Cellular cAMP Content Research Articles

IgG immune complex-induced acute lung injury is ameliorated by cAMP via down-regulation of C/EBP- and AP-1-mediated transcriptions

BackgroundAcute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are life threatening pulmonary diseases, and we are now lack of effective therapeutic methods. Inflammatory responses are essential for initiating ALI/ARDS. Thus, ameliorating inflammatory reaction might be beneficial for treatment of the disease. There are increasing data that phosphodiesterase-4 (PDE4)-selective inhibitors, which may elevate cellular cyclic adenosine 3′, 5′-monophosphate (cAMP) level, could suppress inflammation. However, whether they could be used to treat IgG immune complex (IgG-IC)-associated ALI has not been determined.MethodsALI is induced by treating mice with airway deposition of IgG immune complexes. Cellular cAMP concentrations are elevated by treating mice or macrophages with Rolipram/Roflumilast. The degree of pulmonary injury is reflected by lung permeability, leukocyte accumulation, histological change and expressions of pro-inflammatory mediators. 6-Bnz-cAMP and H-89 are used to regulate protein kinase A (PKA) activity, and 8-pCPT-2′-O-Me-cAMP is applied to activate exchange proteins directly activated by cAMP (Epac). Gene expressions are analyzed by real-time PCR, ELISA or Western blot. CCAAT/enhancer binding protein (C/EBP) and activation protein 1 (AP-1) transcription activities are estimated by measuring the luciferase productions.ResultsIgG-IC-induced ALI is attenuated by the PDE4-selective inhibitor, which is due to reduced expressions of cytokine and chemokines. Interestingly, we find that cAMP downstream effector molecule PKA but not Epac is involved in negative regulation of IgG-IC-mediated pro-inflammatory mediators’ productions. Mechanistically, activation of cAMP-PKA signal axis leads to inactivation of MAPK pathway, resulting in a decrease in C/EBP- and AP-1-mediated transcriptions of pro-inflammatory mediators.ConclusionsOur data demonstrate, for the first time, that cAMP-PKA signal is involved in down-regulation of IgG-IC-associated inflammatory responses via down-regulating MAPK activation, which is critical for transcriptional activities of C/EBP and AP-1. Collectively, our experiments provide theoretical base for the potential application of PDE4-selective inhibitor to clinic for treatment of IgG-IC-related acute lung injury.

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.

Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors.

Communication between PATCHED1 (PTCH1) and SMOOTHENED (SMO) is fundamental to Hedgehog (Hh) signal transduction in development and disease. We describe a real-time cell-based SMO functional assay based on SMO activity-dependent changes in cellular cAMP concentrations. This assay is capable of detecting changes in SMO conformation within minutes of PTCH1 inactivation by Hh ligands. As a result, it expands the range of experimental perturbations that can be used to dissect PTCH1-SMO communication, enabling a deeper mechanistic understanding of a longstanding mystery in Hh signal transduction.

Dynamics of adrenergic signaling in cardiac myocytes and implications for pharmacological treatment

Dense innervation of the heart by the sympathetic nervous system (SNS) allows cardiac output to respond appropriately to the needs of the body under varying conditions, but occasionally the abrupt onset of SNS activity can trigger cardiac arrhythmias. Sympathetic activity leads to the release of norepinephrine (NE) onto cardiomyocytes, activating β1-adrenergic receptors (β1-ARs) and leading to the production of the second messenger cyclic AMP (cAMP). Upon sudden activation of β1-ARs in experiments, intracellular cAMP can transiently rise to a high concentration before converging to a steady state level. Although changes to cellular cAMP concentration are important in modulating the overall cardiovascular response to sympathetic tone, the underlying mechanisms of the cAMP transients and the parameters that control their magnitude are unclear.We reduce a detailed computational model of the β1-adrenergic signaling cascade to a system of two differential equations by eliminating extraneous variables and applying quasi-steady state approximation. The structure of the reduced model reveals that the large cAMP transients associated with abrupt β1-AR activation are generated by the interplay of production/degradation of cAMP and desensitization/resensitization of β1-ARs. The reduced model is used to predict how the dynamics of intracellular cAMP depend on the concentrations of norepinephrine (NE), phosphodiesterases 3 and 4 (PDE3,4), G-protein coupled receptor kinase 2 (GRK2), and β1-AR, in healthy conditions and a simple model of early stages of heart failure.The key findings of the study are as follows: 1) Applying a reduced model of the dynamics of cardiac sympathetic signaling we show that the concentrations of two variables, cAMP and non-desensitized β1-AR, capture the overall dynamics of sympathetic signaling; 2) The key factors influencing cAMP production are AC activity and PDE3,4 activity, while those that directly impact β1-AR phosphorylation are GRK2 and PKA1. Thus, disease states that affect sympathetic control of the heart can be thoroughly assessed by studying AC activity, PDE3,4, GRK2 and PKA activity, as these factors directly impact cAMP production/degradation and β1-AR (de) phosphorylation and are therefore predicted to comprise the most effective pharmaceutical targets in diseases affecting cardiac β1-adrenergic signaling.

