Nanodomain cAMP signalling in cardiac pathophysiology: potential for developing targeted therapeutic interventions.

3', 5'-cyclic adenosine monophosphate (cAMP) mediates the effects of sympathetic stimulation on the rate and strength of cardiac contraction. Beyond this pivotal role, in cardiac myocytes cAMP also orchestrates a diverse array of reactions to various stimuli. To ensure specificity of response, the cAMP signaling pathway is intricately organized into multiple, spatially confined, subcellular domains, each governing a distinct cellular function. In this review, we describe the molecular components of the cAMP signalling pathway, how they organized are inside the intracellular space and how they achieve exquisite regulation of signalling within nanometer-size domains. We delineate the key experimental findings that lead to the current model of compartmentalised cAMP signaling and we offer an overview of our present understanding of how cAMP nanodomains are structured and regulated within cardiac myocytes. Furthermore, we discuss how compartmentalized cAMP signaling is affected in cardiac disease and consider the potential therapeutic opportunities arising from understanding such organization. By exploiting the nuances of compartmentalized cAMP signaling, novel and more effective therapeutic strategies for managing cardiac conditions may emerge. Finally, we highlight the unresolved questions and hurdles that must be addressed to translate these insights into interventions that may benefit patients.

Recruitment of cortical silent responses by forskolin in the anterior cingulate cortex of adult mice.

Recent studies using different experimental approaches demonstrate that silent synapses may exist in the adult cortex including the sensory cortex and anterior cingulate cortex (ACC). The postsynaptic form of long-term potentiation (LTP) in the ACC recruits some of these silent synapses and the activity of calcium-stimulated adenylyl cyclases (ACs) is required for such recruitment. It is unknown if the chemical activation of ACs may recruit silent synapses. In this study, we found that activation of ACs contributed to synaptic potentiation in the ACC of adult mice. Forskolin, a selective activator of ACs, recruited silent responses in the ACC of adult mice. The recruitment was long-lasting. Interestingly, the effect of forskolin was not universal, some silent synapses did not undergo potentiation or recruitment. These findings suggest that these adult cortical synapses are not homogenous. The application of a selective calcium-permeable AMPA receptor inhibitor 1-naphthyl acetyl spermine (NASPM) reversed the potentiation and the recruitment of silent responses, indicating that the AMPA receptor is required. Our results strongly suggest that the AC-dependent postsynaptic AMPA receptor contributes to the recruitment of silent responses at cortical LTP.

Upregulation of phosphodiesterase 7A leads to the downregulation of cAMP-PKA-CREB-BDNF signaling and neuroinflammation in the hippocampus and contributes to concurrent chronic pain and depression in two mouse models.

BACKGROUND: Phosphodiesterases (PDEs) are enzymes that catalyze the hydrolysis of cyclic adenosine monophosphate AMP (cAMP) and/or cyclic guanosine monophosphate (cGMP). PDE inhibitors can mitigate chronic pain and depression when these disorders occur individually; however, there is limited understanding of their role in concurrent chronic pain and depression. We aimed to evaluate the mechanisms of action of PDE using two mouse models of concurrent chronic pain and depression. METHODS: C57BL/6J mice were subjected to partial sciatic nerve ligation (PSNL) to induce chronic neuropathic pain or injected with complete Freund's adjuvant (CFA) to induce inflammatory pain, and both animals showed depression-like behavior. First, we determined the change in PDE expression in both animal models. Next, we determined the effect of PDE7 inhibitor BRL50481 or hippocampal PDE7A knockdown on PSNL- or CFA-induced chronic pain and depression-like behavior. We also investigated the role of cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling and neuroinflammation in the effect of PDE7A inhibition on PSNL- or CFA-induced chronic pain and depression-like behavior. RESULTS: This induction of chronic pain and depression in the two animal models upregulated hippocampal PDE7A. Oral administration of PDE7 inhibitor, BRL50481, or hippocampal PDE7A knockdown significantly reduced mechanical hypersensitivity and depression-like behavior. Hippocampal PDE7 inhibition reversed PSNL- or CFA-induced downregulation of cAMP and BDNF and the phosphorylation of PKA, CREB and p65. cAMP agonist forskolin, reversed these changes and caused milder behavioral symptoms of pain and depression. BRL50481 reversed neuroinflammation in the hippocampus in PSNL mice. CONCLUSIONS: Hippocampal PDE7A mediated concurrent chronic pain and depression in both mouse models by inhibiting cAMP-PKA-CREB-BDNF signaling Inhibiting PDE7A or activating cAMP-PKA-CREB-BDNF signaling are potential strategies to treat concurrent chronic pain and depression.

