Increase In cAMP Concentration Research Articles

Research progress on phosphodiesterase 4 inhibitors in central nervous system diseases.

Phosphodiesterases (PDE) are involved in the regulation of cellular physiological processes and neurological functions, including neuronal plasticity, synapto-genesis, synaptic transmission, memory formation and cognitive functions by catalyzing the hydrolysis of intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Many basic and clinical studies have shown that PDE4 inhibitors block or ameliorate the occurrence and development of central nervous system (CNS) diseases by inhibiting cAMP hydrolysis, increasing cAMP content and enhancing its downstream effects. PDE4 inhibitors have long-term potentiation effect, which can enhance phosphorylation of cAMP response element binding protein (CREB) and upregulate expression of memory related Arc genes in hippocampal neurons, thereby improving cognitive impairment and Alzheimer's disease-like symptoms. They can also delay the occurrence and development of Parkinson's disease by reducing the cytotoxicity induced by α-syn and increasing the effect of miR-124-3p on cell functions. Alteration of PDE4 activity is the molecular basis for psychosis and some cognitive disorders, therefore it is considered as a therapeutic target for schizophrenia. PDE4 inhibitors play a role in depression by inhibiting the advanced glycation end product receptor (RAGE), TLR4 and NLRP3 pathways in the hippocampus, reducing the activation of microglia and the production of IL-1β, down-regulating HMGB1/RAGE signaling pathway and inhibiting inflammatory factors. PDE4 inhibitor plays a role in the treatment of autism spectrum disorder by reducing the damage of cerebellar glial cells, increasing nociceptive threshold, and improving mutual learning and memory deficits. PDE4 inhibitors might be used in the treatment of fragile X syndrome by regulating the level of cAMP and affecting the expression of fragile X mental retardation protein (FMRP). PDE4 inhibitors can also promote the differentiation of oligodendrocyte progenitor cells and enhance myelination, which has potential in the treatment of multiple sclerosis. PDE4 is also related to bipolar disorder, which may be one of the therapeutic targets. At present, several PDE4 inhibitors are in clinical trials for the treatment of CNS diseases. This article reviews and discusses the progress on basic research and clinical trials of PDE4 inhibitors in CNS diseases, providing a reference for the prevention and treatment of CNS diseases and the development of new drugs.

Danshen and Zhizi Compatibility Alleviates Heart Injury and Cardiac Ferroptosis in Myocardial Infarction in Rats by Cyclic Adenosine Monophosphate/Protein Kinase A Signaling

Objective: To investigate the effect and mechanism of the Danshen and Zhizi Compatibility (DZ) on alleviating heart injury and cardiac ferroptosis in rats with myocardial infarction. Methods: A rat model of myocardial infarction was established by ligation of the left anterior descending artery. The rats were equally and randomly divided into 5 groups. The sham group underwent open-chest surgery without arterial ligation, while the other 4 groups underwent surgery, including 3 groups treated with low dose (4 g/kg/d), high dose (8 g/kg/d) DZ and high dose (8 g/kg/d) DZ supplemented with H-89 (0.5 mg/kg/d) respectively. The sham and myocardial infarction group received the same volume of saline. 14 days after surgery, the serum and heart tissues were harvested to detect cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity, heart injury and the level of ferroptosis. Results: G-protein coupled receptors (GPCRs) have a high binding affinity with the main components of DZ, which indicated that DZ probably contributed to ameliorating cardiac injury by activating downstream cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling. Treatment with the high dose of DZ significantly increased cAMP concentration in the serum, PKA activity in the heart tissue and upregulated perilipin (PLIN)5 expression. DZ significantly attenuated heart injury, whereas H-89 reversed the protective effects of DZ. In addition, DZ administration inhibited ferroptosis as evidenced by reduced malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) levels. In addition, DZ increased glutathione (GSH) levels and Glutathione peroxidase (GPX)4 protein expression in heart tissue, whereas H-89 abrogated the regulatory effect of DZ. Conclusion: Our results demonstrated that DZ alleviated heart injury and cardiac ferroptosis in myocardial infarction through the cAMP-PKA signalling pathway.