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides

BackgroundApproximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3′,5′-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced.ResultsTo quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity.ConclusionsWe found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.

Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages.

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.

Regulation of AMPK activity by type 10 adenylyl cyclase: contribution to the mitochondrial biology, cellular redox and energy homeostasis.

The downregulation of AMP-activated protein kinase (AMPK) activity contributes to numerous pathologies. Recent reports suggest that the elevation of cellular cAMP promotes AMPK activity. However, the source of the cAMP pool that controls AMPK activity remains unknown. Mammalian cells possess two cAMP sources: membrane-bound adenylyl cyclase (tmAC) and intracellularly localized, type 10 soluble adenylyl cyclase (sAC). Due to the localization of sAC and AMPK in similar intracellular compartments, we hypothesized that sAC may control AMPK activity. In this study, sAC expression and activity were manipulated in H9C2 cells, adult rat cardiomyocytes or endothelial cells. sAC knockdown depleted the cellular cAMP content and decreased AMPK activity in an EPAC-dependent manner. Functionally, sAC knockdown reduced cellular ATP content, increased mitochondrial ROS formation and led to mitochondrial depolarization. Furthermore, sAC downregulation led to EPAC-dependent mitophagy disturbance, indicated by an increased mitochondrial mass and unaffected mitochondrial biogenesis. Consistently, sAC overexpression or stimulation with bicarbonate significantly increased AMPK activity and cellular ATP content. In contrast, tmAC inhibition or stimulation produced no effect on AMPK activity. Therefore, the sAC-EPAC axis may regulate basal and induced AMPK activity and support mitophagy, cellular energy and redox homeostasis. The study argues for sAC as a potential target in treating pathologies associated with AMPK downregulation.

Prostaglandin E2 inhibits profibrotic function of human pulmonary fibroblasts by disrupting Ca2+ signaling.

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-β (TGF-β) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-β in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-β-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-β-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-β. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

Mechanism of bisphenol AF-induced progesterone inhibition in human chorionic gonadotrophin-stimulated mouse Leydig tumor cell line (mLTC-1) cells.

Bisphenol AF (BPAF) has been shown to inhibit testicular steroidogenesis in male rats. However, the precise mechanisms related to the toxic effects of BPAF on reproduction remain poorly understood. In the present study, a mouse Leydig tumor cell line (mLTC-1) was used as a model to investigate the mechanism of steroidogenic inhibition and to identify the molecular target of BPAF. Levels of progesterone and the concentration of cyclic adenosine monophosphate (cAMP) in cells exposed to BPAF were detected, and expression of key genes and proteins in steroid biosynthesis was assessed. The results showed that BPAF exposure decreased human chorionic gonadotrophin (hCG)-stimulated progesterone production in a dose-dependent manner. The 24-h IC50 (half maximal inhibitory concentration) value for BPAF regarding progesterone production was 70.2 µM. A dramatic decrease in cellular cAMP concentration was also observed. Furthermore, BPAF exposure inhibited expression of genes and proteins involved in cholesterol transport and progesterone biosynthesis. Conversely, the protein levels of steroidogenic acute regulatory protein (StAR) were not altered, and those of progesterone were still decreased upon 22R-hydroxycholesterol treatment of cells exposed to higher doses of BPAF. Together, these data indicate that BPAF exposure inhibits progesterone secretion in hCG-stimulated mLTC-1 cells by reducing expression of scavenger receptor class B type I (SR-B1) and cytochrome P450 (P450scc) due to the adverse effects of cAMP. However, StAR might not be the molecular target in this process.