Cyclic adenosine monophosphate critically modulates cardiac GLP-1 receptor's anti-inflammatory effects.

BACKGROUND: Glucagon-like peptide (GLP)-1 receptor (GLP1R) agonists exert a multitude of beneficial cardiovascular effects beyond control of blood glucose levels and obesity reduction. They also have anti-inflammatory actions through both central and peripheral mechanisms. GLP1R is a G protein-coupled receptor (GPCR), coupling to adenylyl cyclase (AC)-stimulatory Gs proteins to raise cyclic 3`-5`-adenosine monophosphate (cAMP) levels in cells. cAMP exerts various anti-apoptotic and anti-inflammatory effects via its effectors protein kinase A (PKA) and Exchange protein directly activated by cAMP (Epac). However, the precise role and importance of cAMP in mediating GLP1R`s anti-inflammatory actions, at least in the heart, remains to be determined. To this end, we tested the effects of the GLP1R agonist liraglutide on lipopolysaccharide (LPS)-induced acute inflammatory injury in H9c2 cardiac cells, either in the absence of cAMP production (AC inhibition) or upon enhancement of cAMP levels via phosphodiesterase (PDE)-4 inhibition with roflumilast. METHODS & RESULTS: Liraglutide dose-dependently inhibited LPS-induced apoptosis and increased cAMP levels in H9c2 cells, with roflumilast but also PDE8 inhibition further enhancing cAMP production by liraglutide. GLP1R-stimulated cAMP markedly suppressed the LPS-dependent induction of pro-inflammatory tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-6 cytokine expression, of inducible nitric oxide synthase (iNOS) expression and nuclear factor (NF)-kB activity, of matrix metalloproteinases (MMP)-2 and MMP-9 levels and activities, and of myocardial injury markers in H9c2 cardiac cells. The effects of liraglutide were mediated by the GLP1R since they were abolished by the GLP1R antagonist exendin(9-39). Importantly, AC inhibition completely abrogated liraglutide`s suppression of LPS-dependent inflammatory injury, whereas roflumilast significantly enhanced the protective effects of liraglutide against LPS-induced inflammation. Finally, PKA inhibition or Epac1/2 inhibition alone only partially blocked liraglutide`s suppression of LPS-induced inflammation in H9c2 cardiac cells, but, together, PKA and Epac1/2 inhibition fully prevented liraglutide from reducing LPS-dependent inflammation. CONCLUSIONS: cAMP, via activation of both PKA and Epac, is essential for GLP1R`s anti-inflammatory signaling in cardiac cells and that cAMP levels crucially regulate the anti-inflammatory efficacy of GLP1R agonists in the heart. Strategies that elevate cardiac cAMP levels, such as PDE4 inhibition, may potentiate the cardiovascular, including anti-inflammatory, benefits of GLP1R agonist drugs.

The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Cyclic Adenosine Monophosphate Signaling in Chronic Kidney Disease: Molecular Targets and Therapeutic Potentials.

Chronic kidney disease (CKD) is characterized by a steady decline in kidney function and affects roughly 10% of the world's population. This review focuses on the critical function of cyclic adenosine monophosphate (cAMP) signaling in CKD, specifically how it influences both protective and pathogenic processes in the kidney. cAMP, a critical secondary messenger, controls a variety of cellular functions, including transcription, metabolism, mitochondrial homeostasis, cell proliferation, and apoptosis. Its compartmentalization inside cellular microdomains ensures accurate signaling. In kidney physiology, cAMP is required for hormone-regulated activities, particularly in the collecting duct, where it promotes water reabsorption through vasopressin signaling. Several illnesses, including Fabry disease, renal cell carcinoma, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, diabetic nephropathy, autosomal dominant polycystic kidney disease, and renal tubular acidosis, have been linked to dysfunction in the cAMP system. Both cAMP analogs and phosphodiesterase inhibitors have the potential to improve kidney function and reduce kidney damage. Future research should focus on developing targeted PDE inhibitors for the treatment of CKD.

Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases.

Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography-tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents.

