Taurochenodeoxycholic acid (TCDCA) is one of the main effective components of bile acid, playing critical roles in apoptosis and immune responses through the TGR5 receptor. In this study, we reveal the interaction between TCDCA and TGR5 receptor in TGR5-knockdown H1299 cells and the regulation of inflammation via the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding (CREB) signal pathway in NR8383 macrophages. In TGR5-knockdown H1299 cells, TCDCA significantly activated cAMP level via TGR5 receptor, indicating TCDCA can bind to TGR5; in NR8383 macrophages TCDCA increased cAMP content compared to treatment with the adenylate cyclase (AC) inhibitor SQ22536. Moreover, activated cAMP can significantly enhance gene expression and protein levels of its downstream proteins PKA and CREB compared with groups of inhibitors. Additionally, TCDCA decreased tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-12 through nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activity. PKA and CREB are primary regulators of anti-inflammatory and immune response. Our results thus demonstrate TCDCA plays an essential anti-inflammatory role via the signaling pathway of cAMP-PKA-CREB induced by TGR5 receptor.
Animals ; Cell Line ; Cyclic AMP/metabolism* ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Cytokines/metabolism* ; Humans ; Inflammation ; Macrophages ; Rats ; Receptors, G-Protein-Coupled/metabolism* ; Signal Transduction/drug effects* ; Taurochenodeoxycholic Acid/pharmacology*

No abstract available.
Calcium Channels/metabolism* ; Calcium Signaling/physiology* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Pancreas/enzymology* ; Periodicity ; Phosphorylation ; Receptors, Cytoplasmic and Nuclear/metabolism*

OBJECTIVE: To observe the direct intervention effects of electroacupuncture (EA) and non-steroid anti-inflammatory drugs (NSAIDs) on pain memory, and to explore their effects on cAMP/PKA/cAMP pathway in anterior cingulate gyrus (ACC). METHODS: Fifty clean healthy male SD rats were randomly divided into a control group, a model group, an indomethacin group, an EA group and a sham EA group, 10 rats in each group. Except the control group, the pain memory model was established in the remaining four groups by twice injection of carrageenan at foot; 0.1 mL of 2%λ-carrageenan was subcutaneously injected at the left foot of rats; 14 days later, when the pain threshold of rats of each group returned to the basic level, the second injection was performed with the same procedure. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36) for 30 min; the rats in the indomethacin group was treated with indomethacin intragastric administration with the dose of 3 mg/kg; the rats in the sham EA group was treated with EA without electricity at the point 0.3 mm forward "Zusanli" (ST 36) with the depth of 2 mm for 30 min; the rats in the control group was not given any invention. All the above interventions were performed 5 h, 1 d, 2 d and 3 d after the second injection of 2% λ-carrageenan. The left-side paw withdrawal thresholds (PWT) were observed before the first injection, 4 h, 3 d, 5 d after the first injection, before the second injection and 4 h, 1 d, 2 d, 3 d after the second injection. Three days after the second injection, the number of positive cells of cAMP, p-PKA, p-CREB and the number of positive cells of protein co-expression in the right ACC brain area were detected by immunofluorescence, and the relative protein expression of p-PKA and p-CREB were detected by Western blot. RESULTS: Compared with the control group, the PWTs in the model group decreased significantly 4 h, 3 d and 5 d after the first injection and 1 d, 2 d and 3 d after the second injection (<0.05); compared with the control group, the positive expression of cAMP, p-PKA and p-CREB in the right ACC brain area in the model group increased significantly (<0.05), and the number of positive cells of the co-expression of cAMP/p-PKA and p-PKA/p-CREB also increased significantly (<0.05). Compared with the model group, indomethacin group and sham EA group, the PWTs in the EA group were increased significantly 1 d, 2 d and 3 d after the second injection (<0.05); compared with the model group, indomethacin group and sham EA group, the positive expression of p-PKA and p-CREB in the right ACC brain area in the EA group decreased significantly (<0.05), and the number of positive cells of co-expression of cAMP/p-PKA and p-PKA/p-CREB was decreased significantly (<0.05). Compared with the model group and sham EA group, the positive expression of cAMP in the right ACC brain area was decreased in the EA group (<0.05). CONCLUSION EA have a direct intervention effect on pain memory, which have significant advantage over NSAIDs in the treatment of chronic pain. The advantage effect of EA on pain memory may be related to the inhibition of cAMP/PKA/CREB pathway in ACC area.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; therapeutic use ; Cyclic AMP ; metabolism ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Electroacupuncture ; Gyrus Cinguli ; metabolism ; Male ; Pain Threshold ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

This study aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells, with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway. The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases. First, the safe dosage of BAI was screened, and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h. The cells were assigned into a control group, a model group, and high-, medium-, and low-dose(5, 2.5, and 1.25 μmol·L~(-1), respectively) BAI groups. The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts, as well as the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), and cyclic adenosine monophosphate(cAMP) in the cell supernatant, were measured. Western blot was performed to determine the expression of protein kinase A(PKA), phosphorylated cAMP-response element binding protein(p-CREB), and nuclear factor-kappa B p65(NF-κB p65). Hoechst 33342/PI staining was employed to assess cell apoptosis. High and low doses of BAI were used for treatment in the research on the mechanism. The results revealed that BAI at the concentrations of 10 μmol·L~(-1) and below had no impact on normally cultured HMC3 cells. LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells. However, 5, 2.5, and 1.25 μmol·L~(-1) BAI significantly increased the SOD activity and 5 μmol·L~(-1) BAI significantly decreased the MDA content. In addition, BAI ameliorated the M1 polarization of HMC3 cells induced by LPS, as indicated by cellular morphology. The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α, IL-1β, IL-6, and cAMP in the cell supernatant. Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-regulating the expression of NF-κB p65. Hoechst 33342/PI staining results indicated that BAI mitigated the apoptosis of HMC3 cells. Overall, the results indicated that BAI had protective effects on the HMC3 cells induced by LPS, and could inhi-bit inflammatory response and improve cell apoptosis, which might be related to the regulation of the cAMP-PKA-NF-κB/CREB pathway.
Humans ; NF-kappa B/metabolism* ; Microglia ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Superoxide Dismutase/metabolism*

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

OBJECTIVETo investigate the effect and mechanism of emodin for regulating aquapoin-2 (AQP2) in NRK cells cultured in vitro.

METHODSExperiments on NRK cells cultured with alpha-DMEM medium in vitro were conducted in two steps. (1) Cells were randomly divided into 4 groups: the control group, and the three emodin treated groups treated with different dosages of emodin (5, 10 and 20 mg/L) respectively. After 24 h treatment, the location of AQP2 was decided by indirect immunofluorescene, and the AQP2 protein and mRNA expression levels were detected by Western blot and semiquantive RT-PCR. (2) Cells were randomly divided into 4 groups, the control group, and the three treated groups treated respectively with 10 mg/L 8-Bromo-cAMP, 20 mg/L emodin, and 20 mg/L emodin +10 mg/L 8-Bromo-cAMP. The activity of protein kinase A (PKA) in NRK cells after 24 h treatment was determined with non-radioactive detecting method.

RESULTSAQP2 was located at the cell membrane of NRK cells. Western blot and semiquantitive RT-PCR found that AQP2 protein and mRNA expressions were significantly decreased in NRK cells of groups treated by 10 mg/L and 20 mg/L emodin (P < 0.05). PKA activity determination showed significantly decreased phosphorylation level of PKA in NRK cells of groups treated with 20 mg/L emodin group (P < 0.05).

CONCLUSIONEmodin can inhibit the genetic transcription and the translation of AQP2 gene in NRK cells, which demonstrates that the change of AQP2 expression regulated by emodin may be correlated with the diuresis effect of rhubarb, and it is likely that the regulation is going through PKA signal pathway.

Animals ; Aquaporin 2 ; genetics ; metabolism ; Cell Line ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Emodin ; pharmacology ; Kidney ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Signal Transduction ; drug effects

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

OBJECTIVETo explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) tau and the inhibition of its biological activity.

METHODSUltracentrifugation, chromatography, manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant tau, isolate and purify 32P tau peptides and determine phosphorylation sites.

RESULTSPhosphorylation of tau by casein kinase 1 (CK-1), cyclic AMP-dependent protein kinase (PKA) and glycogen synthetase kinase 3 (GSK-3) separately inhibited its biological activity and the inhibition of this activity by (CSK-3 was significantly increased if tau was prephosphorylated by CK-1 or PKA. The most potent inhibition was seen by a combined phosphorylation of tau with PKA and GSK-3. The treatment of tau by PKA and GSK-3 combination induced phosphorylation of tau at Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-356, Ser-404, whereas Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400 were phosphorylated by GSK-3 alone under the same condition.

CONCLUSIONPhosphorylation of tau by PKA plus GSK-3 at Thr-205 might play a key role in tau pathology in AD.

Alzheimer Disease ; metabolism ; Binding Sites ; Casein Kinases ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Humans ; In Vitro Techniques ; Microtubules ; metabolism ; Phosphorylation ; Protein Kinases ; metabolism ; tau Proteins ; metabolism

The aim of the present study was to investigate the effects of cyclic adenosine monophosphate (cAMP) on rat gastric antral circular smooth muscle function. Forskolin, a direct activator of adenylyl cyclase (AC), was used to observe the influences of cAMP. Multi-channel physiological recorder was used to record spontaneous contraction activity of gastric antral circular muscle from Wistar rats. And ELISA method was used to detect the change of cAMP production in perfusate. The results showed that forskolin concentration-dependently suppressed the amplitude and frequency of the spontaneous contraction of the gastric antral muscle, and lowered the baseline of contraction movement significantly. Forskolin concentration-dependently increased the production of cAMP in the perfusate, which showed a significant negative correlation with the contraction amplitude of gastric antral ring muscle. The inhibitory effect of forskolin on spontaneous contraction activity of rat gastric antral circular muscle could be blocked by cAMP-dependent protein kinase (PKA) inhibitor H-89. These results suggest forskolin increases cAMP production and then activates PKA pathway, resulting in the inhibition of the spontaneous contraction activity of rat gastric antral circular smooth muscle.
Adenylyl Cyclases ; metabolism ; Animals ; Colforsin ; pharmacology ; Cyclic AMP ; pharmacology ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Isoquinolines ; pharmacology ; Muscle, Smooth ; drug effects ; Pyloric Antrum ; drug effects ; Rats ; Rats, Wistar ; Sulfonamides ; pharmacology

BACKGROUNDStudies have shown that β-blockers can improve cardiac performance in heart failure (HF) by reversing protein kinase A (PKA)-mediated sarcoplasmic reticulum (SR) Ca2+ leak. However, it is being strongly questioned as to whether the PKA-mediated ryanodine receptor (RyR2) hyper-phosphorylation is a critical regulator of SR Ca2+ leak. In this study, we used a rabbit HF model to investigate whether β-blockers affect SR Ca2+ leak by other potential mechanisms.

METHODSNew Zealand white rabbits were randomly divided in three groups (n=7 in each group): normal group, metoprolol-untreated group and metoprolol-treated group. Cardiac function was determined by echocardiography and hemodynamic assays. The SR Ca2+ leak was measured by a calcium-imaging device, and the expression and activities of related proteins were evaluated by Western blotting and auto-phosphorylation.

RESULTSIn the metoprolol-untreated group, there was significantly increased ventricular cavity size, reduced systolic function, increased SR Ca2+ leak, reduced associated amount of FK506 binding protein 12.6 (FKBP12.6), increased expression and activity of PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII), and increased phosphorylated RyR2 phosphorylation sites (with unchanged RyR2-P2030). In the treated group, there was partly increased ventricular cavity size with preserved systolic function, but no prominent Ca2+ leak, with unchanged expression and activity of PKA, CaMKII and their RyR2 phosphorylation sites.

CONCLUSIONChronic administration of metoprolol prevented the SR Ca2+ leak by restoring not only PKA-dependent but also CaMKII-dependent hyper-phosphorylation of RyR2, which may be one of the potential mechanisms by which β-blockers improve cardiac function and reduce the incidence of fatal arrhythmia in HF.

Animals ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Echocardiography ; Heart Failure ; drug therapy ; metabolism ; Hemodynamics ; drug effects ; Metoprolol ; therapeutic use ; Rats ; Sarcoplasmic Reticulum ; drug effects ; metabolism