Inhibition of Intracellular Type 10 Adenylyl Cyclase Protects Cortical Neurons Against Reperfusion-Induced Mitochondrial Injury and Apoptosis.

Mitochondrial injury significantly contributes to the neuronal death under cerebral ischemia and reperfusion. Within several signaling pathways, cyclic adenosine monophosphate (cAMP) signaling plays a substantial role in mitochondrial injury and cell death. Traditionally, the source of cellular cAMP has been attributed to the membrane-bound adenylyl cyclase, whereas the role of the intracellular localized type 10 soluble adenylyl cyclase (sAC) in neuronal pathology has not been considered. Since neurons express an active form of sAC, we aimed to investigate the role of sAC in reperfusion-induced neuronal apoptosis. For this purpose, the in vitro model of oxygen/glucose deprivation (simulated ischemia, 1h), followed by recovery (simulated reperfusion, 12h) in rat embryonic neurons, was applied. Although ischemia alone had no significant effect on apoptosis, reperfusion led to an activation of the mitochondrial pathway of apoptosis, hallmarked by mitochondrial depolarization, cytochrome c release, and mitochondrial ROS formation. These effects were accompanied by significantly augmented sAC expression and increased cellular cAMP content during reperfusion. Pharmacological suppression of sAC during reperfusion reduced cellular cAMP and ameliorated reperfusion-induced mitochondrial apoptosis and ROS formation. Similarly, sAC knockdown prevented neuronal death. Further analysis revealed a role of protein kinase A (PKA), a major downstream target of sAC, in reperfusion-induced neuronal apoptosis and ROS formation. In conclusion, the results show a causal role of intracellular, sAC-dependent cAMP signaling in reperfusion-induced mitochondrial injury and apoptosis in neurons. The protective effect of sAC inhibition during the reperfusion phase provides a basis for the development of new strategies to prevent the reperfusion-induced neuronal injury.

Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle.

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

Identification of the cAMP phosphodiesterase CpdA as novel key player in cAMP-dependent regulation in Corynebacterium glutamicum.

The second messenger cyclic AMP (cAMP) plays an important role in the metabolism of Corynebacterium glutamicum, as the global transcriptional regulator GlxR requires complex formation with cAMP to become active. Whereas a membrane-bound adenylate cyclase, CyaB, was shown to be involved in cAMP synthesis, enzymes catalyzing cAMP degradation have not been described yet. In this study we identified a class II cAMP phosphodiesterase named CpdA (Cg2761), homologs of which are present in many Actinobacteria. The purified enzyme has a Kmapp value of 2.5 ± 0.3 mM for cAMP and a Vmaxapp of 33.6 ± 4.3 µmol min-1 mg-1 . A ΔcpdA mutant showed a twofold increased cAMP level on glucose and reduced growth rates on all carbon sources tested. A transcriptome comparison revealed 247 genes with a more than twofold altered mRNA level in the ΔcpdA mutant, 82 of which are known GlxR targets. Expression of cpdA was positively regulated by GlxR, thereby creating a negative feedback loop allowing to counteract high cAMP levels. The results show that CpdA plays a key role in the control of the cellular cAMP concentration and GlxR activity and is crucial for optimal metabolism and growth of C. glutamicum.

Aryl-1,3,5-triazine ligands of histamine H4 receptor attenuate inflammatory and nociceptive response to carrageen, zymosan and lipopolysaccharide