The Effect of Bacterial AHL on the Cyclic Adenosine Monophosphate Content in Plants According to High-Performance Liquid Chromatography.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in cells, mediating various stimulation signals such as the growth and development of organisms and stress and participating in regulating various biological processes of cells. This article explores the quantitative determination of cAMP in plants using High-Performance Liquid Chromatography (HPLC) and applies this method to analyzing the changes in cAMP content during the process of plant response to the bacterial quorum sensing signal N-acyl homoserine lactone (AHL). Research has shown that the optimal detection conditions for HPLC are as follows: the chromatographic column is Venusil MP C18 (2), the mobile phase is methanol-water (0.1% trifluoroacetic acid) (v:v, 10:90), the detection wavelength is 259 nm, the column temperature is 35 °C, and the flow rate is 0.8 mL/min. The precision of the standard sample of this method is 98.21%, the precision of the sample is 98.87%, and the recovery rate is 101.067%. The optimal extraction conditions for cAMP in Arabidopsis are to use 15% methanol ultrasonic extraction for 10 min, followed by a 40 °C water bath for 4 h. Bacterial AHL signal processing can significantly stimulate an increase in cAMP levels in Arabidopsis leaves and roots. The establishment of HPLC detection methods for the cAMP content in plants is of great significance for in-depth research on the signal transduction mechanisms of plant-bacterial interactions.

Quantitative phosphoproteomics reveals novel roles of cAMP in plants.

3',5'-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3',5'-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular "sponge" that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Tale of cAMP as a second messenger in auxin signaling and beyond.

The 3',5'-cyclic adenosine monophosphate (cAMP) is a versatile second messenger in many mammalian signaling pathways. However, its role in plants remains not well-recognized. Recent discovery of adenylate cyclase (AC) activity for transport inhibitor response 1/auxin-signaling F-box proteins (TIR1/AFB) auxin receptors and the demonstration of its importance for canonical auxin signaling put plant cAMP research back into spotlight. This insight briefly summarizes the well-established cAMP signaling pathways in mammalian cells and describes the turbulent and controversial history of plant cAMP research highlighting the major progress and the unresolved points. We also briefly review the current paradigm of auxin signaling to provide a background for the discussion on the AC activity of TIR1/AFB auxin receptors and its potential role in transcriptional auxin signaling as well as impact of these discoveries on plant cAMP research in general.

Chronic psychological stress aggravates psoriasis-like skin inflammation via overactivation of ß2-adrenoceptor and nuclear factor kappa B pathways.

The relationship between psoriasis severity and psychological stress has been described in several studies. However, the mechanism by which chronic stress exacerbates psoriasis is not completely understood. This study aimed at investigating whether chronic psychological stress can aggravate psoriasis-like skin inflammation. Mice were subjected to a restraint stress model and topically treated with imiquimod (IMQ). Differentiated human keratinocytes were treated with high epinephrine levels and IMQ in vitro. Stress aggravated macroscopic features and the increase in epidermal thickness induced by IMQ in mouse skin. The increase in NF-κB and IL-17A expression induced by IMQ was potentiated by chronic stress in mouse skin. The skin of stressed mice treated with IMQ showed higher levels of ß2-adrenergic receptors (ß2-AR). In human keratinocytes, high epinephrine levels exacerbated the increase in the levels of ß2-AR and IL-17A induced by IMQ. ß-AR antagonist reversed the effects of chronic stress in IMQ-induced inflammation both in vivo and in vitro. In conclusion, stress-stimulated overactivation of the ß2-AR and NF-κB pathways potentiates a Th1/Th17 profile leading to an exacerbation of psoriasis.

Novel Phenylethanoid Glycosides Improve Hippocampal Synaptic Plasticity via the Cyclic Adenosine Monophosphate-CREB-Brain-Derived Neurotrophic Growth Factor Pathway in APP/PS1 Transgenic Mice.

