Elevation Of Intracellular cAMP Levels Research Articles

The impact of GLP-1 signalling on the energy metabolism of pancreatic islet β-cells and extrapancreatic tissues

Glucagon-like peptide-1 signalling impacts glucose homeostasis and appetite thereby indirectly affecting substrate availability at the whole-body level. The incretin canonically produces an insulinotropic effect, thereby lowering blood glucose levels by promoting the uptake and inhibiting the production of the sugar by peripheral tissues. Likewise, GLP-1 signalling within the central nervous system reduces the appetite and food intake, whereas its gastric effect delays the absorption of nutrients, thus improving glycaemic control and reducing the risk of postprandial hyperglycaemia. We review the molecular aspects of the GLP-1 signalling, focusing on its impact on intracellular energy metabolism. Whilst the incretin exerts its effects predominantly via a Gs receptor, which decodes the incretin signal into the elevation of intracellular cAMP levels, the downstream signalling cascades within the cell, acting on fast and slow timescales, resulting in an enhancement or an attenuation of glucose catabolism, respectively.

Dexamethasone attenuates neuropathic pain through spinal microglial expression of dynorphin A via the cAMP/PKA/p38 MAPK/CREB signaling pathway

This study aimed to elucidate the opioid mechanisms underlying dexamethasone-induced pain antihypersensitive effects in neuropathic rats. Dexamethasone (subcutaneous and intrathecal) and membrane-impermeable Dex-BSA (intrathecal) administration dose-dependently inhibited mechanical allodynia and thermal hyperalgesia in neuropathic rats. Dexamethasone and Dex-BSA treatments increased expression of dynorphin A in the spinal cords and primary cultured microglia. Dexamethasone specifically enhanced dynorphin A expression in microglia but not astrocytes or neurons. Intrathecal injection of the microglial metabolic inhibitor minocycline blocked dexamethasone-stimulated spinal dynorphin A expression; intrathecal minocycline, the glucocorticoid receptor antagonist Dex-21-mesylate, dynorphin A antiserum, and κ-opioid receptor antagonist GNTI completely blocked dexamethasone-induced mechanical antiallodynia and thermal antihyperalgesia. Additionally, dexamethasone elevated spinal intracellular cAMP levels, leading to enhanced phosphorylation of PKA, p38 MAPK and CREB. The specific adenylate cyclase inhibitor DDA, PKA inhibitor H89, p38 MAPK inhibitor SB203580 and CREB inhibitor KG-501 completely blocked dexamethasone-induced anti-neuropathic pain and increased microglial dynorphin A exprression. In conclusion, this study reveal that dexamethasone mitigateds neuropathic pain through upregulation of dynorphin A in spinal microglia, likely involving the membrane glucocorticoid receptor/cAMP/PKA/p38 MAPK/CREB signaling pathway.

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.

Lovastatin impairs cellular proliferation and enhances hyaluronic acid production in fibroblast-like synoviocytes

IntroductionStatins have demonstrated chondroprotective effects by reducing inflammation and mitigating extracellular matrix degradation. However, statins are also reported to be cytotoxic to several types of cells. Early-onset osteoarthritis (OA) is characterized by synovial inflammation, which adversely affects hyaluronan (HA) production in fibroblast-like synoviocytes (FLSs). Nevertheless, the precise effects of statins on the synovium remain unclear. MethodsThis study investigated the impact of lovastatin on human FLSs, and HA secretion-related genes, signaling pathways, and production were evaluated. ResultsThe findings revealed that high doses of lovastatin (20 or 40 μM) decreased FLS viability and increased cell death. FLS proliferation ceased when cultured in a medium containing 5 or 10 μM lovastatin. mRNA expression analysis demonstrated that lovastatin (5 and 10 μM) upregulated the gene level of hyaluronan synthase 1 (HAS1), HAS2, and proteoglycan 4 (PRG4), but not HAS3. While the expression of multidrug resistance-associated protein 5 transporter gene remained unaffected, both inward-rectifying potassium channel and acid-sensing ion channel 3 were upregulated. Western blot further confirmed that lovastatin increased the production of HAS1 and PRG4, and activated the PKC-α, ERK1/2, and p38-MAPK signaling pathways. Additionally, lovastatin elevated intracellular cAMP levels and HA production in FLSs. ConclusionLovastatin impairs cellular proliferation but enhances HA production in human FLSs.

