGL) in osteoblasts. This paper provides evidences that a transcriptional repressor like ICER might modulate TRANCE mRNA expression by rolipram in osteoblasts.
3', 5'-Cyclic-Nucleotide Phosphodiesterase/*antagonists & inhibitors ; Animals ; Animals, Outbred Strains ; Cyclic AMP-Dependent Protein Kinases/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression/drug effects ; Mice ; Osteoblasts/*drug effects/metabolism ; Phosphodiesterase Inhibitors/*pharmacology ; Research Support, Non-U.S. Gov't ; Rolipram/*pharmacology ; Transcription Factors/genetics/*metabolism ; p38 Mitogen-Activated Protein Kinases/metabolism

The aim of this project is to establish a GLP-1 signaling pathway targeted cell model, for screening the new class of GLP-1 receptor agonists as anti-diabetic candidates. Firstly construct a recombined plasmid with multi-copied specific response element (RIP-CRE) regulated by GLP-1 signaling pathway and E-GFP reporter gene. Transient transfect this recombined plasmid into islet cell NIT-1, then detect the responsibility of transfected cell to GLP-1 analogue, Exendin 4. For secondly, use stable transfection and monocloning cell culture to obtain a GLP-1 signaling-specific cell line. It indicates that this cell model can response to Exendin 4, which response can be completely inhibited by GLP-1 receptor antagonist, Exendin 9-39, further showing GLP-1 receptor specific activity with a cAMP-PKA-independently mechanism. Establishment of this novel cell model can be used in high-throughput drug screening of peptides or small molecular GLP-1 analogues.
Animals ; Cell Line ; Cyclic AMP Response Element Modulator ; pharmacology ; Cyclic AMP-Dependent Protein Kinases ; antagonists & inhibitors ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; Genes, Reporter ; Glucagon-Like Peptide-1 Receptor ; Green Fluorescent Proteins ; metabolism ; Hypoglycemic Agents ; agonists ; antagonists & inhibitors ; metabolism ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Isoquinolines ; pharmacology ; Peptide Fragments ; pharmacology ; Peptides ; antagonists & inhibitors ; pharmacology ; Plasmids ; Rats ; Receptors, Glucagon ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; Sulfonamides ; pharmacology ; Transfection ; Venoms ; pharmacology

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.
Bucladesine/pharmacology ; Carbazoles/pharmacology ; Cell Line, Tumor ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*metabolism ; Dibutyryl Cyclic GMP/pharmacology ; Enzyme Inhibitors/pharmacology ; Humans ; Indoles/pharmacology ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Pyrroles/pharmacology ; Research Support, Non-U.S. Gov't ; rhoA GTP-Binding Protein/*metabolism

In the injured brain, microglia is known to be activated and produce proinflammatory mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). We investigated the role of protein kinase A (PKA) in microglial activation by both plasminogen and gangliosides in rat primary microglia and in the BV2 immortalized murine microglial cell line. Both plasminogen and gangliosides induced IL-1beta, TNF-alpha and iNOS mRNA expression, and that this expression was inhibited by the addition of the PKA inhibitors, KT5720 and H89. Both plasminogen and gangliosides activated PKA and increased the DNA binding activity of the cAMP response element- binding protein (CREB). Furthermore, KT5720 and H89 reduced the DNA binding activities of CREB and NF-kappaB in plasminogen-treated cells. These results suggest that PKA plays an important role in plasminogen and gangliosides- induced microglial activation.
Animals ; Carbazoles/pharmacology ; Cell Line ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology ; DNA-Binding Protein, Cyclic AMP-Responsive/metabolism ; DNA-Binding Proteins/metabolism ; Gangliosides/pharmacology/*physiology ; Gene Expression Regulation ; Indoles/pharmacology ; Interleukin-1/genetics ; Isoquinolines/pharmacology ; Mice ; Microglia/drug effects/*enzymology/*immunology ; NF-kappa B/metabolism ; Nitric-Oxide Synthase/genetics ; Plasminogen/pharmacology/*physiology ; Pyrroles/pharmacology ; RNA, Messenger/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Sulfonamides/pharmacology ; Tumor Necrosis Factor-alpha/genetics

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.
Animals ; Apoptosis/drug effects ; Cell Line, Tumor ; Cyclic AMP-Dependent Protein Kinases/*antagonists & inhibitors/metabolism ; DNA Damage/drug effects ; DNA Repair/drug effects ; Gamma Rays ; Gene Expression Regulation, Neoplastic/radiation effects ; Genes, bcl-2 ; Humans ; Mice ; Neoplasm Proteins/genetics/metabolism ; Neoplasms/enzymology/*genetics ; Radiation Tolerance/*genetics ; Research Support, Non-U.S. Gov't