INTRODUCTION: Alzheimer's disease (AD) is a major public health concern worldwide, but there are still no drugs available that treat it effectively. Previous studies have shown that phenylethanoid glycosides have pharmacological effects, which include anti-AD properties, but the underlying mechanisms by which they ameliorate AD symptoms remain unknown. METHODS: In this study, we used an APP/PS1 AD mouse model to explore the function and mechanisms underlying savatiside A (SA) and torenoside B (TB) in the treatment of AD. SA or TB (100 mg·kg-1·d-1) was orally administered to 7-month-old APP/PS1 mice for 4 weeks. Cognitive and memory functions were measured using behavioral experiments (including the Morris water maze test and the Y-maze spontaneous alternation test). Molecular biology experiments (including Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays) were used to detect any corresponding changes in signaling pathways. RESULTS: The results showed that SA or TB treatment could significantly reduce cognitive impairment in APP/PS1 mice. We also showed that chronic treatment with SA/TB could prevent spine loss, synaptophysin immunoreactivity, and neuronal loss in mice, thereby improving synaptic plasticity and moderating learning and memory deficits. SA/TB administration also promoted the expression of synaptic proteins in APP/PS1 mouse brains and upregulated phosphorylation of proteins in the cyclic adenosine monophosphate (cAMP)/CREB/brain-derived neurotrophic growth factor (BDNF) pathway that are responsible for synaptic plasticity. Additionally, chronic SA/TB treatment increased the levels of BDNF and nerve growth factor (NGF) in the brains of APP/PS1 mice. Both astrocyte and microglia volumes, as well as the generation of amyloid ß, were also decreased in SA/TB-treated APP/PS1 mice compared to control APP/PS1 mice. CONCLUSION: In summary, SA/TB treatment was associated with activation of the cAMP/CREB/BDNF pathway and increased BDNF and NGF expression, indicating that SA/TB improves cognitive functioning via nerve regeneration. SA/TB is a promising candidate drug for the treatment of AD.

Increased expression of glucagon-like peptide-1 and cystic fibrosis transmembrane conductance regulator in the ileum and colon in mouse treated with metformin.

Metformin, an oral medication, is prescribed to patients with type 2 diabetes mellitus. Although the efficacy, safety, and low economic burden of metformin on patients have long been recognized, approximately 5% of the patients treated with this drug develop severe diarrhea and discontinue the treatment. We previously reported that 1,000 mg·kg-1·day-1 of metformin induced diarrhea in diabetic obese (db/db) mice and wood creosote (traditional medication for diarrhea) ameliorated the symptoms. In this study, we attempted to elucidate the molecular mechanisms by which metformin induces diarrhea. Cystic fibrosis transmembrane conductance regulator (CFTR) is a key ion (chloride) channel in cyclic adenosine monophosphate (cAMP)-induced diarrhea. Metformin treatment increased bile flow (bile acids and bilirubin) in the ileum of mice. In addition, the treatment was accompanied by an increase in mRNA and protein levels of CFTR in the mucosa of the ileum and colon in both wild-type (C57BL/6J) and db/db mice. Glucagon-like peptide-1 (GLP-1), as well as cholic acid, induces CFTR mRNA expression in human colon carcinoma Caco-2 cells through cAMP signaling. Although wood creosote (10 mg/kg) ameliorated diarrhea symptoms, it did not alter the mRNA levels of Glp-1 or Cftr. Similar to overeating, metformin upregulated GLP-1 and CFTR expression, which may have contributed to diarrhea symptoms in mice. Although we could not identify db/db mouse-specific factors associated with metformin-induced diarrhea, these factors may modulate colon function. Wood creosote may not interact with these factors but ameliorates diarrhea symptoms.

Molecular mechanisms mediate roflumilast protective effect against isoprenaline-induced myocardial injury.

BACKGROUND: Myocardial necrosis is one of the most common cardiac and pathological diseases. Unfortunately, using the available medical treatment is not sufficient to rescue the myocardium. So that, we aimed in our model to study the possible cardioprotective effect of roflumilast (ROF) in an experimental model of induced myocardial injury using a toxic dose of isoprenaline (ISO) and detecting the role of vascular endothelial growth factor/endothelial nitric oxide synthase (VEGF/eNOS) and cyclic guanosine monophosphate/cyclic adenosine monophosphate/ sirtuin1 (cGMP/cAMP/SIRT1) signaling cascade. MATERIALS AND METHODS: Animals were divided into five groups; control, ISO given group (150 mg/kg) i.p. on the 4th and 5th day, 3 ROF co-administered groups in different doses (0.25, 0.5, 1 mg/kg/day) for 5 days. RESULTS: Our data revealed that ISO could induce cardiac toxicity as manifested by significant increases in troponin I, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), malondialdehyde (MDA), tumor necrosis factor alpha (TNFα), and cleaved caspase-3 with toxic histopathological changes. Meanwhile, there were significant decreases in reduced glutathione (GSH), total antioxidant capacity (TAC), VEGF, eNOS, cGMP, cAMP and SIRT1. However, co-administration of ROF showed significant improvement and normalization of ISO induced cardiac damage. CONCLUSION: We concluded that ROF successfully reduced ISO induced myocardial injury and this could be attributed to modulation of PDE4, VEGF/eNOS and cGMP/cAMP/SIRT1 signaling pathways with antioxidant, anti-inflammatory, and anti-apoptotic properties.