Divergent Pharmacology and Biased Signaling of the Four Melanocortin-4 Receptor Isoforms in Rainbow Trout (Oncorhynchus mykiss).

The melanocortin-4 receptor (MC4R) is essential for the modulation of energy balance and reproduction in both fish and mammals. Rainbow trout (Oncorhynchus mykiss) has been extensively studied in various fields and provides a unique opportunity to investigate divergent physiological roles of paralogues. Herein we identified four trout mc4r (mc4ra1, mc4ra2, mc4rb1, and mc4rb2) genes. Four trout Mc4rs (omMc4rs) were homologous to those of teleost and mammalian MC4Rs. Multiple sequence alignments, a phylogenetic tree, chromosomal synteny analyses, and pharmacological studies showed that trout mc4r genes may have undergone different evolutionary processes. All four trout Mc4rs bound to two peptide agonists and elevated intracellular cAMP levels dose-dependently. High basal cAMP levels were observed at two omMc4rs, which were decreased by Agouti-related peptide. Only omMc4rb2 was constitutively active in the ERK1/2 signaling pathway. Ipsen 5i, ML00253764, and MCL0020 were biased allosteric modulators of omMc4rb1 with selective activation upon ERK1/2 signaling. ML00253764 behaved as an allosteric agonist in Gs-cAMP signaling of omMc4rb2. This study will lay the foundation for future physiological studies of various mc4r paralogs and reveal the evolution of MC4R in vertebrates.

Developmental expression of CREB1 and NFATC2 in pig embryos.

The CREB1 gene encodes the cAMP response element binding protein 1 (CREB1), a leucine zipper transcription factor that regulates cellular gene expression in response to elevated levels of intracellular cAMP. When activated by phosphorylation, CREB1 binds to the cAMP response element (CRE) of the promoters of its target genes. CREB1 is an essential component in many physiological processes, and its function is correlated to neurodevelopment, plasticity and cell survival, and learning and memory. The NFATC2 gene codes for the nuclear factor of activated T-cells 2 protein. The NFATC2 protein is a DNA-binding protein that functions as an inducer of gene transcription during immune response. The aim of the present study was to examine the developmental expression of porcine CREB1 and NFACT2 transcripts. The expression of CREB1 and NFACT2 mRNA was examined by quantitative real-time RT-PCR. For the CREB1 transcript, we found significant reduction in transcript levels in the brain stem and basal ganglia during porcine embryo development, determined from day 60 to day 115 of gestation. In contrast, a significant increase in CREB1 mRNA was detected in the lungs during embryo development. No significant changes in the NFATC2 transcript were detected in porcine brain tissue during embryo development. Differential CREB1 mRNA expression was found in pig brain tissues during embryo development.

Polyoxidovanadates [MoVIVV9O28]5- and [H2PtIVVV9O28]5- interact with CHO cell plasma membrane lipids causing aggregation and activation of a G protein-coupled receptor

Mono substituted heteropolyoxidovanadates, when compared to effects of a corresponding isopolyoxidovanadate (POV), were found to be more effective initiators of signal transduction by a G protein-coupled receptor (GPCR), specifically the luteinizing hormone receptor (LHR). Here we report that LHRs signal productively when CHO cells expressing the receptor are treated with two heteropolyoxidovanadates PtIV in monoplatino(IV)nonavanadate(V) ([H2PtVIVV9O28]5-, V9Pt), and MoIV in monomolybdo(VI)nonavanadate(V) (Mo[VIVV9O28]5-, V9Mo). Both substituted decavanadate derivatives were more effective than decavanadate which is more charged, has greater stability and forms the [V10O28]6- anion (V10) in cell culture medium at pH 7.4. For viable CHO cells expressing 10 k or 32 k LHR/cell and treated with 11 μM V9Pt and 13 μM V9Mo, mono substituted heteropolyoxidovanadates significantly decreased the packing of plasma membrane lipids for about 1 h. This brief change in membrane structure was accompanied by increased aggregation of LHR and cell signaling as indicated by increased intracellular levels of cAMP. More pronounced changes in lipid packing and LHR signaling were associated with short acting heteropolyoxidovanadates than with the more stable V10. When LHR was overexpressed, V9Pt and V9Mo had little or no effect on membrane lipid packing or receptor aggregation and the LHR was constitutively activated as indicated by elevated intracellular cAMP levels. Speciation of V9Pt and V9Mo in H2O and cell medium was monitored using 51V NMR spectroscopy and confirmed that V9Pt and V9Mo had greater effects on CHO cells despite decomposing more rapidly in the cell growth medium. Thus, under conditions that promote CHO cell growth, V9Pt and V9Mo, despite their smaller molecular charge and their reduced stability, favor LHR signaling over that induced by V10. Importantly, under the same experimental conditions, CHO cells treated with V9Pt and V9Mo do not exhibit as strong toxic effects observed for cells treated with the longer lived V10. In summary, unlike the longer lived V10 which is more growth inhibitory to cells, monosubstituted heteropolyoxidovanadates are more effective in transiently initiating signaling by a G protein-coupled receptor but, because of rapid hydrolysis, inhibit cell growth less.