Potential PDE4B inhibitors as promising candidates against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an RNA virus belonging to the coronavirus family responsible for coronavirus disease 2019 (COVID-19). It primarily affects the pulmonary system, which is the target of chronic obstructive pulmonary disease (COPD), for which many new compounds have been developed. In this study, phosphodiesterase 4 (PDE4) inhibitors are being investigated. The inhibition of PDE4 enzyme produces anti-inflammatory and bronchodilator effects in the lung by inducing an increase in cAMP concentrations. Piclamilast and rolipram are known selective inhibitors of PDE4, which are unfortunately endowed with common side effects, such as nausea and emesis. The selective inhibition of the phosphodiesterase 4B (PDE4B) subtype may represent an intriguing technique for combating this highly contagious disease with fewer side effects. In this article, molecular docking studies for the selective inhibition of the PDE4B enzyme have been carried out on 21 in-house compounds. The compounds were docked into the pocket of the PDE4B catalytic site, and in most cases, they were almost completely superimposed onto piclamilast. Then, in order to enlarge our study, drug-likeness prediction studies were performed on the compounds under study.

SAT001 Membrane Progesterone Receptors Induce Glycogenolysis In Uterine Epithelial Cells

Abstract Disclosure: M. Berg: None. M. Dean: None. In humans and cattle, 40-60% of pregnancies fail, with most losses occurring prior to or during implantation. Throughout this time, the developing embryo needs glucose to survive. The demand for glucose increases dramatically at the morula stage as the embryo enters the uterus. Yet, how glucose is regulated to match the changing needs of the embryo is unclear. Glycogen is composed of thousands of glucose molecules and is present in the uterine epithelium of many species. Our lab has shown that the glycogen content of the bovine uterine epithelium was lower during the luteal phase than at estrus. Therefore, our objective was to elucidate the role of progesterone in glycogenolysis of uterine epithelial cells. Bovine uterine epithelial (BUTE) cells were treated with insulin-like growth factor 1 (IGF1) to stimulate glycogenesis, followed by treatment for 48 hours. Progesterone decreased glycogen levels in BUTE cells by 99% (P=0.0002). RU486 did not block progesterone's effect, indicating that the effect of progesterone was not mediated by nuclear progesterone receptors (nPR). Thus, we hypothesized that the effect of progesterone is mediated by membrane progesterone receptors (mPRs). RT-PCR confirmed that BUTE cells expressed all five mPRs (α, β, γ, δ, and ε). Like progesterone, a specific mPR agonist (Org OD 02-0) decreased glycogen levels in BUTE cells by 99% (P<0.0001). Indicating a potential for mPRs to regulate glycogen in vivo, immunohistochemistry showed that the bovine uterine epithelium expressed high levels of mPRα. These results validate that progesterone is acting through mPRs to stimulate glycogen breakdown in BUTE cells. Once progesterone binds to the mPR, two signaling pathways can be activated, cAMP and AMPK. Neither progesterone nor Org OD 02-0 changed intracellular cAMP concentrations. In agreement, the adenylyl cyclase activator forskolin increased cAMP concentration but did not decrease glycogen. BUTE cells treated with progesterone had increased pAMPK levels at 24 hours compared to the control (P=0.0001). BUTE cells treated with an AMPK activator (D942) had a decrease in glycogen (P=0.0051). Supporting these results in vivo, pAMPK levels in the uterine epithelium were high in the bovine uterine epithelium during the luteal phase when glycogen levels were low. In a human model, progesterone and Org OD 02-0 decreased glycogen levels to a similar extent in Ishikawa cells that express mPRs but not nPRs (P=0.0027). In conclusion, progesterone acting through the mPR and AMPK to stimulate glycogenolysis may play a crucial role in providing glucose to endometrial tissue or the growing embryo. Presentation: Saturday, June 17, 2023

Parathyroid Hormone-Related Protein Promotes the Proliferation of Patient-Derived Glioblastoma Stem Cells via Activating cAMP/PKA Signaling Pathway.

Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its heterogeneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM. Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP. Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

Potential for modulation of platelet function via adenosine receptors during inflammation.

Traditionally, platelets are known to play an important role in haemostasis and thrombosis; however, they serve also as important modulators of inflammation and immunity. Platelets secrete adhesion molecules and cytokines, interact with leukocytes and endothelium, and express toll-like receptors involved in a direct interaction with pathogens. Platelets express A2A and A2B subtypes of receptors for adenosine. The activation of these receptors leads to an increase in cAMP concentration in the cytoplasm, thereby resulting in inhibited secretion of pro-inflammatory mediators and reduced cell activation. Therefore, platelet adenosine receptors could be a potential target for inhibiting platelet activation and thus down-regulating inflammation or immunity. The biological effects of adenosine are short-lasting, because the compound is rapidly metabolized; hence, its lability has triggered efforts to synthesize new, longer-lasting adenosine analogues. In this article, we have reviewed the literature regarding the pharmacological potential of adenosine and other agonists of A2A and A2B receptors to affect platelet function during inflammation. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Metabolome shifts triggered by chlorine sanitisation induce Escherichia coli on fresh produce into the viable but nonculturable state

Facing the increasing occurrence of “big six” Escherichia coli outbreaks linked to fresh produce, chlorine-based sanitisers are widely used for fresh produce decontamination in recent years. However, latest finding that chlorine may induce E. coli cells into a viable not nonculturable (VBNC) state is bringing a new challenge to the fresh produce industry. VBNC cells are undetectable by the plate count test, and yet they retain pathogenicity and are more antibiotic-resistant than culturable cells. As a result, their eradication is critical to ensure the safety of fresh produce. Understanding VBNC cells at the metabolic level may provide a breakthrough for their eradication. Therefore, this study was carried out to collect the VBNC pathogenic E. coli (O26:H11, O121:H19, and O157:H7) cells from chlorine-treated pea sprouts and characterise them using NMR-based metabolomics. From the globally increased metabolite contents detected in the VBNC E. coli cells as compared to the culturable cells, mechanisms underlying E. coli’s VBNC induction were elucidated. These include rendering the energy generation scheme to become more compatible with the lowered energy needs, disaggregating protein aggregates to release amino acids for osmoprotection and later resuscitation, as well as increasing cAMP content to downregulate RpoS. These identified metabolic characteristics can inspire future development of targeted measures for VBNC E. coli cell inhibition. Our methods can also be applied to other pathogens to help lower the risk of overall foodborne diseases.

Maltose-Enhanced Exopolysaccharide Synthesis of Lactiplantibacillus plantarum through CRP-like Protein

Carbon sources alter the synthesis of exopolysaccharides (EPS) in Lactiplantibacillus plantarum. Maltose increased the EPS production of L. plantarum 163 6.5-fold. Subsequently, EPS production, transcriptome, and proteome were analyzed using glucose or maltose to further clarify the regulatory mechanism. A cAMP receptor protein (UniProtKB: F9UNI5) has been identified to control EPS synthesis in the presence of cAMP by binding to the EPS synthesis promoter Pcps4A-J. Overexpression of the cAMP synthesis gene cyaA increased cAMP content and EPS production 4.5- and 2.2-fold, respectively. Furthermore, yogurt produced with L. plantarum 163-cyaA had a similar viscosity to that of commercial Greek yogurt; it had 20 and 83.7% greater viscosity than that produced with L. plantarum 163 with maltose and glucose, respectively. These findings indicated that L. plantarum 163-cyaA has potential applications in the production of functional fermented dairy products.