Xenon's Sedative Effect Is Mediated by Interaction with the Cyclic Nucleotide-Binding Domain (CNBD) of HCN2 Channels Expressed by Thalamocortical Neurons of the Ventrobasal Nucleus in Mice.

Previous studies have shown that xenon reduces hyperpolarization-activated cyclic nucleotide-gated channels type-2 (HCN2) channel-mediated current (Ih) amplitude and shifts the half-maximal activation voltage (V1/2) in thalamocortical circuits of acute brain slices to more hyperpolarized potentials. HCN2 channels are dually gated by the membrane voltage and via cyclic nucleotides binding to the cyclic nucleotide-binding domain (CNBD) on the channel. In this study, we hypothesize that xenon interferes with the HCN2 CNBD to mediate its effect. Using the transgenic mice model HCN2EA, in which the binding of cAMP to HCN2 was abolished by two amino acid mutations (R591E, T592A), we performed ex-vivo patch-clamp recordings and in-vivo open-field test to prove this hypothesis. Our data showed that xenon (1.9 mM) application to brain slices shifts the V1/2 of Ih to more hyperpolarized potentials in wild-type thalamocortical neurons (TC) (V1/2: -97.09 [-99.56--95.04] mV compared to control -85.67 [-94.47--82.10] mV; p = 0.0005). These effects were abolished in HCN2EA neurons (TC), whereby the V1/2 reached only -92.56 [-93.16- -89.68] mV with xenon compared to -90.03 [-98.99--84.59] mV in the control (p = 0.84). After application of a xenon mixture (70% xenon, 30% O2), wild-type mice activity in the open-field test decreased to 5 [2-10] while in HCN2EA mice it remained at 30 [15-42]%, (p = 0.0006). In conclusion, we show that xenon impairs HCN2 channel function by interfering with the HCN2 CNBD site and provide in-vivo evidence that this mechanism contributes to xenon-mediated hypnotic properties.

Drug repurposing for targeting cyclic nucleotide transporters in acute leukemias - A missed opportunity.

While current treatment regimens for acute leukemia can dramatically improve patient survival, there remains room for improvement. Due to its roles in cell differentiation, cell survival, and apoptotic signaling, modulation of the cyclic AMP (cAMP) pathway has provided a meaningful target in hematological malignancies. Several studies have demonstrated that gene expression profiles associated with increased pro-survival cAMP activity or downregulation of various pro-apoptotic factors associated with the cAMP pathway are apparent in acute leukemia patients. Previous work to increase leukemia cell intracellular cAMP focused on the use of cAMP analogs, stimulating cAMP production via transmembrane-associated adenylyl cyclases, or decreasing cAMP degradation by inhibiting phosphodiesterase activity. However, targeting cyclic nucleotide efflux by ATP-binding cassette (ABC) transporters represents an unexplored approach for modulation of intracellular cyclic nucleotide levels. Preliminary studies have shown that inhibition of cAMP efflux can stimulate leukemia cell differentiation, cell growth arrest, and apoptosis, indicating that targeting cAMP efflux may show promise for future therapeutic development. Furthermore, inhibition of cyclic nucleotide transporter activity may also contribute multiple anticancer benefits by reducing extracellular pro-survival signaling in malignant cells. Hence, several opportunities for drug repurposing may exist for targeting cyclic nucleotide transporters.

Regulator of G Protein Signaling Contributes to the Development and Aflatoxin Biosynthesis in Aspergillus flavus through the Regulation of Gα Activity.

Heterotrimeric G-proteins play crucial roles in growth, asexual development, and pathogenicity of fungi. The regulator of G-protein signaling (RGS) proteins function as negative regulators of the G proteins to control the activities of GTPase in Gα subunits. In this study, we functionally characterized the six RGS proteins (i.e., RgsA, RgsB, RgsC, RgsD, RgsE, and FlbA) in the pathogenic fungus Aspergillus flavus. All the aforementioned RGS proteins were also found to be functionally different in conidiation, aflatoxin (AF) biosynthesis, and pathogenicity in A. flavus. Apart from FlbA, all other RGS proteins play a negative role in regulating both the synthesis of cyclic AMP (cAMP) and the activation of protein kinase A (PKA). Additionally, we also found that although RgsA and RgsE play a negative role in regulating the FadA-cAMP/PKA pathway, they function distinctly in aflatoxin biosynthesis. Similarly, RgsC is important for aflatoxin biosynthesis by negatively regulating the GanA-cAMP/PKA pathway. PkaA, which is the cAMP-dependent protein kinase catalytic subunit, also showed crucial influences on A. flavus phenotypes. Overall, our results demonstrated that RGS proteins play multiple roles in the development, pathogenicity, and AF biosynthesis in A. flavus through the regulation of Gα subunits and cAMP-PKA signals. IMPORTANCE RGS proteins, as crucial regulators of the G protein signaling pathway, are widely distributed in fungi, while little is known about their roles in Aspergillus flavus development and aflatoxin. In this study, we identified six RGS proteins in A. flavus and revealed that these proteins have important functions in the regulation of conidia, sclerotia, and aflatoxin formation. Our findings provide evidence that the RGS proteins function upstream of cAMP-PKA signaling by interacting with the Gα subunits (GanA and FadA). This study provides valuable information for controlling the contamination of A. flavus and mycotoxins produced by this fungus in pre- and postharvest of agricultural crops.

Selective reduction of neurotransmitter release by cAMP-dependent pathways in mouse detrusor.

Parasympathetic nerve-mediated contractions of detrusor smooth muscle are generated by ATP and acetylcholine (ACh) release from efferent nerve terminals. In humans, ACh is responsible for detrusor contractions in normal human bladders, whereas ATP has an additional role in overactive bladder pathologies. The ATP metabolite, adenosine, relaxes nerve-mediated contractions, with a potential action via presynaptic adenosine A1 receptor activation and subsequent suppression of neuronal ATP release. We investigated the effect of A1 receptor activation and downstream cAMP-dependent pathways on nerve-mediated ATP and ACh release, and detrusor contraction in mouse detrusor. Bladders from male C57BL/6 mice (12 wk) were used for in vitro experiments. Upon electrical field stimulation of intact preparations (detrusor and mucosal layers), ATP or ACh release was measured simultaneously with tension recordings. Activation of A1 receptors by adenosine or exogenous agonists reduced the lower frequency component of nerve-mediated contractions and neuronal ATP release. The A1 receptor antagonist abolished these effects. A1 receptor activation inhibits adenylyl cyclase (AC) activity and cAMP generation. The effect of A1 receptor activation was mimicked by a PKA antagonist but not by modulators of exchange proteins activated by cAMP, demonstrating that modulation of nerve-mediated ATP release is via PKA. Adenosine had no effect on ACh release or the higher frequency component of nerve-mediated contractions. Differential regulation of neurotransmitter release is possible at the detrusor nerve-muscle junction, as demonstrated by A1 receptor activation, and downstream inhibition of AC, cAMP generation, and PKA. The ability to specifically attenuate ATP release offers a potential to target purinergic motor pathways enhanced in overactive bladder pathologies.

GLP1R inhibits the progression of endometrial carcinoma through activation of cAMP/PKA pathway.

BACKGROUND: This study strived to explore the role and mechanism of glucagon-like peptide-1 receptor (GLP1R) in endometrial carcinoma (EC). METHODS: In detail, after transfection of GLP1R overexpression vector and small interfering RNA targeting PKA, the mRNA expressions of GLP1R and PKA in EC cells (Ishikawa and RL95-2) were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The cell biological behaviors, including proliferation, migration, invasion, and apoptosis, were detected using 5-ethynyl-2'-deoxyuridine (EdU), wound healing, transwell, and flow cytometry assays, respectively. The cyclic adenosine monophosphate (cAMP) content and related protein expressions (GLP1R, p-PKA, and PKA) were determined by enzyme-linked immunosorbent assay (ELISA) and western blot. The effects of GLP1R and PKA on tumorigenesis were evaluated by measuring the tumor volume and weight of mice bearing EC. RESULT: According to the results, GLP1R expression was downregulated in EC tissues and cells, and there was a positive correlation between GLP1R and PKA expressions. Upregulation of GLP1R promoted apoptosis and activated the cAMP/PKA signaling pathway in EC cells, while hindering the EC cell proliferation, invasion, migration, and the growth of tumor in mice. However, these effects were blunted by downregulation of PKA, which also accelerated the progression of EC in vitro and in vivo via inhibiting the activation of cAMP/PKA signaling pathway. CONCLUSION: Collectively, upregulation of GLP1R impeded EC progression via inducing the activation of cAMP/PKA signaling pathway, which may be a potential treatment for EC.