Group 3 innate lymphocytes (ILC3s) upregulate IL-22 in response to elevated intracellular cAMP levels

Group 3 innate lymphocytes (ILC3s) are important immune cells within mucosal tissues and protect against bacterial infections. They can be activated in response to the innate cytokines IL-23 or IL-1β, which rapidly increases their production of effector molecules that regulate barrier functions. Pathogens can subvert these anti-bacterial effects to evade mucosal defenses to infect the host. Bacillus anthracis, the causative agent of anthrax, produces two major toxins that can modulate the immune response. We have previously shown that lethal toxin downmodulates the function of ILC3s. On the other hand, edema toxin has been shown promote T helper 17 (Th17) cell differentiation, adaptive counterparts of ILC3s, via elevation of cyclic adenosine monophosphate (cAMP). We hypothesized that edema toxin may also modulate ILC3 function. In this study, we show that edema toxin has the opposite effect of lethal toxin; edema toxin directly activates ILC3s independently of innate cytokine stimulation. Treatment of a mouse ILC3-like cell line with edema toxin, a potent adenylate cyclase, upregulated production of the cytokine IL-22, a major effector molecule of ILC3s and a critical factor in maintaining mucosal barriers. Forskolin treatment phenocopied the effect observed with edema toxin and led to an increase in CREB phosphorylation in ILC3s. This observation has potential implications for a role for cAMP signaling in the activation of ILC3s.

Endoplasmic Reticulum Stress Contributed to Dipyridamole-Induced Impaired Autophagic Flux and Glioma Apoptosis.

Elevation of intracellular cAMP levels has been implicated in glioma cell proliferation inhibition, differentiation, and apoptosis. Inhibition of phosphodiesterase is a way to elevate intracellular cAMP levels. The present study aimed to investigate the anti-glioma potential of dipyridamole, an inhibitor of phosphodiesterase. Upon treatment with dipyridamole, human U87 glioma cells decreased cell viability, clonogenic colonization, migration, and invasion, along with Noxa upregulation, Endoplasmic Reticulum (ER) stress, impaired autophagic flux, Yes-associated Protein 1 (YAP1) phosphorylation, and YAP1 reduction. Pharmacological and genetic studies revealed the ability of dipyridamole to initiate Noxa-guided apoptosis through ER stress. Additionally, the current study further identified the biochemical role of YAP1 in communicating with ER stress and autophagy under situations of dipyridamole treatment. YAP1 promoted autophagy and protected glioma cells from dipyridamole-induced apoptotic cell death. Dipyridamole impaired autophagic flux and rendered glioma cells more vulnerable to apoptotic cell death through ER stress-inhibitable YAP1/autophagy axis. The overall cellular changes caused by dipyridamole appeared to ensure a successful completion of apoptosis. Dipyridamole also duplicated the biochemical changes and apoptosis in glioma T98G cells. Since dipyridamole has additional biochemical and pharmacological properties, further research centered on the anti-glioma mechanisms of dipyridamole is still needed.

Caffeine-induced inversion of prostaglandin E2 effects on hepatic stellate cell activation

Background and aimsLiver inflammation leads to the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. The present study aimed to investigate the effects of prostaglandin E2 (PGE2), which is biosynthesized by Kupffer cells, hepatocytes, and HSCs during inflammation, on HSC activation, including its combinatory effect with caffeine. MethodsHSCs isolated from mice were activated by culturing in a medium supplemented with 10% fetal bovine serum for 7 days on plastic plates. The activation of HSCs was evaluated by immunofluorescence of α-smooth muscle actin in HSCs. Comprehensive gene expression analysis was performed using mRNA-sequencing to compare HSCs cultured for 1 or 7 days, with or without PGE2, caffeine, or both. ResultsPGE2 (1 μM) facilitated the activation of HSCs but inhibited the HSC activation in the presence of caffeine (3 mM). Comprehensive gene expression analysis revealed that HSCs treated with PGE2 in the presence of caffeine were classified in the same class as HSCs cultured for 1 day, i.e., quiescent HSCs. In contrast, PGE2 did not exhibit an inhibitory effect on HSC activation when co-treated with any isoform-specific phosphodiesterase inhibitors. Although the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine suppressed the elevation of intracellular cAMP level induced by PGE2 in the presence of caffeine, it had no effect on the inhibition of HSC activation by PGE2 plus caffeine. ConclusionThe effect of PGE2 on HSC activation is changed from facilitatory to inhibitory when combined with caffeine, suggesting that caffeine may effectively suppress liver fibrosis during inflammation.

Regulation of Brown Adipose Tissue and Beige Fat by the Adenosine A2B Receptor 8.5.5

Pandemic obesity and its associated co-morbidities like type 2 diabetes, coronary heart disease or stroke is one of the biggest contemporary health problems and there is an urgent need for novel pharmacological treatment. Brown adipose tissue (BAT) is specialized in dissipating energy in the form of heat by uncoupling ATP synthesis due to its unique uncoupling protein 1 (UCP1). BAT abundance is correlated with leanness in humans and, thereby, BAT is a pharmaceutical target for an anti-obesity therapy. Adenosine is a novel alternative activator of human and brown adipocytes. The enigmatic Gs-protein-coupled adenosine A2B receptor is abundantly expressed in murine BAT. Here, we analyzed the role of the A2B receptor in BAT function in a model of diet-induced obesity. Adipose tissue-specific ablation of A2B reduced BAT energy expenditure (EE) in mice, whereas specific pharmacological stimulation of A2B ameliorated obesity concomitant with improved glucose homeostasis. Additionally, A2B activation promoted browning of inguinal WAT together with a reduced pro-inflammatory phenotype. In humans, high A2B expression correlated with high BAT activity and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment of primary human brown adipocytes resulted in elevated intracellular cAMP levels and increased oxygen consumption, whereas A2B stimulation of primary human white adipocytes promoted upregulation of thermogenic genes. Taken together, our results propose the adenosine A2B receptor as novel target with great potential to increase EE from BAT/brown adipocytes along with promoting browning of white fat. 8.5.5

A most versatile kinase: The catalytic subunit of PKA in T-cell biology.

The cAMP-dependent protein kinase, more commonly referred to as protein kinase A (PKA), is one of the most-studied enzymes in biology. PKA is ubiquitously expressed in mammalian cells, can be activated in response to a plethora of biological stimuli, and phosphorylates more than 250 known substrates. Indeed, PKA is of central importance to a wide range of organismal processes, including energy homeostasis, memory formation and immunity. It serves as the primary effector of the second-messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP), which is believed to have mostly inhibitory effects on the adaptive immune response. In particular, elevated levels of intracellular cAMP inhibit the activation of conventional T cells by limiting signal transduction through the T-cell receptor and altering gene expression, primarily in a PKA-dependent manner. Regulatory T cells have been shown to increase the cAMP levels in adjacent T cells by direct and indirect means, but the role of cAMP within regulatory T cells themselves remains incompletely understood. Paradoxically, cAMP has been implicated in promoting T-cell activation as well, adding another functional dimension beyond its established immunosuppressive effects. Furthermore, PKA can phosphorylate the NF-κB subunit p65, a transcription factor that is essential for T-cell activation, independently of cAMP. This phosphorylation of p65 drastically enhances NF-κB-dependent transcription and thus is likely to facilitate immune activation. How these immunosuppressive and immune-activating properties of PKA balance in vivo remains to be elucidated. This review provides a brief overview of PKA regulation, its ability to affect NF-κB activation, and its diverse functions in T-cell biology.

Epac1 (Exchange Protein Directly Activated by cAMP 1) Upregulates LOX-1 (Oxidized Low-Density Lipoprotein Receptor 1) to Promote Foam Cell Formation and Atherosclerosis Development

The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1's function in atherosclerosis development, a triple knockout mouse model (LTe) was generated by crossing Epac1-/- mice with atherosclerosis-prone LDb mice lacking both Ldlr and Apobec1. Deletion of Epac1 led to a significant reduction of atherosclerotic lesion formation as measured by postmortem staining, accompanied by attenuated macrophage/foam cell infiltrations within atherosclerotic plaques as determined by immunofluorescence staining in LTe animals compared with LDb littermates. Primary bone marrow-derived macrophages were isolated from Epac1-null and wild-type mice to investigate the role of Epac1 in lipid uptake and foam cell formation. ox-LDLs (oxidized low-density lipoproteins) stimulation of bone marrow-derived macrophages led to elevated intracellular cAMP and Epac1 levels, whereas an Epac-specific agonist, increased lipid accumulation in wild-type, but not Epac1-null, bone marrow-derived macrophages. Mechanistically, Epac1 acts through PKC (protein kinase C) to upregulate LOX-1 (ox-LDL receptor 1), a major scavenger receptor for ox-LDL uptake, exerting a feedforward mechanism with ox-LDL to increase lipid uptake and propel foam cell formation and atherogenesis. Our study demonstrates a fundamental role of cAMP/Epac1 signaling in vascular remodeling by promoting ox-LDL uptake and foam cell formation during atherosclerosis lesion development. Therefore, Epac1 represents a promising, unexplored therapeutic target for atherosclerosis.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-β Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832—a pan-PDE inhibitor, 869—a TRPA1 modulator, and 145—a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type β1 (TGF-β1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-β pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

Differential modulation of endothelial cell function by fresh frozen plasma

AimsIn vivo studies suggest a positive influence of fresh frozen plasma (FFP) on endothelial properties and vascular barrier function, leading to improved outcomes in animal sepsis models as well as in major abdominal surgery. However, those effects are incompletely described. It was our aim to evaluate in vitro effects of FFP on endothelial key functions and to identify underlying mechanisms. Materials and methodsHuman pulmonary microvascular endothelial cells (HPMECs) were prestimulated with LPS, followed by incubation with FFP. Permeability for FITC-dextran was assessed, and intercellular gap formation was visualized. NF-κB nuclear translocation and expression of pro-inflammatory, pro-adhesion, and leakage-related genes were evaluated, and monocyte adhesion to ECs was assessed. Intracellular cAMP levels as well as phosphorylation of functional proteins were analyzed. In patients undergoing major abdominal surgery, Syndecan-1 serum levels were assessed prior to and following FFP transfusion. Key findingsPost-incubation of HPMVECs with FFP increased intracellular cAMP levels that had been decreased by preceding LPS stimulation. On one hand, this reduced endotoxin-mediated upregulation of IL-8, ICAM-1, VCAM-1, VEGF, and ANG-2. Impaired phosphorylation of functional proteins was restored, and intercellular cohesion and barrier function were rescued. On the other hand, NF-κB nuclear translocation as well as monocyte adhesion was markedly increased by the combination of LPS and FFP. Syndecan-1 serum levels were lower in surgery patients that were transfused with FFP compared to those that were not. SignificanceOur data provide evidence for a differential modulation of crucial endothelial properties by FFP, potentially mediated by elevation of intracellular cAMP levels.

Effect of Roflumilast in airways disorders via dual inhibition of phosphodiesterase and Ca2+-channel

The bronchodilator effects of Roflumilast “a selective phosphodiesterase type-4 (PDE4)” inhibitor studied in this experimental protocol. The spiral strips of isolated guinea-pig tracheal chains mounted in organ bath and maintained in Krebs solution ventilated with carbogen at 32 °C and in Ca++ restricted krebs solution. PDE inhibitory activity was evaluated by recording dose response curves using inhibitory effect of isoprenaline on CCh induced contractions. For confirmation of PDE inhibition the intracellular cAMP levels were also estimated. Roflumilast resulted a sharp inhibition in contractile responses of carbachol (CCh, 1 µM) and K+ (80 mM) and the results were almost similar to verapamil. In Ca++ restricted Krebs solution, a rightward shift in the Ca++ response curves observed in the tracheal chain strips which were pretreated with Roflumilast (0.001–0.003 mg/mL) and the maximum response was suppressed, similarly as with verapamil. PDE inhibitory effect of Roflumilast evaluated by recording dose-dependent (0.03–0.1 mg/mL) responses, the isoprenaline-induced inhibitory dose response curves shifted leftward similar to papaverine (PDE inhibitor). Pretreatment with Roflumilast exhibited elevated intracellular cAMP levels in tracheal strips. Findings of the experiment conclude bronchodilatory influence of Roflumilast via PDE and Ca++ channel inhibition. Results of current experiment offers comprehensive mechanistic background of Roflumilast in future as therapeutic bronchodilator for hyperactive bronchial airway diseases.

Elevated intracellular cAMP concentration mediates growth suppression in glioma cells

Supressed levels of intracellular cAMP have been associated with malignancy. Thus, elevating cAMP through activation of adenylyl cyclase (AC) or by inhibition of phosphodiesterase (PDE) may be therapeutically beneficial. Here, we demonstrate that elevated cAMP levels suppress growth in C6 cells (a model of glioma) through treatment with forskolin, an AC activator, or a range of small molecule PDE inhibitors with differing selectivity profiles. Forskolin suppressed cell growth in a PKA-dependent manner by inducing a G2/M phase cell cycle arrest. In contrast, trequinsin (a non-selective PDE2/3/7 inhibitor), not only inhibited cell growth via PKA, but also stimulated (independent of PKA) caspase-3/-7 and induced an aneuploidy phenotype. Interestingly, a cocktail of individual PDE 2,3,7 inhibitors suppressed cell growth in a manner analogous to forskolin but not trequinsin. Finally, we demonstrate that concomitant targeting of both AC and PDEs synergistically elevated intracellular cAMP levels thereby potentiating their antiproliferative actions.

The phosphodiesterase 10 inhibitor papaverine exerts anti-inflammatory and neuroprotective effects via the PKA signaling pathway in neuroinflammation and Parkinson\u2019s disease mouse models

BackgroundNeuroinflammation plays a pivotal role in the pathogenesis of Parkinson’s disease (PD). Thus, the development of agents that can control neuroinflammation has been suggested as a promising therapeutic strategy for PD. In the present study, we investigated whether the phosphodiesterase (PDE) 10 inhibitor has anti-inflammatory and neuroprotective effects in neuroinflammation and PD mouse models.MethodsPapaverine (PAP) was utilized as a selective inhibitor of PDE10. The effects of PAP on the expression of pro-inflammatory molecules were examined in lipopolysaccharide (LPS)–stimulated BV2 microglial cells by ELISA, RT-PCR, and Western blot analysis. The effects of PAP on transcription factors were analyzed by the electrophoretic mobility shift assay, the reporter gene assay, and Western blot analysis. Microglial activation and the expression of proinflammatory molecules were measured in the LPS- or MPTP-injected mouse brains by immunohistochemistry and RT-PCR analysis. The effect of PAP on dopaminergic neuronal cell death and neurotrophic factors were determined by immunohistochemistry and Western blot analysis. To assess mouse locomotor activity, rotarod and pole tests were performed in MPTP-injected mice.ResultsPAP inhibited the production of nitric oxide and proinflammatory cytokines in LPS-stimulated microglia by modulating various inflammatory signals. In addition, PAP elevated intracellular cAMP levels and CREB phosphorylation. Treatment with H89, a PKA inhibitor, reversed the anti-inflammatory effects of PAP, suggesting the critical role of PKA signaling in the anti-inflammatory effects of PAP. We verified the anti-inflammatory effects of PAP in the brains of mice with LPS-induced systemic inflammation. PAP suppressed microglial activation and proinflammatory gene expression in the brains of these mice, and these effects were reversed by H89 treatment. We further examined the effects of PAP on MPTP-injected PD model mice. MPTP-induced dopaminergic neuronal cell death and impaired locomotor activity were recovered by PAP. In addition, PAP suppressed microglial activation and proinflammatory mediators in the brains of MPTP-injected mice.ConclusionsPAP has strong anti-inflammatory and neuroprotective effects and thus may be a potential candidate for treating neuroinflammatory disorders such as PD.

Citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone induces BDNF via cAMP/ERK/CREB signaling and reduces phosphodiesterase activity in C6 cells.

Brain-derived neurotrophic factor (BDNF) is associated with onset of several central nervous system disorders, e.g., Parkinson's disease, Alzheimer's disease, depression, epilepsy, and chronic pain. In our previous in vivo studies using ischemic and depression mouse models, we revealed that citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) exerts neuroprotective effects by enhancing the expression of BDNF in astrocytes within the hippocampus. Therefore, in the present study, we examined the mechanism of BDNF induction by HMF in vitro using rat C6 glioma cells. C6 glioma cells were treated with HMF (10 μM) or HMF + U0126 (10 μM), HMF + H89 (1 μM), or HMF + K252a (200 nM) for 48 h. The protein level of mature BDNF (m-BDNF), phosphorylated-ERK (p-ERK) and phosphorylated-cAMP-response element binding protein (p-CREB) were measured using western blot analysis. To clarify the mechanism of HMF for increasing m-BDNF, the inhibitory effect of phosphodiesterase 4B (PDE4B) and PDE4D, and intracellular cAMP levels were examined by ELISA. Our findings revealed that the m-BDNF-inducing activity of HMF was abolished by U0126 but not by H89 or K252a. HMF was found to phosphorylate (activate) ERK and cAMP-response element binding protein (CREB), a BDNF transcription factor. HMF inhibited PDE4B and PDE4D activity. Moreover, 10 μM HMF elevated intracellular cAMP levels in C6 cells. These findings suggest that HMF might exert its neuroprotective effects by inducing m-BDNF expression in C6 cells, model cell line of astrocytes, via the activation of cAMP/ERK/CREB signaling and inhibiting PDE4B or PDE4D.

72 - The tyrosine nitration of PKG-1α inhibits its regulatory function in the pulmonary endothelium

Protein tyrosine nitration secondary to peroxynitrite generation is involved in the pathogenesis of vascular diseases. We have previously shown that tyrosine nitration is associated with the development of acute lung injury (ALI). Protein kinase PKG-1a, which integrates cAMP/cGMP signaling and is important for the endothelial barrier maintenance, can be inhibited by nitration at Tyr247. We studied a tyrosine nitration effect on PKG-1a regulatory function in the pulmonary endothelium. Since cyclic nucleotide-specific phosphodiesterases (PDEs) can be modulated by phosphorylation, we explored the functional link between nitration-mediated PKG-1a inhibition and PDE(s) activation. LPS-challenged mice were utilized as ALI model. The peroxynitrite scavenger, MnTMPyP was used to attenuate protein nitration. Cyclic nucleotide levels, PKA/PKG/PDEs activities and PKG nitration were evaluated. PKG-1a-dependent phosphorylation sites on PDE3A were elucidated by mass-spectrometry. LPS-treatment decreased cAMP and increased cGMP levels in mouse lungs. PKA/PKG activities were also decreased. Nitrosative stress played a pivotal role in these changes. We identified an increase in PKG-1a nitration at Tyr247 and PDE3A activity increase. PKG activation in vitro induced an increase in PDE3A serine phosphorylation that correlated with a decrease in its activity. Mass-spectrometry identified a novel PKG-1a-dependent phosphorylation site, Ser654, in PDE3A. We confirmed PKG-1a-PDE3A interaction, and PKG-dependent PDE3A phosphorylation. This phosphorylation suppressed PDE3A activity and elevated intracellular cAMP levels. Ser654Ala-PDE3A mutant overexpression abolished PKG-1a-dependent inhibitory effect and prevented cAMP increase. We designed a peptide aimed to protect PKG-1a from Tyr247 nitration. This peptide attenuated the endothelial barrier disruption in LPS-challenged mice. We have identified a novel mechanism of cyclic nucleotide cross-talk regulated by PKG-dependent inhibitory phosphorylation of PDE3A. In ALI, PKG-1a Tyr247 nitration abolishes PDE3A phosphorylation leading to cAMP signaling impairment and endothelial dysfunction. Prevention of PKG-1a nitration significantly improved the barrier-protective mechanisms in ALI model. These data suggest a therapeutic approach of PKG activity protection in ALI treatment.