Objective and designHistamine H4 receptor (H4R) offers a great potential for new therapeutic strategies for the treatment of inflammation-based diseases. The aim of this study is to present the pharmacological profile of two recently synthesized ligands of H4R with particular reference to their anti-inflammatory and analgesic activity.Materials and subjectsWe used mice and rats in the in vivo tests. We also used murine RAW 264.7 cells and isolated guinea-pig ileum in in vitro test.TreatmentsIn the in vivo tests, animals were pre-treated with the increasing doses of investigated compounds (12.5, 25 and 50 mg/kg) and reference compounds: JNJ7777120 (25 mg/kg), indomethacin (10 mg/kg). Macrophages were pre-treated with two concentrations of tested compounds 100 and 10 µM.MethodsWe examined anti-inflammatory and analgesic effects of the new H4R antagonists in the in vivo models of inflammation induced by carrageenan or zymosan. We assessed the level of cAMP and release of cytokines, ROS and NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Moreover, we assessed the affinity of the investigated compounds for histamine H1 receptor in functional studies.ResultsBoth investigated compounds reduced paw edema, mechanical and thermal hyperalgesia in the carrageenan-induced acute inflammation. Moreover, administration of the investigated compounds resulted in decreased granulocyte influx and attenuated nociceptive reaction in the zymosan-induced peritonitis model. In the same model of inflammation, the investigated compounds reduced vascular permeability; however, this effect was observed only after the highest applied dose. Furthermore, the test compounds had no impact on cell viability in the experiments on RAW 264.7 macrophages. In these cells, stimulated with LPS, the test compounds decreased reactive oxygen species (ROS) production. They increased the cellular concentration of cAMP and attenuated the production of inflammatory cytokines such as TNFα and IL-1β. All results were comparable to those obtained for the reference compound JNJ7777120 with the exception of the impact on NO production. Nevertheless, this effect was similar to that obtained for the other reference compound rolipram, which is a phosphodiesterase 4 (PDE 4) inhibitor. Further experiments revealed that both of the investigated compounds possessed relatively low affinity for histamine H1 receptor and do not inhibit the activity of the PDE 4B1 enzyme. In addition, all the effects of the investigated compounds in in vivo experiments were observed at doses that did not cause neurologic deficits in rotarod test and did not reduce spontaneous locomotor activity.ConclusionsOur results demonstrate the anti-inflammatory and analgesic activity of the new aryl-1,3,5-triazine derivatives, which are primarily H4R–dependent.

Abstract 66: Discovery of a Novel Pharmaceutical Class as Potential Heart Failure Treatment

Utilizing an unbiased in vivo phenotypic chemical screening platform in zebrafish embryos, our laboratory has identified a number of novel compounds with high selectivity for a wide range of cellular targets, including kinases (CK2a, DRAK2, DYRK2 and bone morphogenetic protein receptors), GPCRs (lysophosphatidic acid receptor 1, and extracellular proton sensor), p300 histone acetyltransferase, and phosphodiesterase-4 (PDE4). While the compounds we discovered have therapeutic implications for a wide range of diseases, our translational work has focused on addressing the cardiovascular diseases. Heart failure (HF) is a leading cause of disability and mortality in US, affecting about 6 million Americans, and the incidence of heart failure is anticipated to increase substantially in the coming decades. Yet, current HF pharmaceuticals are palliative, and the outlook for HF drug pipeline is uncertain. Within this backdrop, we recently discovered eggmanone, an extraordinarily selective PDE4 inhibitor which has no known off-target. An usual feature of eggmanone is that it increases cAMP levels specifically in distinct cellular microdomains, without raising the total cellular cAMP content. In isolated mouse cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), eggmanone increases cardiac contractility by targeting a discrete myocyte microdomain without causing significant changes in myocyte calcium cycling. Importantly, eggmanone enhances systolic function in mice with failing hearts without increasing the heart rate. These results raise the exciting possibility that a microdomain-specific PDE4 inhibitor like eggmanone may be useful as an inotropic therapy for HF which avoids the pitfalls of traditional PDE inhibitors, whose utility has been limited by proarrhythmia, tachyphylaxis and cardiotoxicity.

0335 : Contribution of large conductance Ca(2+)-activated K(+) channels to vasorelaxation induced by cAMP-phosphodiesterases inhibitors is blunted in coronary arteries from rats with heart failure