ENOS Location is a Fundamental Mechanism for Inactivation of Inflammation‐induced Hyperpermeability

Inflammation disrupts the endothelial barrier and increases microvascular permeability (hyperpermeability), leading to tissue edema. Mechanisms for the onset of hyperpermeability have been the focus of many studies. However, the major pathological consequences are related to impairment in terminating hyperpermeability, rather than its onset, and to sustained inflammation. Therefore, we are innovatively studying mechanisms involved in the inactivation of hyperpermeability and restoration of endothelial barrier and normal microvascular permeability. We demonstrated that agonist‐induced translocation of eNOS (endothelial nitric oxide synthase) from cell membrane to cytosol initiates hyperpermeability. We hypothesize that agonist signaling for hyperpermeability also initiates a delayed increase in [cAMP], causing translocation of eNOS and Epac1 (exchange protein activated by cAMP) from cytosol to the cell membrane, which inactivates hyperpermeability. We tested whether platelet‐activating factor (PAF) induced hyperpermeability first and in delayed manner increased cAMP concentration to selectively stimulate Epac1 leading to translocation of eNOS from cytosol back to the cell membrane. In addition, we studied whether vasodilator‐stimulated phosphoprotein (VASP) is involved in the inactivation of hyperpermeability since PAF‐induced hyperpermeability in VASP‐KO endothelial cells remains elevated after Epac1 stimulation. We used human microvascular endothelial cells (HMVEC) and ECV‐304 cells transfected with green fluorescent protein (GFP)‐conjugated eNOS or the constructs GFPeNOSG2A (which anchors eNOS to the cytoplasm, ECV‐GFPeNOS‐G2A) and GFPeNOS‐CAAX (which anchors eNOS to the plasma membrane, ECV‐GFPeNOS‐CAAX) to study the restoration of endothelial barrier after hyperpermeability induced by PAF or vascular endothelial growth factor (VEGF). The time‐correlation between [NO] and [cAMP] in PAF or VEGF‐stimulated HMVEC and ECV‐eNOSGFP indicates that NO increase precedes [cAMP] increase. The increase in [cAMP] in HMVEC and ECV‐eNOSGFP correlates with a close proximity between eNOS, Epac1 and VASP, and both eNOS and Epac1 translocate to the cell membrane. None of these observations occur when eNOS is anchored to the cell membrane or cytosol. Furthermore, stimulation of Epac1 in HMVEC and ECV‐eNOSGFP inactivates their hyperpermeability response to PAF or VEGF. Stimulation of Epac1 in ECV‐GFPeNOS‐CAAX, after PAF application, inhibits hyperpermeability, while Epac1 stimulation does not reduce PAF‐elicited hyperpermeability in ECV‐GFPeNOS‐G2A. Stimulation of Epac1, after application of PAF, in the in vivo male and female hamster cheek pouch prevents hyperpermeability. In conclusion, our results support the concept that eNOS, cAMP, Epac1 and VASP work in synchrony to terminate hyperpermeability by translocating eNOS back to the cell membrane, and the overarching concept that location of eNOS is an important determinant in the regulation of microvascular permeability.

Extracellular vesicle-induced cyclic AMP signaling

Second messenger signaling is required for cellular processes. We previously reported that extracellular vesicles (EVs) from stimulated cultured endothelial cells contain the biochemical second messenger, cAMP. In the current study, we sought to determine whether cAMP-enriched EVs induce second messenger signaling pathways in naïve recipient cells. Our results indicate that cAMP-enriched EVs increase cAMP content sufficient to stimulate PKA activity. The implications of our work are that EVs represent a novel intercellular mechanism for second messenger, specifically cAMP, signaling.

MicroRNA miR-133a-3p Facilitates Adrenergic Proarrhythmic Ectopy in Rat Pulmonary Vein Myocardium by Increasing cAMP Content.

Cardiac-specific microRNA miR-133a-3p modulates adrenergic signaling. Adrenergic receptors and their intracellular pathways are the key players in proarrhythmic ectopy derived from the myocardial sleeves of the pulmonary veins. We studied the effect of miR-133a-3p on ectopy induced by norepinephrine in myocardial tissue of rat pulmonary veins. Using microelectrode technique, we revealed facilitation of proarrhythmic pattern of spontaneous bursts of action potentials induced by norepinephrine in tissue preparations of the pulmonary veins isolated from rats in 24 h after injection of a transfection mixture containing miR-133a-3p (1 mg/kg) in vivo. According to ELISA data, the cAMP level in the pulmonary vein myocardium of rats receiving miR-133a-3p was 2-fold higher than in control animals. Bioinformatic analysis showed that mRNA of protein phosphatases and some phosphodiesterases are most probable targets of miR-133a-3p. The proarrhythmic effect of miR-133a-3p can be related to inhibition of the expression of phosphodiesterases accompanied by cAMP accumulation and increased intracellular β-adrenergic signaling.

PDE4B Is a Homeostatic Regulator of Cyclic AMP in Dendritic Cells.

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings, but how cells compensate for such decreases is unknown. We have used a unique system—murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein Gαs—to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in Gαs-depleted DCs. PDE4B expression is dynamic, falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations, respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations.

Suppression of FoxO1 mRNA by β2 -adrenoceptor-cAMP signaling through miR-374b-5p and miR-7a-1-3p in C2C12 myotubes.

β2‐Adrenoceptor (β2‐AR) signaling decreases the transcriptional activity of forkhead box O (FoxO), but the underlying mechanisms remain incompletely understood. Here, we investigated how β2‐AR signaling regulates the protein abundance of FoxO and its transcriptional activity in skeletal muscle. We observed that stimulation of β2‐AR with its selective agonist, clenbuterol, rapidly decreased FoxO1 mRNA expression, and this was accompanied by a decrease in either FoxO1 protein level or FoxO transcriptional activity. We subsequently observed that miR‐374b‐5p and miR‐7a‐1‐3p were rapidly upregulated in response to β2‐AR stimulation. Transfection with mimics of either miRNA successfully decreased FoxO1 mRNA. Moreover, because β2‐AR stimulation increased cAMP concentration, pretreatment with an adenylyl cyclase inhibitor canceled out these effects of β2‐AR stimulation. These results suggest that β2‐AR stimulation results in rapid upregulation of miR‐374b‐5p and miR‐7a‐1‐3p in myotubes, which in turn results in a decrease in FoxO1 mRNA expression via the β2‐AR–cAMP signaling pathway.

History-dependent dopamine release increases cAMP levels in most basal amygdala glutamatergic neurons to control learning.

Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. This learning depends on intracellular plasticity signals such as cyclic adenosine monophosphate (cAMP), which is regulated by activation of dopamine receptors. We examine the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We perform real-time tracking and manipulation of cAMP in BA neurons invitro and invivo. Optogenetically evoked release of dopamine drives proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. This cAMP response decreases across trials with short intertrial intervals owing to depression of dopamine release. No such depression is evident when photostimulating cAMP production directly. cAMP and protein kinase A responses to repeated appetitive or aversive stimuli also exhibit pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

Taurochenodeoxycholic Acid Increases cAMP Content via Specially Interacting with Bile Acid Receptor TGR5

Taurochenodeoxycholic acid (TCDCA) is one of the main components of bile acids (BAs). TCDCA has been reported as a signaling molecule, exerting anti-inflammatory and immunomodulatory functions. However, it is not well known whether those effects are mediated by TGR5. This study aimed to elucidate the interaction between TCDCA and TGR5. To achieve this aim, first, the TGR5 eukaryotic vector was constructed. The expression level of TGR5 in 293T cells was determined by immunofluorescence, real-time quantitative PCR (RT-PCR, qPCR), and Western blot. The luciferase assay, fluorescence microscopy, and enzyme-linked immunosorbent assay (ELISA) were recruited to check the interaction of TCDCA with TGR5. TCDCA treatment in 293T cells resulted in TGR5 internalization coupled with a significant increase in cAMP luciferase expression. Our results demonstrated that TCDCA was able to bind to the TGR5 receptor and activate it. These results provide an excellent potential therapeutic target for TCDCA research. Moreover, these findings also provide theoretical evidence for further TCDCA research.

Cyclic Adenosine Monophosphate Eliminates Sex Differences in Estradiol-Induced Elastin Production from Engineered Dermal Substitutes.

Lack of adult cells’ ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17β-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-β levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17β-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17β-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17β-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes.

Taurochenodeoxycholic acid mediates cAMP-PKA-CREB signaling pathway.

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.

Extracellular ATP modulates podocyte function through P2Y purinergic receptors and pleiotropic effects on AMPK and cAMP/PKA signaling pathways

Podocytes and their foot processes interlinked by slit diaphragms, constitute a continuous outermost layer of the glomerular capillary and seem to be crucial for maintaining the integrity of the glomerular filtration barrier. Purinergic signaling is involved in a wide range of physiological processes in the renal system, including regulating glomerular filtration. We evaluated the role of nucleotide receptors in cultured rat podocytes using non-selective P2 receptor agonists and agonists specific for the P2Y1, P2Y2, and P2Y4 receptors. The results showed that extracellular ATP evokes cAMP-dependent pathways through P2 receptors and influences remodeling of the podocyte cytoskeleton and podocyte permeability to albumin via coupling with RhoA signaling. Our findings highlight the relevance of the P2Y4 receptor in protein kinase A–mediated signal transduction to the actin cytoskeleton. We observed increased cAMP concentration and decreased RhoA activity after treatment with a P2Y4 agonist. Moreover, protein kinase A inhibitors reversed P2Y4-induced changes in RhoA activity and intracellular F-actin staining. P2Y4 stimulation resulted in enhanced AMPK phosphorylation and reduced reactive oxygen species generation. Our findings identify P2Y–PKA–RhoA signaling as the regulatory mechanism of the podocyte contractile apparatus and glomerular filtration. We describe a protection mechanism for the glomerular barrier linked to reduced oxidative stress and reestablished energy balance.

MiR-34a is upregulated in AIP-mutated somatotropinomas and promotes octreotide resistance.

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and have a heavy treatment burden. Growth hormone (GH)-secreting PAs (somatotropinomas) cause acromegaly-gigantism. Genetic forms of somatotropinomas due to germline AIP mutations (AIPmut+) have an early onset and are aggressive and resistant to treatment with somatostatin analogs (SSAs), including octreotide. The molecular underpinnings of these clinical features remain unclear. We investigated the role of miRNA dysregulation in AIPmut+ vs AIPmut- PA samples by array analysis. miR-34a and miR-145 were highly expressed in AIPmut+ vs AIPmut- somatotropinomas. Ectopic expression of AIPmut (p.R271W) in Aip-/- mouse embryonic fibroblasts (MEFs) upregulated miR-34a and miR-145, establishing a causal link between AIPmut and miRNA expression. In PA cells (GH3), miR-34a overexpression promoted proliferation, clonogenicity, migration and suppressed apoptosis, whereas miR-145 moderately affected proliferation and apoptosis. Moreover, high miR-34a expression increased intracellular cAMP, a critical mitogenic factor in PAs. Crucially, high miR-34a expression significantly blunted octreotide-mediated GH inhibition and antiproliferative effects. miR-34a directly targets Gnai2 encoding Gαi2, a G protein subunit inhibiting cAMP production. Accordingly, Gαi2 levels were significantly lower in AIPmut+ vs AIPmut- PA. Taken together, somatotropinomas with AIP mutations overexpress miR-34a, which in turn downregulates Gαi2 expression, increases cAMP concentration and ultimately promotes cell growth. Upregulation of miR-34a also impairs the hormonal and antiproliferative response of PA cells to octreotide. Thus, miR-34a is a novel downstream target of mutant AIP that promotes a cellular phenotype mirroring the aggressive clinical features of AIPmut+ acromegaly.

Underlying molecular mechanism in the modulation of the ram sperm acrosome reaction by progesterone and 17β-estradiol

Steroid hormones progesterone (P4) and 17β-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 μM in the media induced an increase in Ca2+i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H2DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification.