Upregulation Of cAMP Research Articles

A mini-review: phosphodiesterases in charge to balance intracellular cAMP during T-cell activation.

T-cell activation is a pivotal process of the adaptive immune response with 3',5'-cyclic adenosine monophosphate (cAMP) as a key regulator of T-cell activation and function. It governs crucial control over T-cell differentiation and production of pro-inflammatory cytokines, such as IFN-γ. Intriguingly, levels of intracellular cAMP differ between regulatory (Treg) and conventional T-cells (Tcon). During cell-cell contact, cAMP is transferred via gap junctions between these T-cell subsets to mediate the immunosuppressive function of Treg. Moreover, the activation of T-cells via CD3 and CD28 co-stimulation leads to a transient upregulation of cAMP. Elevated intracellular cAMP levels are balanced precisely by phosphodiesterases (PDEs), a family of enzymes that hydrolyze cyclic nucleotides. Various PDEs play distinct roles in regulating cAMP and cyclic guanosine monophosphate (cGMP) in T-cells. Research on PDEs has gained growing interest due to their therapeutic potential to manipulate T-cell responses. So far, PDE4 is the best-described PDE in T-cells and the first PDE that is currently targeted in clinical practice to treat autoimmune diseases. But also, other PDE families harbor additional therapeutic potential. PDE2A is a dual-substrate phosphodiesterase which is selectively upregulated in Tcon upon activation. In this Mini-Review, we will highlight the impact of cAMP regulation on T-cell activation and function and summarize recent findings on different PDEs regulating intracellular cAMP levels in T-cells.

Encystation stimuli sensing is mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia

Protozoan parasites use cAMP signaling to precisely regulate the place and time of developmental differentiation, yet it is unclear how this signaling is initiated. Encystation of the intestinal parasite Giardia lamblia can be activated by multiple stimuli, which we hypothesize result in a common physiological change. We demonstrate that bile alters plasma membrane fluidity by reducing cholesterol-rich lipid microdomains, while alkaline pH enhances bile function. Through depletion of the cAMP producing enzyme Adenylate Cyclase 2 (AC2) and the use of a newly developed Giardia-specific cAMP sensor, we show that AC2 is necessary for encystation stimuli-induced cAMP upregulation and activation of downstream signaling. Conversely, over expression of AC2 or exogenous cAMP were sufficient to initiate encystation. Our findings indicate that encystation stimuli induce membrane reorganization, trigger AC2-dependent cAMP upregulation, and initiate encystation-specific gene expression, thereby advancing our understanding of a critical stage in the life cycle of a globally important parasite.

Rosemary (Rosmarinus officinalis L.) hydrosol based on serotonergic synapse for insomnia

Ethnopharmacological relevanceRosemary (Rosmarinus officinalis L.) has been widely used as a traditional remedy for insomnia, depression and anxiety in China and Western countries. Modern pharmacological studies have shown that rosemary has important applications in neurological disorders. However, the mechanism of action of rosemary hydrosol in the treatment of insomnia is not known. Aims of the studyInsomnia is closely linked to anxiety and depression, and its pathogenesis is related to biology, psychology, and sociology. Rosemary is a natural plant that has been used to treat insomnia and depression and has good biological activity, but its material basis and mechanism for the treatment of insomnia are not clear. Here, we report on the role of aqueous extracts of rosemary in the treatment of insomnia. Materials and methodsThe study was based on network pharmacology, using a combination of RNA-sequencing, “quantity-effect” weighting coefficients, and pharmacodynamic experiments. DL-4-chlorophenylalanine (PCPA) was intraperitoneally injected into SD rats to replicate the insomnia model with a blank, model, diazepam, and rosemary hydrosol low-, medium-, and high-dose groups were set up for the experiment. The key pathways in the treatment of insomnia with rosemary hydrosol were analyzed by molecular docking, open field assay, ELISA, western-Blot, Rt-PCR, and immunohistochemical assay. ResultsRosemary hydrosol was analyzed by GC-MS to identify 19 components. 1579 differential genes were obtained by RNA-Seq analysis, 533 targets for rosemary hydrosol and 2705 targets for insomnia, and 29 key targets were obtained by intersection. The KEGG results were ranked by “quantity-effect” weighting coefficients, resulting in serotonergic synapse was the key pathway for the treatment of insomnia with rosemary hydrosol. Molecular docking results showed that 1,7,7-trimethylbicyclo[2.2.1] heptan-2-one, 3-methyl-4-isopropylphenol, caryophyllene, and citronellol of rosemary hydrosol acted synergistically to achieve a therapeutic effect on insomnia. Caryophyllene acts on the HTR1A target by upregulating 5-HT1AR, leading to increased 5-HT release, and upregulation of ADCY5, cAMP, PKA and GABAA at serotonergic synapses; citronellol upregulated ADCY5 and 1,7,7-trimethylbicyclo[2.2.1] heptan-2-one, and 3-methyl-4-isopropylphenol up-regulated GABAA to improve insomnia symptoms. In open-field experiments, ELISA kits (5-HT, GABA, and DA), Western-blotting, Rt-PCR and immunohistochemical assay experiments, insomnia rats in the low-, medium- and high-dose groups of rosemary hydrosol showed different degrees of improvement compared with the model group. ConclusionsIt was shown that rosemary hydrosol may exert its therapeutic effects on insomnia through serotonergic synapses by combining RNA-Seq, “quantity-effect” weighting coefficients network pharmacology and pharmacodynamic experiments. We have provided a preliminary theoretical study for the development of rosemary hydrosol additive into a beverage for the treatment of insomnia, but it needs to be studied in depth. This study was conducted in rats and the results have limitations and may not apply to humans.

The Role of Cyclic Adenosine Monophosphate (cAMP) in Modulating Glucocorticoid Receptor Signaling and Its Implications on Glucocorticoid-Related Collagen Loss.

Glucocorticoid receptors (GRs) play a pivotal role in the stress response of the body, but overactivation can disrupt normal physiological functions. This study explores the role of cyclic adenosine monophosphate (cAMP) in GR activation and the associated mechanisms. We initially used the human embryonic kidney 293 cell line (HEK293) and found that cAMP enhancement, using forskolin and 3-isobutyl-1-methylxanthine (IBMX), did not alter glucocorticoid signaling under normal conditions, as evidenced by glucocorticoid response element (GRE) activity and the translocation of GR. However, in stressful conditions induced by dexamethasone, a synthetic glucocorticoid, cAMP was found to lessen glucocorticoid signaling within a short time frame but amplify it over an extended period in HEK293 cells. Bioinformatic analysis revealed that cAMP upregulation triggers the extracellular signal-regulated kinase (ERK) pathway, which influences GR translocation and ultimately regulates its activity. This stress-modulating function of cAMP was also investigated in the Hs68 dermal fibroblast line, known for its susceptibility to glucocorticoids. We found that cAMP enhancement via forskolin reduces GRE activity and reverses collagen loss in Hs68 cells exposed to dexamethasone. These findings underline the context-specific role of cAMP signaling in managing glucocorticoid signaling and its potential therapeutic application in treating stress-related pathological conditions like skin aging characterized by collagen reduction.

High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property–dependent manner

Optical control of G protein-coupled receptor (GPCR) signaling is a highly valuable approach for comprehensive understanding of GPCR-based physiologies and controlling them precisely. However, optogenetics for GPCR signaling is still developing and requires effective and versatile tools with performance evaluation from their molecular properties. Here, we systematically investigated performance of two bistable opsins that activate Gi/Go-type G protein (mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP)) in optical control in vivo using Caenorhabditis elegans. Transgenic worms expressing MosOpn3, which binds 13-cis retinal to form photopigments, in nociceptor neurons showed light-induced avoidance responses in the presence of all-trans retinal, a retinal isomer ubiquitously present in every tissue, like microbial rhodopsins and unlike canonical vertebrate opsins. Remarkably, transgenic worms expressing MosOpn3 were ~7,000 times more sensitive to light than transgenic worms expressing ChR2 in this light-induced behavior, demonstrating the advantage of MosOpn3 as a light switch. LamPP is a UV-sensitive bistable opsin having complete photoregenerative ability by green light. Accordingly, transgenic worms expressing LamPP in cholinergic motor neurons stopped moving upon violet light illumination and restored coordinate movement upon green light illumination, demonstrating color-dependent control of behavior using LamPP. Furthermore, we applied molecular engineering to produce MosOpn3-based tools enabling light-dependent upregulation of cAMP or Ca2+ levels and LamPP-based tool enabling clamping cAMP levels color dependently and context independently, extending their usability. These findings define the capacity of two bistable opsins with similar retinal requirement as ChR2, providing numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.

CAMP and voltage modulate rat auditory mechanotransduction by decreasing the stiffness of gating springs

Hair cells of the auditory and vestibular systems transform mechanical input into electrical potentials through the mechanoelectrical transduction process (MET). Deflection of the mechanosensory hair bundle increases tension in the gating springs that open MET channels. Regulation of MET channel sensitivity contributes to the auditory system's precision, wide dynamic range and, potentially, protection from overexcitation. Modulating the stiffness of the gating spring modulates the sensitivity of the MET process. Here, we investigated the role of cyclic adenosine monophosphate (cAMP) in rat outer hair cell MET and found that cAMP up-regulation lowers the sensitivity of the channel in a manner consistent with decreasing gating spring stiffness. Direct measurements of the mechanical properties of the hair bundle confirmed a decrease in gating spring stiffness with cAMP up-regulation. In parallel, we found that prolonged depolarization mirrored the effects of cAMP. Finally, a limited number of experiments implicate that cAMP activates the exchange protein directly activated by cAMP to mediate the changes in MET sensitivity. These results reveal that cAMP signaling modulates gating spring stiffness to affect auditory sensitivity.

Myricetin improves cognitive function in SAMP8 mice and upregulates brain-derived neurotrophic factor and nerve growth factor

IntroductionConsidering that neurodegeneration is an irreversible process, an efficient, low-burden approach to prevent dementia is strongly needed. Here, we show that the daily intake of myricetin normalised cognitive dysfunction in senescence-accelerated mouse prone 8 (SAMP8) mice. MethodsSAMP8 mice were fed a diet supplemented with myricetin and novel object recognition tests and Y-maze tests were performed. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brains of SAMP8 mice were measured. The phosphorylation level of cAMP-response-element-binding protein (CREB) level in brains of SAMP8 mice were evaluated. Also, SH-SY5Y cells were treated with myricetin and cAMP levels were measured. ResultsIn SAMP8 mice, neurotrophins, including BDNF and NGF, were downregulated relative to levels in their normal counterparts. In addition, myricetin intake upregulated the phosphorylation of CREB, the major transcription factor for BDNF and NGF. Also, myricetin induced cAMP upregulation, and CREB phosphorylation via a cAMP-dependent protein kinase-dependent manner in SH-SY5Y cells. ConclusionTaken together, myricetin improves cognitive function in SAMP8 mice and upregulates BDNF and NGF.

Silencing of A-kinase anchor protein 4 inhibits the metastasis and growth of non-small cell lung cancer

ABSTRACT Non-small cell lung cancer (NSCLC) is one of the most malignant tumors. The treatment of advanced NSCLC can be challenging due to drug resistance. The discovery of novel cancer-testis antigens to develop new strategies for advanced metastatic NSCLC is required. AKAP4 is an oncogene discovered in some malignant tumors, and its molecular function of AKAP4 in NSCLC is unknown. This study aimed to explore the potential function of AKAP4 in the development and progression of NSCLC. AKAP-4 was found to be significantly upregulated in both clinical NSCLC tissues and NSCLC cell lines. Cell viability and migration were suppressed, apoptosis was induced, and tube formation was inhibited by the knockdown of AKAP-4, accompanied by the downregulation of VEGF, N-cadherin, EphA2, and MMP-2, and upregulation of c-AMP, PKA, and E-cadherin. In vivo xenograft experiments revealed that tumor growth was inhibited by the knockdown of AKAP4, accompanied by the activation of c-AMP/PKA signaling and inhibition of epithelial-mesenchymal transition progression. Our results show that AKAP4 might be an important target for treating NSCLC because of its function in promoting the migration and proliferation of NSCLC cells.

Peripheral ablation of type III adenylyl cyclase induces hyperalgesia and eliminates KOR-mediated analgesia in mice.

Ca2+/calmodulin-stimulated group I adenylyl cyclase (AC) isoforms AC1 and AC8 have been involved in nociceptive processing and morphine responses. However, whether AC3, another member of group I ACs, is involved in nociceptive transmission and regulates opioid receptor signaling remains elusive. Here, we report that conditional KO of AC3 (AC3 CKO) in L3 and L4 DRGs robustly facilitated the mouse nociceptive responses, decreased voltage-gated potassium (Kv) channel currents, and increased neuronal excitability. Furthermore, we report AC3 CKO eliminated the analgesic effect of κ-opioid receptor (KOR) agonist and its inhibition on Kv channel by classical Gαi/o signaling or nonclassical direct interaction of KOR and AC3 proteins. Interestingly, significantly upregulated AC1 level and cAMP concentration were detected in AC3-deficient DRGs. Inhibition of AC1 completely reversed cAMP upregulation, neuronal excitability enhancement, and nociceptive behavioral hypersensitivity in AC3-CKO mice. Our findings suggest a crucial role of peripheral AC3 in nociceptive modulation and KOR opioid analgesia.

Osthole-Mediated Inhibition of Neurotoxicity Induced by Ropivacaine via Amplification of the Cyclic Adenosine Monophosphate Signaling Pathway.

BackgroundRopivacaine is widely used for clinical anesthesia and postoperative analgesia. However, the neurotoxicity induced by ropivacaine in a concentration- and duration-dependent manner, and it is difficult to prevent neurotoxicity. Osthole inhibits phosphodiesterase-4 activity by binding to its catalytic site to prevent cAMP hydrolysis. The aim of this present study is to explore the precise molecular mechanism of osthole-mediated inhibition of neurotoxicity induced by ropivacaine.Methods:SH-SY5Y cell viability and apoptosis were measured in different concentration and duration. Protein concentration was determined in each signaling pathway. The molecular mechanism of osthole-mediated inhibition of ropivacaine-caused neurotoxicity was evaluated.ResultsThe study demonstrated that osthole inhibits SH-SY5Y cells neurotoxicity in a duration- and concentration-dependent manner. Moreover, ropivacaine significantly increased the expression of caspase-3 by promoting the phosphorylation of p38. Osthole-induced upregulation of cAMP activated cAMP-dependent signaling pathway, sequentially leading to elevated cyclic nucleotide response element-binding protein levels, which inhibits P38-dependent signaling and decreases apoptosis of SH-SY5Y.ConclusionsThis study display the evidence confirmed the molecular mechanism by which osthole amplification of cAMP-dependent signaling pathway, and overexpression of cyclic nucleotide response element-binding protein inhibits P38-dependent signaling and decreases ropivacaine-induced SH-SY5Y apoptosis.

Upregulation of cAMP prevents antibody-mediated thrombus formation in COVID-19

AbstractThromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. The exact mechanisms of COVID-19-associated hypercoagulopathy, however, remain elusive. Recently, we observed that platelets (PLTs) from patients with severe COVID-19 infection express high levels of procoagulant markers, which were found to be associated with increased risk for thrombosis. In the current study, we investigated the time course as well as the mechanisms leading to procoagulant PLTs in COVID-19. Our study demonstrates the presence of PLT-reactive IgG antibodies that induce marked changes in PLTs in terms of increased inner-mitochondrial transmembrane potential (Δψ) depolarization, phosphatidylserine (PS) externalization, and P-selectin expression. The IgG-induced procoagulant PLTs and increased thrombus formation were mediated by ligation of PLT Fc-γ RIIA (FcγRIIA). In addition, contents of calcium and cyclic-adenosine-monophosphate (cAMP) in PLTs were identified to play a central role in antibody-induced procoagulant PLT formation. Most importantly, antibody-induced procoagulant events, as well as increased thrombus formation in severe COVID-19, were inhibited by Iloprost, a clinically approved therapeutic agent that increases the intracellular cAMP levels in PLTs. Our data indicate that upregulation of cAMP could be a potential therapeutic target to prevent antibody-mediated coagulopathy in COVID-19 disease.

Findings from Studies Are Congruent with Obesity Having a Viral Origin, but What about Obesity-Related NAFLD?

Infection has recently started receiving greater attention as an unusual causative/inducing factor of obesity. Indeed, the biological plausibility of infectobesity includes direct roles of some viruses to reprogram host metabolism toward a more lipogenic and adipogenic status. Furthermore, the probability that humans may exchange microbiota components (virome/virobiota) points out that the altered response of IFN and other cytokines, which surfaces as a central mechanism for adipogenesis and obesity-associated immune suppression, is due to the fact that gut microbiota uphold intrinsic IFN signaling. Last but not least, the adaptation of both host immune and metabolic system under persistent viral infections play a central role in these phenomena. We hereby discuss the possible link between adenovirus and obesity-related nonalcoholic fatty liver disease (NAFLD). The mechanisms of adenovirus-36 (Ad-36) involvement in hepatic steatosis/NAFLD consist in reducing leptin gene expression and insulin sensitivity, augmenting glucose uptake, activating the lipogenic and pro-inflammatory pathways in adipose tissue, and increasing the level of macrophage chemoattractant protein-1, all of these ultimately leading to chronic inflammation and altered lipid metabolism. Moreover, by reducing leptin expression and secretion Ad-36 may have in turn an obesogenic effect through increased food intake or decreased energy expenditure via altered fat metabolism. Finally, Ad-36 is involved in upregulation of cAMP, phosphatidylinositol 3-kinase, and p38 signaling pathways, downregulation of Wnt10b expression, increased expression of CCAAT/enhancer binding protein-beta, and peroxisome proliferator-activated receptor gamma 2 with consequential lipid accumulation.

DAla2-GIP-GLU-PAL Protects Against Cognitive Deficits and Pathology in APP/PS1 Mice by Inhibiting Neuroinflammation and Upregulating cAMP/PKA/CREB Signaling Pathways.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive decline in cognitive function. Type 2 diabetes mellitus (T2DM) is an important risk factor for AD. Glucose-dependent insulinotropic polypeptide (GIP) has been identified to be effective in T2DM treatment and neuroprotection. The present study investigated the neuroprotective effects and possible mechanisms of DAla2GIP-Glu-PAL, a novel long-lasting GIP analogue, in APP/PS1 AD mice. Multiple behavioral tests were performed to examine the cognitive function of mice. In vivo hippocampus late-phase long-term potentiation (L-LTP) was recorded to reflect synaptic plasticity. Immunohistochemistry and immunofluorescence were used to examine the Aβ plaques and neuroinflammation in the brain. IL-1β, TNF-α, and cAMP/PKA/CREB signal molecules were also detected by ELISA or western blotting. DAla2GIP-Glu-PAL increased recognition index (RI) of APP/PS1 mice in novel object recognition test, elevated spontaneous alternation percentage of APP/PS1 mice in Y maze test, and increased target quadrant swimming time of APP/PS1 mice in Morris water maze test. DAla2GIP-Glu-PAL treatment enhanced in vivo L-LTP of APP/PS1 mice. DAla2GIP-Glu-PAL significantly reduced Aβ deposition, inhibited astrocyte and microglia proliferation, and weakened IL-1β and TNF-α secretion. DAla2GIP-Glu-PAL also upregulated cAMP/PKA/CREB signal transduction and inhibited NF-κB activation in the hippocampus of APP/PS1 mice. DAla2GIP-Glu-PAL can improve cognitive behavior, synaptic plasticity, and central pathological damage in APP/PS1 mice, which might be associated with the inhibition of neuroinflammation, as well as upregulation of cAMP-/PKA/CREB signaling pathway. This study suggests a potential benefit of DAla2GIP-Glu-PAL in the treatment of AD.

Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm.

Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20-23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.

Effect of cAMP Signaling Regulation in Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 (ID2) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2, Osterix, and IBSP, which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature.

Biological Activity of Porcine Gastric Mucin on Stress Resistance and Immunomodulation.

Purified porcine gastric mucin (PGM) is an alternative biomaterial to native mucin which displays multifunctional properties for exploring a wide range of biomedical applications. The present study evaluated the in vitro (RAW 264.7 macrophage cells) and in vivo (zebrafish embryos and larvae) bioactivities of PGM. The median lethal concentration (LC50) of PGM was 197.9 µg/mL for embryos, while it was non-toxic to RAW 264.7 cells, even at 500 µg/mL. Following PGM exposure (100 µg/mL), a higher embryo hatching rate (59.9%) was observed at 48 h post fertilization, compared to the control (30.6%). Protective effects of PGM from pathogenic Aeromonas hydrophila were demonstrated by high larvae survival rates of 85.0% and 94.0% at 50 and 100 μg/mL of PGM exposure, respectively. Heat tolerance effect of PGM (50 and 100 µg/mL) on larvae (40 °C for 48 h) was confirmed by 75% and 100% of survival rates, respectively. Additionally, PGM reduced the A. hydrophila–induced reactive oxygen species (ROS) generation in larvae. The qRT-PCR results in PGM exposed larvae exhibited induction of immune-related genes (tlr5a and tlr5b, myd88, c-rel, il1β, tnf-α, il6, il10, cxcl18b, ccl34a.4, defbl1, hamp, ctsd, muc2.1, muc5.1, muc5.2, and muc5.3), stress response (hsp70, hsp90aa1.1, and hsp90ab1), and antioxidant genes (cat and sod1). Moreover, our results revealed that PGM involved in the regulation of transcriptional gene induction increases Hsp90 protein in the zebrafish larvae. Furthermore, upregulation of Il6, Il10, Tnfα, Ccl3, Defa-rs2, Defa21 and Camp and antioxidant genes (Sod2 and Cat) were observed in PGM-exposed RAW 264.7 cells. Overall findings confirmed the activation of immune responses, disease resistance against pathogenic bacteria, heat tolerance, and ROS-scavenging properties by PGM, which may provide insights into new applications for PGM as a multifunctional immunomodulator.

Bmp2 regulates Serpinb6b expression via cAMP/PKA/Wnt4 pathway during uterine decidualization.

Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8‐Br‐cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock‐down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up‐regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.

Assessment of Morphine Tolerance Using cAMP‐GloSensor&[trade] Real‐time Detection Assay in SH‐SY5Y Human Neuroblastoma Cells

PurposeActivation of GPCRs by morphine triggers downstream effects including inhibition of adenylyl cyclase and decrease in 3′,5′‐cyclic adenosine monophosphate (cAMP). cAMP upregulation is observed in morphine tolerance and withdrawal. Endothelin‐A receptor (ETAR) antagonist, BQ123 potentiates morphine analgesia, reverses tolerance, and reduces withdrawal symptoms. However, signaling mechanisms involved in these phenomena are unknown. The objective of this study was to determine morphine‐ and BQ123‐induced changes in cAMP signaling in SH‐SY5Y neuroblastoma cells which endogenously express mu‐opioid receptors (MORs) and ETARs.MethodsSH‐SY5Y cells (ATCC®) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37°C/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104 cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and 30% transfection efficiency was achieved. Stably transfected SH‐SY5Y cell line was established using GloSensor™ −23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Promega’s real‐time detection system in live cells, the cAMP‐GloSensor™ assay was used to measure cAMP responses. Stably transfected cells were incubated with equilibrium buffer [cAMP GloSensor™ reagent (2% (v/v)), 88% CO2 independent medium, 10% FBS]. cAMP levels were determined by changes in luminescence using an EnSpire™ multimode microplate reader (PerkinElmer). In acute studies, cells were treated for 2 h with morphine (10–10−5 M) and BQ123 (1 μM). In chronic studies, cells were treated for 24 h with morphine (1 μM) in the presence and absence of BQ123 (1 μM). After induction of tolerance for 24 h, two challenge doses of morphine (10 μM and 20 μM) were tested. Isoproterenol was used as a positive control (1–10−8 M). Each experiment was repeated in triplicate and data was analyzed in GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA).ResultsA concentration‐dependent increase in cAMP was observed with isoproterenol in SH‐SY5Y cells stably transfected with −23F cAMP plasmid. Acute morphine treatment (10–10−5 M for 2 h) produced concentration‐dependent decrease in cAMP, while acute treatment with BQ123 (1–10−5 M) increased cAMP levels. Of interest, BQ123 (1 μM) appeared to enhance morphine‐induced decrease in cAMP response. Chronic morphine treatment (1 μM for 24 h) increased cAMP response, indicative of tolerance. Cells treated with combination of morphine (1 μM) and BQ123 (1 μM) for 24 h showed dose‐dependent inhibition of cAMP levels with challenge doses of morphine.ConclusionTo better understand the mechanism by which ETAR antagonists reverse morphine tolerance and reduce withdrawal symptoms, signaling mechanisms were explored in SH‐SY5Y cells endogenously expressing ETA receptors and MORs. We found that BQ123 appears to enhance morphine‐induced cAMP signaling (measured by Glo‐Sensor™ cAMP assay) through Gi‐coupled MORs in both acute and chronic studies, which support our previous work. We will further explore the involvement of beta‐arrestin and GPCR kinases (GRKs) in these phenomena.Support or Funding InformationChicago College of Pharmacy Faculty Research Grant; College of Graduate Studies, Biomedical Sciences, Midwestern University, Downers Grove IL.

Antiplatelet effects of scoparone through up-regulation of cAMP and cGMP on U46619-induced human platelets

Antiplatelet effects of scoparone through up-regulation of cAMP and cGMP on U46619-induced human platelets

Potential role of the cAMP/PKA/CREB signalling pathway in hypoxic preconditioning and effect on propofol‑induced neurotoxicity in the hippocampus of neonatal rats.

Hypoxic preconditioning (HPC) is neuroprotective against ischaemic brain injury; however, the roles of potential anti-apoptotic signals in this process have not been assessed. To elucidate the molecular mechanisms involved in HPC-induced neuroprotection, the effects of HPC on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signalling pathway and apoptosis in Sprague-Dawley pups (postnatal day 7) treated with propofol were investigated. Western blot and histological analyses demonstrated that HPC exerts multiple effects on the hippocampus, including the upregulation of cAMP and phosphorylation of CREB. These effects were partially blocked by intracerebroventricular injection of the protein kinase antagonist H89 (5 µmol/5 µl). Notably, the level of cleaved caspase-3 was significantly downregulated by treatment with the cAMP agonist Sp-cAMP (20 nmol/5 µl). The results indicate that propofol increased the level of cleaved caspase-3 and Bax by suppressing the activity of cAMP-dependent proteins and Bcl-2; thus, HPC prevents propofol from triggering apoptosis via the cAMP/PKA/CREB signalling pathway.