3’-5’-cyclic adenosine monophosphate (cAMP) is an important mediator of vasorelaxation. In vascular smooth muscle, cellular cAMP concentrations are mainly regulated by type 3 and 4 phosphodiesterases (PDE3 & PDE4). Large conductance Ca(2+)-activated K(+) (BK) channels are reported to mediate cAMP-induced relaxation. Here, we sought to clarify whether BK channels are key players in the regulation of tone by PDE activities in rat coronary arteries (CA), and if this contribution is altered in congestive heart failure (CHF). CA were isolated from rats with CHF sacrificed 22 weeks after surgical stenosis of the ascending aorta. Age-matched « sham » animals were used as controls. CA were isolated and mounted on a myograph. Maximal contraction was obtained using high K(+) solution. Relaxations induced by inhibitors of PDE4 (Ro-20 1724, Ro, 10μM) or the PDE3 (Cilolstamide, Cil, 1μM) were studied in CA contracted with the thromboxane analogue U46619 (1-2μM) and incubated or not with iberiotoxin (IBTX, 100nM), a selective BK channel blocker. CA isolated from rats displaying robust signs of CHF showed a 78% reduction of maximal contraction (P<0.001, N=5-7) and reduced relaxing response to acetylcholine (–55%, P< 0.01), compared to CA from sham rats. Ro and Cil relaxed sham CA to respectively 31±13% and 14±5% of U46619 tone (N=6). In the presence of IBTX, these responses decreased to 3±1% (N=5, P<0.05) and 4±3% (N=4). In CHF rats, Ro relaxed CA to 7±4% and Cil to 27±4% (N=4), whereas IBTX had no effect either on responses to Ro (17±8%, N=5) or Cil (26±6%, N=4). Simultaneous addition of Cil and Ro relaxed sham CA to 79±10% (N=7), an effect reduced by IBTX (20±9%, N=6, P<0.01). Interestingly, Cil and Ro together relaxed CHF CA to 69±9% but this was not altered by IBTX (69±14%, N=5-6). These results show that, in rat CA, BK channels are important effectors of relaxing responses to PDE3 and PDE4 inhibition. In CHF, specific contribution of BK channels to these responses is lost.

Is there a Role of Intracellular Magnesium in Prevention of Heart Failure?

Heart failure, characterized by the reduction in left and /or right ventricular function, is the most common cause of mortality and morbidity in almost around the world. This syndrome is induced by β1-adrenergic receptor (β1-AR) desensitization, which in turn is caused by the reduction in cellular cAMP concentration. Cellular cAMP concentration is modulated by adenylyl cyclases (ACs), a family of enzymes that are strongly activated by intracellular magnesium and circulating levels of the stress-related hormones. Short-term activation of β1-AR signalling pathway by increased levels of these hormones induces an increase in intracellular Mg2+ and cAMP concentrations, which improves cardiac function. Nevertheless, long-term activation of β1-AR signalling pathway by elevated levels of these hormones reduces intracellular Mg2+ and cAMP levels, which deteriorates cardiac function. Intracellular cAMP signalling has a circadian rhythm (CR) in mammals, and CR may modulate diverse phenomena. The loss of CR may per se be a cause of severe diseases. Magnesium can regulate intracellular cAMP production and through this may modulate the CR. Here we propose that longterm activation of β1-AR signalling pathway, and /or the use of diuretics, can induce a progressive reduction of intracellular of magnesium and a subsequent gradual reduction of intracellular cAMP levels. This in turn can cause β1-AR desensitization, the loss of CR of the hormones related with HF, and, finally, HF development. We suggest the therapeutic implications of this mechanism in the prevention of HF.

A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases.

The universal second messenger cAMP is generated upon stimulation of Gs protein-coupled receptors, such as the β2 -adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as β2 -adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer's disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization.

Fluorescence resonance energy transfer microscopy (FRET).

FRET (Förster Resonance Energy Transfer) microscopy breaks the resolution limit of light to let us investigate the conformation and function of proteins within living cells. Intensity-based methods are the most popular and direct approach to detect FRET. Among them, detection of sensitized emission signals and ratio imaging of specially designed FRET sensors are routinely used in modern cell biology laboratories. In this chapter, we provide protocols for both these techniques. We guide the reader through the mathematical corrections necessary to calculate the sensitized emission image. We illustrate this approach with an example of studying the interaction of nexin (SNX1) proteins. In the ratio FRET protocol, we focus on monitoring changes in cellular concentration of cAMP with an EPAC-based FRET sensor.

CAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation.

Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca2+ concentration. Inhibition of cytosolic Ca2+ rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30–60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1‐dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP‐effectors PKA and Epac (with PKI and ESI‐09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase.

Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway

Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H(+)-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H(+)-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H(+)-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H(+)-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway.