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Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. Here, we explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) we identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. We propose that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition.

Tetramethylpyrazine is used in the treatment of many neurological diseases because of its neuroprotective effect. Here, we demonstrate that administration of tetramethylpyrazine effectively reverses memory deficits induced by scopolamine. Moreover, tetramethylpyrazine preserves postsynaptic protein synthesis and restores cAMP/PKA/CREB pathway signaling deficits. Our study not only explores the actions of tetramethylpyrazine on synapses, but also provides novel evidence for the possible therapeutic use of tetramethylpyrazine in dementia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Elevated levels of amyloid beta (Aβ) peptide, hyperphosphorylation of tau protein, and inflammation are pathological hallmarks in Alzheimer's disease (AD). Phosphodiesterase 7 (PDE7) regulates the inflammatory response through the cyclic adenosine monophosphate signaling cascade, and thus plays a central role in AD. The aim of this study was to evaluate the efficacy of an inhibitor of PDE7, named S14, in a mouse model of AD. We report that APP/Ps1 mice treated daily for 4 weeks with S14 show: (1) significant attenuation in behavioral impairment; (2) decreased brain Aβ deposition; (3) enhanced astrocyte-mediated Aβ degradation; and (4) decreased tau phosphorylation. These effects are mediated via the cyclic adenosine monophosphate/cyclic adenosine monophosphate response element-binding protein signaling pathway, and inactivation of glycogen synthase kinase (GSK)3. Our data support the use of PDE7 inhibitors, and specifically S14, as effective therapeutic agents for the prevention and treatment of AD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The orexigenic hormone ghrelin acts like a hunger signal, released by the stomach in response to nutritional status. However, ghrelin and its receptor in the kidney may play other biological roles. Kemp and colleagues identify that ghrelin stimulates renal Na+ absorption through cAMP-dependent trafficking of ENaC in the cortical collecting duct. While ghrelin seems to be a physiological regulator of ENaC, future studies are necessary to clarify its physiological and pathological roles in sodium homeostasis.

Anthracyclines, discovered 50 years ago, are antibiotics widely used as antineoplastic agents and are among the most successful anticancer therapies ever developed to treat a wide range of cancers, including hematological malignancies, soft tissue sarcomas and solid tumors. However, some anthracyclines, including doxorubicin, exhibit major signs of cardiotoxicity that may ultimately lead to heart failure (HF). Despite intensive research on doxorubicine-induced cardiotoxicity, the underlying mechanisms responsible for doxorubicin-induced cardiotoxicity have not been fully elucidated yet. Published literature so far has focused mostly on mitochondria dysfunction with consequent oxidative stress, Ca(2+) overload, and cardiomyocyte death as doxorubicin side effects, leading to heart dysfunction. This review focuses on the current understanding of the molecular mechanisms underlying doxorubicin-induced cardiomyocyte death (i.e.: cardiomyocyte death, mitochondria metabolism and bioenergetic alteration), but we will also point to new directions of possible mechanisms, suggesting potent prior or concomitant alterations of specific signaling pathways with molecular actors directly targeted by the anticancer drugs itself (i.e. calcium homeostasis or cAMP signaling cascade). The mechanisms of anticancer cardiac toxicity may be more complex than just mitochondria dysfunction. Partnership of both basic and clinical research is needed to promote new strategies in diagnosis, therapies with concomitant cardioprotection in order to achieve cancer treatment with acceptable cardiotoxicity along life span., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The main distal nephron segment sodium transporters are the distal tubule chlorothiazide-sensitive sodium chloride cotransporter (NCC) and the collecting duct amiloride-sensitive epithelial sodium channel (ENaC). The infusion of ghrelin into the renal interstitium stimulates distal nephron-dependent sodium reabsorption in normal rats, but the mechanism is unknown. Here we localize renal ghrelin receptors (GR) to the cortical collecting duct (CCD). Ghrelin significantly increased phosphorylated serum/glucocorticoid-regulated kinase-1 (pSGK1), a major upstream signaling intermediate regulating ENaC. To test whether increased apical membrane αENaC induced the antinatriuresis, ghrelin was infused in the presence of acute and chronic amiloride, a selective inhibitor of ENaC. In the presence of amiloride, renal interstitial ghrelin failed to reduce urine sodium excretion, suggesting that ghrelin-induced sodium reabsorption is dependent on intact ENaC activity. While the main sodium transporter of the CCD is ENaC, NCC is also present. In response to renal interstitial ghrelin infusion, neither total nor phosphorylated NCC levels are altered. Ghrelin-induced sodium reabsorption persisted in the presence of chlorothiazide (selective inhibitor of NCC), indicating that intact NCC activity is not necessary for ghrelin-induced antinatriuresis. Finally, renal interstitial ghrelin infusion significantly increased interstitial cAMP levels and adenylyl cyclase blockade abolished ghrelin-induced antinatriuresis. Thus, GRs expressed in the CCD regulate sodium reabsorption by cAMP-induced trafficking of ENaC to the apical membrane.

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only ∼30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRP(Mt)), consistent with the relatively low dependence on cAMP of CRP(Mt) binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In the heart, adrenergic stimulation activates the β-adrenergic receptors coupled to the heterotrimeric stimulatory Gs protein, followed by subsequent activation of adenylyl cyclase, elevation of cyclic AMP levels, and protein kinase A (PKA) activation. One of the main targets for PKA modulation is the cardiac L-type Ca²⁺ channel (CaV1.2) located in the plasma membrane and along the T-tubules, which mediates Ca²⁺ entry into cardiomyocytes. β-Adrenergic receptor activation increases the Ca²⁺ current via CaV1.2 channels and is responsible for the positive ionotropic effect of adrenergic stimulation. Despite decades of research, the molecular mechanism underlying this modulation has not been fully resolved. On the contrary, initial reports of identification of key components in this modulation were later refuted using advanced model systems, especially transgenic animals. Some of the cardinal debated issues include details of specific subunits and residues in CaV1.2 phosphorylated by PKA, the nature, extent, and role of post-translational processing of CaV1.2, and the role of auxiliary proteins (such as A kinase anchoring proteins) involved in PKA regulation. In addition, the previously proposed crucial role of PKA in modulation of unstimulated Ca²⁺ current in the absence of β-adrenergic receptor stimulation and in voltage-dependent facilitation of CaV1.2 remains uncertain. Full reconstitution of the β-adrenergic receptor signaling pathway in heterologous expression systems remains an unmet challenge. This review summarizes the past and new findings, the mechanisms proposed and later proven, rejected or disputed, and emphasizes the essential issues that remain unresolved.

Background: Familial testicular germ cell tumors (FTGCTs) are hypothesized to result from the combined interaction of multiple low-penetrance genes. We reported inactivating germline mutations of the cAMP-binding phosphodiesterase 11A (PDE11A) as modifiers of FTGCT risk. Recent genome-wide association studies have identified single-nucleotide polymorphisms in the KITLG gene, the ligand for the cKIT tyrosine kinase receptor, as strong modifiers of susceptibility to both familial and sporadic testicular germ cell tumors., Design: We studied 94 patients with FTGCTs and 50 at-risk male relatives from 63 unrelated kindreds, in whom the PDE11A gene had been sequenced by investigating the association between KITLG genome-wide association study single-nucleotide polymorphisms rs3782179 and rs4474514 and FTGCT risk in these patients and in 692 controls. We also examined cAMP and c-KIT signaling in testicular tissues and cell lines and extended the studies to 2 sporadic cases, one with a PDE11A defect and one without, as a comparison., Results: We found a higher frequency of the KITLG risk alleles in FTGCT patients who also had a PDE11A sequence variant, compared with those with a wild-type PDE11A sequence. In NTERA-2 and Tcam-2 cells transfected with the mutated forms of PDE11A (R52T, F258Y, Y727C, R804H, V820M, R867G, and M878V), cAMP levels were significantly higher, and the relative phosphodiesterase activity was lower than in the wild-type cells. KITLG expression was consistently increased in the presence of PDE11A-inactivating defects, both at the RNA and protein levels, in familial testicular germ cell tumors. The 2 sporadic cases that were studied, one with a PDE11A defect and another without, agreed with the data in FTGTCT and in the cell lines., Conclusions: Patients with FTGCT and PDE11A defects also carry KITLG risk alleles more frequently. There may be an interaction between cAMP and c-KIT signaling in predisposition to testicular germ cell tumors.

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

Background: We have previously shown that ethanol (EtOH) increases cellular apoptosis to developing neurons via the effects on oxidative stress of neurons directly and via increasing production of microglia-derived factors. To study further the mechanism of EtOH action on neuronal apoptosis, we determined the effects of 2 well-known PKA activators, dibutyryl cAMP (dbcAMP) and brain-derived neurotrophic factor (BDNF), on EtOH-activated oxidative stress and apoptotic processes in the hypothalamic neurons in the presence and absence of microglial cells' influence., Methods: In enriched neuronal cells from fetal rat hypothalami treated with EtOH or with conditioned medium from EtOH-treated microglia, we measured cellular apoptosis by the free nucleosome assay and the levels of cAMP, BDNF, O²⁻, reactive oxygen species (ROS), nitrite, glutathione (GSH), and catalase following treatment with EtOH or EtOH-treated microglial culture conditioned medium. Additionally, we tested the effectiveness of dbcAMP and BDNF in preventing EtOH or EtOH-treated microglial conditioned medium on cellular apoptosis and oxidative stress in enriched hypothalamic neuronal cell in primary cultures., Results: Neuronal cell cultures following treatment with EtOH or EtOH-activated microglial conditioned medium showed decreased production levels of cAMP and BDNF. EtOH also increased apoptotic death as well as oxidative status, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidants, in neuronal cells. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Treatment with BDNF or dbcAMP decreased EtOH or EtOH-activated microglial conditioned medium-induced changes in the levels of intracellular free radicals, ROS and O²⁻, nitrite, GSH, and catalase., Conclusions: These data support the possibility that EtOH by acting directly and via increasing the production of microglial-derived factors reduces cellular levels of cAMP and BDNF to increase cellular oxidative status and apoptosis in hypothalamic neuronal cells in primary cultures., (Copyright © 2013 by the Research Society on Alcoholism.)

Background and Objective: Alveolar fluid clearance (AFC) is important for the resolution of acute lung injury (ALI). The role of cystic fibrosis transmembrane conductance regulator (CFTR) in AFC has not been entirely elucidated in animal models of ALI. The aim of this study was to investigate the role of CFTR and its mechanisms in AFC in normal and ALI mice., Methods: Seventy mice were randomly divided into 14 groups and ALI was established by intratracheal instillation of lipopolysaccharide (LPS). After 48 h, CFTR activator CFTRact-16 or inhibitor CFinh-172 with or without β-agonist was instillated intratracheally and AFC was measured with radioisotopic tracer., Results: Although there was no effects of CFTRact-16 on AFC in mice with or without isoproterenol, CFinh-172 markedly decreased isoproterenol-stimulated AFC in both normal (P < 0.01) and LPS-induced ALI mice (P < 0.01) and there was significantly decreased basal AFC in ALI mice (P < 0.05)., Conclusions: These results provide direct functional evidence for CFTR in cAMP-mediated AFC in both normal and ALI mice., (© 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.)

Objective: To review the functions of these intracellular signals in their regulation of retinal ganglion cell (RGC) axon regeneration., Data Sources: Relevant articles published in English or Chinese from 1970 to present were selected from PubMed. Searches were made using the terms "intrinsic determinants, axon regeneration, RGC, optic nerve regeneration, and central nervous system axon regeneration.", Study Selection: Articles studying the mechanisms controlling RGC and central nervous system (CNS) axon regeneration were reviewed. Articles focusing on the intrinsic determinants of axon regeneration were selected., Results: Like other CNS neurons of mammals, RGCs undergo a developmental loss in their ability to grow axons as they mature, which is a critical contributing factor to the failure of nerve regeneration and repair after injury. This growth failure can be attributed, at least in part, by the induction of molecular programs preventing cellular overgrowth and termination of axonal growth upon maturation. Key intracellular signals and transcription factors, including B cell lymphoma/leukemia 2, cyclic adenine monophosphate, mammalian target of rapamycin, and Krüppel-like transcription factors, have been identified to play central roles in this process., Conclusions: Intense effort and substantial progress have been made to identify the various intrinsic growth pathways that regulate RGC axon regeneration. More work is needed to elucidate the mechanisms of and the interrelationship between the actions of these factors and to successfully achieve regeneration and repair of the severed RGC axons.

In previous studies, we have found that extracellular guanosine can stimulate endogenous progenitor/stem cell proliferation in the spinal cord following chronic injury and in the subventricular zone of the brains of rats afflicted with Parkinson's Disease. In this study, using neural stem cells isolated from one-day old rats, we found that guanosine could stimulate neural stem cell proliferation, and that the proliferation was not due to the guanosine metabolism mechanism since guanine, which is interconverted by an ecto-purine nucleoside phosphorylase from guanosine, has no stimulating effect on the proliferation of neural stem cells. We determined that second messenger cAMP was involved in the pathway as results showed that 100 microM guanosine stimulated cAMP accumulation. Using western blot analysis, we found that 100 microM guanosine can activate the phosphorylation of CREB without changing the total amount of CREB. In conclusion, guanosine can stimulate neural stem cell proliferation, and the cAMP-CREB pathway is involved in this biological effect.

Catecholamines regulate the β-adrenoceptor/cyclic AMP-regulated protein kinase A (cAMP/PKA) pathway. Deregulation of this pathway can cause apoptotic cell death and is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. Here we demonstrate that the β-adrenoceptor/cAMP/PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member Bim in tissues such as the thymus and the heart. In these cell types, the catecholamine-mediated apoptosis is abrogated by loss of Bim. Induction of Bim is driven by the transcriptional co-activator CBP (CREB-binding protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the Bim promoter site. Our findings have implications for understanding pathophysiology associated with a deregulated neuroendocrine system and for developing novel therapeutic strategies for these diseases.

Background: The induction of tolerance and protective immunity to microbes is significantly influenced by host- and microbiota-derived metabolites, such as histamine., Objective: We sought to identify the molecular mechanisms for histamine-mediated modulation of pattern recognition receptor signaling., Methods: Human monocyte-derived dendritic cells (MDDCs), myeloid dendritic cells, and plasmacytoid dendritic cells were examined. Cytokine secretion, gene expression, and transcription factor activation were measured after stimulation with microbial ligands and histamine. Histamine receptor 2 (H₂R)-deficient mice, histamine receptors, and their signaling pathways were investigated., Results: Histamine suppressed MDDC chemokine and proinflammatory cytokine secretion, nuclear factor κB and activator protein 1 activation, mitogen-activated protein kinase phosphorylation, and T(H)1 polarization of naive lymphocytes, whereas IL-10 secretion was enhanced in response to LPS and Pam3Cys. Histamine also suppressed LPS-induced myeloid dendritic cell TNF-α secretion and suppressed CpG-induced plasmacytoid dendritic cell IFN-α gene expression. H₂R signaling through cyclic AMP and exchange protein directly activated by cyclic AMP was required for the histamine effect on LPS-induced MDDC responses. Lactobacillus rhamnosus, which secretes histamine, significantly suppressed Peyer patch IL-2, IL-4, IL-5, IL-12, TNF-α, and GM-CSF secretion in wild-type but not H₂R-deficient animals., Conclusion: Both host- and microbiota-derived histamine significantly alter the innate immune response to microbes through H₂R., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background & Aims: The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts., Methods: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses., Results: Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation., Conclusions: Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Abnormal pigmentation, particularly hyperpigmentation, is major issue of concern for people with colored skin. Several hypopigmenting agents, which exert their action by inhibiting tyrosinase activity and/or transcription, have been used for treatment. However, results have been discouraging. To manage abnormal pigmentation properly, the mechanisms of melanogenesis should be understood. Endogenous and exogenous factors affect melanogenesis via intracellular machineries. cAMP and PKC are critical factors of important transduction pathways and cross-talk between them could amplify the melanogenic effect. Here, factors involved in melanogenesis regulation via cAMP and/or PKC pathways are reviewed with their action mechanisms.

Background: Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change., Methods: In an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied., Results: Dose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP., Conclusions: cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

Introduction: Levosimendan is approved for left heart failure and is also used in right heart failure to reduce right ventricular afterload. Despite the fact that pulmonary arteries (PAs) and pulmonary veins (PVs) contribute to cardiac load, their responses to levosimendan are largely unknown., Materials and Methods: Levosimendan-induced vasorelaxation of PAs and PVs was studied in precision-cut lung slices from guinea pigs by videomicroscopy; baseline luminal area was defined as 100%. Intracellular cAMP- and cGMP-levels were measured by ELISA and NO end products were determined by the Griess reaction., Results: Levosimendan relaxed control PVs (116%) and those pre-constricted with an endothelinA-receptor agonist (119%). PAs were only relaxed if pre-constricted (115%). Inhibition of KATP-channels (glibenclamide), adenyl cyclase (SQ 22536) and protein kinase G (KT 5823) largely attenuated the levosimendan-induced relaxation in control PVs, as well as in pre-constricted PAs and PVs. Inhibition of BKCa (2+)-channels (iberiotoxin) and Kv-channels (4-aminopyridine) only contributed to the relaxant effect of levosimendan in pre-constricted PAs. In both PAs and PVs, levosimendan increased intracellular cAMP- and cGMP-levels, whereas NO end products remained unchanged. Notably, basal NO-levels were higher in PVs. The KATP-channel activator levcromakalim relaxed PAs dependent on cAMP/PKA/PKG and increased cAMP-levels in PAs., Discussion: Levosimendan initiates complex and divergent signaling pathways in PAs and PVs. Levosimendan relaxes PAs and PVs primarily via KATP-channels and cAMP/cGMP; in PAs, BKCa (2+)- and Kv-channels are also involved. Our findings with levcromakalim do further suggest that in PAs the activation of KATP-channels leads to the production of cAMP/PKA/PKG. In conclusion, these results suggest that levosimendan might reduce right ventricular afterload by relaxation of PAs as well as pulmonary hydrostatic pressure and pulmonary edema by relaxation of PVs.

Successful colonization and survival in variable environments require a competitive advantage during the initial growth phase after experiencing nutrient changes. Starved yeast cells anticipate exposure to glucose by activating the Hxt5p (hexose transporter 5) glucose transporter, which provides an advantage during early phases after glucose resupply. cAMP and glucose FRET (fluorescence resonance energy transfer) sensors were used to identify three signalling pathways that co-operate in the anticipatory Hxt5p activity in glucose-starved cells: as expected the Snf1 (sucrose nonfermenting 1) AMP kinase pathway, but, surprisingly, the sugar-dependent G-protein-coupled Gpr1 (G-protein-coupled receptor 1)/cAMP/PKA (protein kinase A) pathway and the Pho85 (phosphate metabolism 85)/Plc (phospholipase C) 6/7 pathway. Gpr1/cAMP/PKA are key elements of a G-protein-coupled sugar response pathway that produces a transient cAMP peak to induce growth-related genes. A novel function of the Gpr1/cAMP/PKA pathway was identified in glucose-starved cells: during starvation the Gpr1/cAMP/PKA pathway is required to maintain Hxt5p activity in the absence of glucose-induced cAMP spiking. During starvation, cAMP levels remain low triggering expression of HXT5, whereas cAMP spiking leads to a shift to the high capacity Hxt isoforms.

The maintenance of endothelial barrier is critical for the vascular homeostasis and is maintained by the interaction of adherens junction (AJ) and tight junction (TJ) proteins between adjacent cells. This interaction is stabilized by actin cytoskeleton forming cortical actin ring. Here, we developed a novel vascular leakage blocker, Sac-1004 and investigated its mechanism of action in endothelial cells (ECs). Sac-1004 inhibited endothelial hyperpermeability induced by vascular endothelial growth factor, histamine and thrombin via stabilization of cortical actin ring and AJ proteins at the cell-cell junction. Treatment of Sac-1004 in ECs increased cAMP levels and activated Rac, both of which are known to strengthen endothelial barrier. Furthermore, Sac-1004 induced phosphorylation of cortactin and its localization at cell membrane that is essential for the stabilization of cortical actin ring. These effects of Sac-1004 on ECs were significantly abrogated by dideoxyadenosine (adenylyl cyclase inhibitor) and NSC23766 (Rac inhibitor). Taken together, our findings indicate that Sac-1004 blocks vascular leakage by enhancing endothelial integrity via the cAMP/Rac/cortactin pathway and imply the potential usefulness of Sac-1004 in the development of therapeutic means for vascular leakage-related diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Purpose: Prostaglandin E₂ (PGE₂) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells., Experimental Approach: bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE₂ with or without agonists/antagonists of PGE₂ receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry., Key Results: PGE₂ significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE₂ effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N⁶-Bnz-cAMP). PGE₂-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11-7082 and MG-132) attenuated the induction of ICAM-1 by PGE₂. PGE₂, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti., Conclusion and Implications: Our findings suggest that PGE₂ induces ICAM-1 expression via EP4 receptor and Epac/Akt/NF-κB signalling pathway in bEnd.3 brain endothelial cells, supporting its pathophysiological role in brain inflammation., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

Rationale: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events., Objective: We investigated the mechanisms underlying the vascular effects of C2238-αANP., Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-αANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-αANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-αANP, as evaluated by intracellular cGMP levels. In contrast, C2238-αANP, but not T2238-αANP, markedly reduced intracellular cAMP levels in an NPR-C-dependent manner. Accordingly, C2238-αANP showed higher affinity binding to NPR-C, than T2238-αANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-αANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-αANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-αANP. Overall, these data indicate that C2238-αANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-αANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results., Conclusions: C2238-αANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers.

Background: cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation., Results: Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP., Conclusions: Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes., Significance: The method is applicable to other gene-tractable intertwined systems. Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.

The beta1-adrenoceptors (β(1)AR) and beta-2 (β(2)AR) adrenoceptors represent the predominant pathway for sympathetic control of myocardial function. Diverse mechanisms have evolved to translate signalling via these two molecules into differential effects on physiology. In this review, we discuss how the functions of the βAR are organized from the level of secondary messengers to the whole heart to achieve this. Using novel microscopy and bio-imaging methods researchers have uncovered subtle organization of the control of cyclic adenosine monophosphate (cAMP), the predominant positively inotropic pathway for the βAR. The β(2)AR in particular is demonstrated to give rise to highly compartmentalized, spatially confined cAMP signals. Organization of β(2)AR within the T-tubule and caveolae of cardiomyocytes concentrates this receptor with molecules which buffer and shape its cAMP signal to give fine control. This situation is undermined in various forms of heart failure. Human and animal models of heart failure demonstrate disruption of cellular micro-architecture which contributes to the change in response to cardiac βARs. Loss of cellular structure has proved key to the observed loss of confined β(2)AR signalling. Some pharmacological and genetic treatments have been successful in returning failing cells to a more structured phenotype. Within these cells it has been possible to observe the partial restoration of normal β(2)AR signalling. At the level of the organ, the expression of the two βAR subtypes varies between regions with the β(2)AR forming a greater proportion of the βAR population at the apex. This distribution may contribute to regional wall motion abnormalities in Takotsubo cardiomyopathy, a syndrome of high sympathetic activity, where the phosphorylated β(2)AR can signal via Gi protein to produce negatively inotropic effects.

Follicle-stimulating hormone (FSH) activates FSH receptors (FSHR) in granulosa cells to induce follicle differentiation, growth and estradiol production. FSH is used clinically to treat female infertility and is administered by injection. To increase patient convenience and compliance, compound homogeneity and composition, low molecular weight (LMW), orally bioavailable, FSHR agonists are now being developed to replace FSH. In this study, we present the signaling mechanisms of a newly developed LMW dihydropyridine agonist of the FSHR, Org 214444-0. Org 214444-0 is shown to be a stereoselective, nanomolar potent FSHR agonist and selective over the structurally related LHR and TSHR. Org 214444-0 is an allosteric agonist interacting with the transmembrane region of the FSHR. When co-incubated with FSH, Org 214444-0 augments FSH's potency in binding (6.5-fold) and adenylyl cyclase/cAMP activation (3.5-fold) in a concentration-dependent manner. Like FSH, Org 214444-0 induces FSHR internalization and is only marginally effective in stimulating phospholipase C. Moreover, Org 214444-0 stimulates cAMP and estradiol production in human granulosa cells in culture and supports the follicular phase in mature female rats. We conclude that Org 214444-0 is a bonafide FSHR agonist., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Th9 cells are a novel Th cell subset that produces IL-9 and is involved in type I hypersensitivity such as airway inflammation. Although its critical roles in asthma have attracted interest, the physiological regulatory mechanisms of Th9 cell differentiation and function are largely unknown. Asthma is easily affected by psychological factors. Therefore, we investigated one of the physiological mediators derived from the nervous system, calcitonin gene-related peptide (CGRP), in asthma and Th9 cells because CGRP and activation of the cAMP/protein kinase A (PKA) pathway by CGRP are known to be important regulators in several immune responses and allergic diseases. In this study, we demonstrated that the CGRP/cAMP/PKA pathway promotes IL-9 production via NFATc2 activation by PKA-dependent glycogen synthase kinase-3β inactivation. Moreover, CGRP also induces the expression of PU.1, a critical transcriptional factor in Th9 cells, which depends on PKA, but not NFATc2. Additionally, we demonstrated the physiological importance of CGRP in IL-9 production and Th9 differentiation using an OVA-induced airway inflammation model and T cell-specific CGRP receptor-deficient mice. The present study revealed a novel regulatory mechanism comprising G protein-coupled receptor ligands and nervous system-derived substances in Th9 cell differentiation and type I hypersensitivity.

The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Because mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity, and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK, and phospho-CREB are higher in rapid eye movement (REM) sleep compared with awake mice but are not elevated in non-REM sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity, and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation.

3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger critically involved in the regulation of heart function. It has been shown to act in discrete subcellular signalling compartments formed by differentially localized receptors, phosphodiesterases and protein kinases. Cardiac diseases such as hypertrophy or heart failure are associated with structural and functional remodelling of these microdomains which leads to changes in cAMP compartmentation. In this review, we will discuss recent key findings which provided new insights into cAMP compartmentation in cardiomyocytes with a particular focus on its alterations in heart disease., (Acta Physiologica © 2013 Scandinavian Physiological Society.)

In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.

Olfactory receptor neurons (ORNs), which undergo lifelong neurogenesis, have been studied extensively to understand how neurons form precise topographical networks. Neural projections from ORNs are principally guided by the genetic code, which directs projections from ORNs that express a specific odorant receptor to the corresponding glomerulus in the olfactory bulb. In addition, ORNs utilise spontaneous firing activity to establish and maintain the neural map. However, neither the process of generating this spontaneous activity nor its role as a guidance cue in the olfactory bulb is clearly understood. Utilising extracellular unit-recordings in mouse olfactory epithelium slices, we demonstrated that the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels in the somas of ORNs depolarise their membranes and boost their spontaneous firing rates by sensing basal cAMP levels; the odorant-sensitive cyclic nucleotide-gated (CNG) channels in cilia do not. The basal cAMP levels were maintained via the standing activation of β-adrenergic receptors. Using a Tet-off system to over-express HCN4 channels resulted in the enhancement of spontaneous ORN activity and dramatically reduced both the size and number of glomeruli in the olfactory bulb. This phenotype was rescued by the administration of doxycycline. These findings suggest that cAMP plays different roles in cilia and soma and that basal cAMP levels in the soma are directly converted via HCN channels into a spontaneous firing frequency that acts as an intrinsic guidance cue for the formation of olfactory networks.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

Aims: Heart failure (HF) is characterized by regionalized contractile alterations resulting in loss of the transmural contractile gradient across the left ventricular free wall. We tested whether a regional alteration in mitochondrial oxidative metabolism during HF could affect myofilament function through protein kinase A (PKA) signaling., Results: Twelve weeks after permanent left coronary artery ligation that induced myocardial infarction (MI), subendocardial (Endo) cardiomyocytes had decreased activity of complex I and IV of the mitochondrial electron transport chain and produced twice more superoxide anions than sham Endo and subepicardial cells. This effect was associated with a reduced antioxidant activity of superoxide dismutase and Catalase only in MI Endo cells. The myofilament contractile properties (Ca(2+) sensitivity and maximal tension), evaluated in skinned cardiomyocytes, were also reduced only in MI Endo myocytes. Conversely, in MI rats treated with the antioxidant N-acetylcysteine (NAC) for 4 weeks, the generation of superoxide anions in Endo cardiomyocytes was normalized and the contractile properties of skinned cardiomyocytes restored. This effect was accompanied by improved in vivo contractility. The beneficial effects of NAC were mediated, at least, in part, through reduction of the PKA activity, which was higher in MI myofilaments, particularly, the PKA-mediated hyperphosphorylation of cardiac Troponin I., Innovation: The Transmural gradient in the mitochondrial content/activity is lost during HF and mediates reactive oxygen species-dependent contractile dysfunction., Conclusions: Regionalized alterations in redox signaling affect the contractile machinery of sub-Endo myocytes through a PKA-dependent pathway that contributes to the loss of the transmural contractile gradient and impairs global contractility.

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

Since the discovery nearly 60 years ago, cAMP is envisioned as one of the most universal and versatile second messengers. The tremendous feature of cAMP to tightly control highly diverse physiologic processes, including calcium homeostasis, metabolism, secretion, muscle contraction, cell fate, and gene transcription, is reflected by the award of five Nobel prizes. The discovery of Epac (exchange protein directly activated by cAMP) has ignited a new surge of cAMP-related research and has depicted novel cAMP properties independent of protein kinase A and cyclic nucleotide-gated channels. The multidomain architecture of Epac determines its activity state and allows cell-type specific protein-protein and protein-lipid interactions that control fine-tuning of pivotal biologic responses through the "old" second messenger cAMP. Compartmentalization of cAMP in space and time, maintained by A-kinase anchoring proteins, phosphodiesterases, and β-arrestins, contributes to the Epac signalosome of small GTPases, phospholipases, mitogen- and lipid-activated kinases, and transcription factors. These novel cAMP sensors seem to implement certain unexpected signaling properties of cAMP and thereby to permit delicate adaptations of biologic responses. Agonists and antagonists selective for Epac are developed and will support further studies on the biologic net outcome of the activation of Epac. This will increase our current knowledge on the pathophysiology of devastating diseases, such as diabetes, cognitive impairment, renal and heart failure, (pulmonary) hypertension, asthma, and chronic obstructive pulmonary disease. Further insights into the cAMP dynamics executed by the Epac signalosome will help to optimize the pharmacological treatment of these diseases.

The cellular form of prion protein PrP(C) is highly expressed in the brain, where it can be converted into its abnormally folded isoform PrP(Sc) to cause neurodegenerative diseases. Its predominant synaptic localization suggests a crucial role in synaptic signaling. Interestingly, PrP(C) is developmentally regulated and its high expression in the immature brain could be instrumental in regulating neurogenesis and cell proliferation. Here, PrP(C)-deficient (Prnp(0/0)) mice were used to assess whether the prion protein is involved in synaptic plasticity processes in the neonatal hippocampus. To this aim, calcium transients associated with giant depolarizing potentials, a hallmark of developmental networks, were transiently paired with mossy fiber activation in such a way that the two events were coincident. While this procedure caused long-term potentiation (LTP) in wild-type (WT) animals, it caused long-term depression (LTD) in Prnp(0/0) mice. Induction of LTP was postsynaptic and required the activation of cAMP-dependent protein kinase A (PKA) signaling. The induction of LTD was presynaptic and relied on G-protein-coupled GluK1 receptor and protein lipase C. In addition, at emerging CA3-CA1 synapses in WT mice, but not in Prnp(0/0) mice, pairing Schaffer collateral stimulation with depolarization of CA1 principal cells induced LTP, known to be PKA dependent. Postsynaptic infusion of a constitutively active isoform of PKA catalytic subunit Cα into CA1 and CA3 principal cells in the hippocampus of Prnp(0/0) mice caused a persistent synaptic facilitation that was occluded by subsequent pairing. These data suggest that PrP(C) plays a crucial role in regulating via PKA synaptic plasticity in the developing hippocampus.

Besides playing a pathogenic role in Alzheimer disease, amyloid-beta peptides are normally produced in low amounts in the brain, and several lines of evidence suggest that they can modulate synaptic plasticity and memory. As cyclic adenosine monophosphate (cAMP) is known to be involved in the same processes and the blockade of its degradation by phosphodiesterase 4 inhibitors has consistently shown beneficial effects on cognition, we investigated the possible correlation between this second messenger and Aβ peptides in neuronal N2a cells overexpressing the amyloid-β precursor protein (APP). We herein report that the elevation of endogenous cAMP by rolipram increased APP protein expression and both its amyloidogenic and nonamyloidogenic processing. The effects of rolipram were reproduced by both the cAMP membrane-permeant analog 8Br-cAMP and the forskolin-induced activation of adenylyl cyclase but were not affected by the PKA inhibitor H-89. Our results demonstrate that, in neuronal cells, APP metabolism is physiologically modulated by cAMP and suggest that this might represent an additional mechanism through which the second messenger could influence memory functions., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Background and Purpose: The Ca(v) 3.2 isoform of T-type Ca(2+) channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE(2) -induced hyperalgesia. Here we examined and analysed Ca(v) 3.2 sensitization via the PGE(2) /cAMP pathway in NG108-15 cells that express Ca(v) 3.2 and produce cAMP in response to PGE(2) , and its impact on mechanical nociceptive processing in rats., Experimental Approach: In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Ca(v) 3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats., Key Results: In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE(2) increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE(2) was abolished by RQ-00015986-00, an EP(4) receptor antagonist. AKAP150 was co-immunoprecipitated with Ca(v) 3.2, regardless of stimulation with db-cAMP, and Ca(v) 3.2 was phosphorylated by db-cAMP or PGE(2) . In rats, intraplantar (i.pl.) administration of db-cAMP or PGE(2) caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl(2) , known to inhibit Ca(v) 3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE(2) -induced mechanical hyperalgesia., Conclusion and Implications: Our findings suggest that PGE(2) causes AKAP-dependent phosphorylation and sensitization of Ca(v) 3.2 through the EP(4) receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Since herkinorin is the first non-opioid mu agonist derived from salvinorin A that has the ability to induce cerebral vascular dilatation, we hypothesized that herkinorin could have similar vascular dilatation effect via the mu and kappa opioid receptors and the cAMP pathway. The binding affinities of herkinorin to kappa and mu opioid receptors were determined by in-vitro competition binding assays. The cerebral arteries were monitored in piglets equipped with a closed cranial window and the artery responses were recorded before and every 30s after injection of artificial cerebrospinal fluid (CSF) in the presence or absence of the investigated drugs: herkinorion, norbinaltorphimine (NTP), a kappa opioid receptor antagonist, β-funaltrexamine (β-FNA), a mu opioid receptor antagonist, or Rp-8-Br-cAMPS (Rp-cAMPS), an inhibitor of protein kinase A (PKA). CSF samples were collected before and 10 min after herkinorin and NTP administration for the measurement of cAMP levels. Data were analyzed by repeated-measures analysis of variance. Our results show that herkinorin binds to both kappa and mu opioid receptors. Its vasodilation effect is totally abolished by NTP, but is not affected by β-FNA. The levels of cAMP in the CSF elevate after herkinorin administration, but are abolished with NTP administration. The cerebral vasodilative effect of herkinorin is also blunted by Rp-cAMPS. In conclusion, as a non-opioid kappa and mu opioid receptor agonist, herkinorin exhibits cerebral vascular dilatation effect. The dilatation is mediated though the kappa opioid receptor rather than the mu opioid receptor. cAMP signaling also plays an important role in this process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate the activity of protein kinase A (PKA) and protein kinase G (PKG), respectively. This process helps maintain circulating platelets in a resting state. Here we studied the role of cAMP and cGMP in the regulation of megakaryocyte (MK) differentiation and platelet formation. Cultured, platelet-producing MKs were differentiated from fetal livers harvested from 13.5 days postcoital mouse embryos. MK development was accompanied by a dramatic increase in cAMP production and expression of soluble guanylate cyclase, PKG, and PKA as well as their downstream targets vasodilator-stimulated phosphoprotein (VASP) and MENA. Stimulation of prostaglandin E(1) receptor/adenylyl cyclase or soluble guanylate cyclase/PKG in cultured MKs increased VASP phosphorylation, indicating that these components share a common signaling pathway. To dissect out the role of cyclic nucleotides in MK differentiation, cAMP/PKA and cGMP/PKG signaling were alternately blocked in cultured MKs. Down-regulation of cAMP pathway effectors decreased MK numbers and ploidy. Notably, cGMP levels increased at the beginning of MK development and returned to basal levels in parallel with MK maturation. However, inhibition of cGMP pathway effectors had no effect on MK development. In addition, platelet release from mature MKs was enhanced by cGMP and inhibited by cAMP. Our data suggest that cAMP plays an important role in MK differentiation, while cAMP and cGMP have opposite effects on platelet production. Identifying the signaling pathways that underpin MK development and proplatelet formation will provide greater insights into thrombopoiesis and may potentially yield useful therapeutic targets., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

To examine the functional regulation of serotonin transporter (SERT) by cAMP, we examined whether SERT uptake activity was affected by dibutyryl cAMP (dbcAMP), a cAMP analog, in SERT-transfected RN46A cells derived from embryonic rat raphe neurons. Long-term exposure (> 4 h) of dbcAMP (1 mM) to SERT-expressing RN46A cells significantly up-regulated SERT activity. In addition, a selective PKA activator, but not a selective EPAC activator, increased the serotonin uptake activity of SERT, suggesting that this regulation was mainly mediated via PKA. Time-dependent up-regulation of SERT activity by dbcAMP was accompanied by neural differentiation of RN46A cells. Further investigation of dbcAMP-induced up-regulation of SERT revealed that dbcAMP elevated SERT protein levels without affecting SERT mRNA transcription. The chase assay for residual SERT protein revealed that dbcAMP slowed its degradation rate. Immunohistochemical analysis revealed that plasma membrane-localized SERT was more abundant in dbcAMP-treated cells than in non-treated cells, suggesting that dbcAMP up-regulated SERT by decreasing its degradation and increasing its plasma membrane expression. These results raise the possibility that the elevation of intracellular cAMP up-regulated SERT function through a mechanism linked to the differentiation of RN46A cells and show the importance of SERT function during the developmental process of the serotonergic nervous system.

In diabetic retinopathy, vision loss is usually secondary to macular edema, which is thought to depend on the functional integrity of the blood-retina barrier. The levels of advanced glycation end products in the vitreous correlate with the progression of diabetic retinopathy. Natriuretic peptides (NP) are expressed in the eye and their receptors are present in the retinal pigment epithelium (RPE). Here, we investigated the effect of glycated-albumin (Glyc-alb), an advanced glycation end product model, on RPE-barrier function and the ability of NP to suppress this response. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of ARPE-19 and human fetal RPE (hfRPE) monolayers. The monolayers were treated with 0.1-100 μg/ml Glyc-alb in the absence or presence of 1 pM to 100 nM apical atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or C-type natriuretic peptide (CNP). Glyc-alb induced a significant reduction in TEER within 2 hours. This response was concentration-dependent (EC(50)= 2.3 μg/ml) with a maximal reduction of 40 ± 2% for ARPE-19 and 27 ± 7% for hfRPE at 100 μg/ml 6 hours post-treatment. One hour pretreatment with ANP, BNP, or CNP blocked the reduction in TEER induced by Glyc-alb (100 μg/ml). The suppression of the Glyc-alb response by NP was dependent on the generation of cyclic guanosine monophosphate and exhibited a rank order of agonist potency consistent with the activation of natriuretic-peptide-receptor-2 (NPR2) subtype (CNP >> BNP ≥ ANP). Our data demonstrate that Glyc-alb is effective in reducing RPE-barrier function, and this response is suppressed by NP. Moreover, these studies support the idea that NPR2 agonists can be potential candidates for treating retinal edema in diabetic patients.

We evaluated the efficacy, potency, and selectivity of the three most commonly used adenylate cyclase (AC) inhibitors in a battery of cell lines constructed to study signaling via three discrete cAMP sensors identified in neuroendocrine cells. SQ22,536 [9-(tetrahydrofuryl)-adenine] and 2',5'-dideoxyadenosine (ddAd) are effective and potent AC inhibitors in HEK293 cells expressing a cAMP response element (CRE) reporter gene, and MDL-12,330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride] is not. Neuroscreen-1 (NS-1) cells were used to assess the specificity of the most potent AC inhibitor, SQ22,536, to block downstream cAMP signaling to phosphorylate CREB (via PKA); to activate Rap1 (via Epac); and to activate ERK signaling leading to neuritogenesis (via the newly described neuritogenic cAMP sensor NCS). SQ22,536 failed to inhibit the effects of cAMP analogs 8-Br-cAMP and 8-CPT-2'-O-Me-cAMP on PKA-mediated CREB activation/phosphorylation and Epac-mediated Rap1 activation, indicating that it does not inhibit these cAMP pathways beyond the level of AC. On the other hand, SQ22,536, but not ddAd, inhibited the effects of cAMP analogs 8-Br-cAMP and 8-CPT-cAMP on ERK phosphorylation and neuritogenesis, indicating that it acts not only as an AC blocker, but also as an inhibitor of the NCS. The observed off-target actions of SQ22,536 are specific to cAMP signaling: SQ22,536 does not block the actions of compounds not related to cAMP signaling, including ERK induction by PMA, and ERK activation and neuritogenesis induced by NGF. These data led us to indicate a second target for SQ22,536 that should be considered when interpreting its effects in whole cell and in vivo experiments.

Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the biosynthesis of cholesterol, fatty acid, and triglyceride. They control the expression of crucial genes involved in lipogenesis and uptake. In this review, we summarize the processing of SREBPs and their regulation by insulin, cAMP, and vitamin A, and the relationship between miRNA and lipid metabolism. We also discuss the recent functional studies on SREBPs. These discoveries suggest that inhibition of SREBP can be a novel strategy to treat metabolic diseases, such as type II diabetes, insulin resistance, fatty liver, and atherosclerosis.

Interleukin 4 (IL-4) is a pleiotropic cytokine involved in many functions during the development as well as in adult life. Previous work from our group demonstrated, in vitro, that this interleukin is able to prevent rat retinal ganglion cells death after axotomy. The aim of the present study was to investigate the signaling pathways involved in this trophic effect, particularly the cAMP pathway and also to demonstrate the expression of IL-4 in retinas at different stages of post natal development. Our results show that the trophic effect of IL-4 on rat retinal ganglion cells is dependent on the activation of Janus Kinase 3, Protein Kinase A, c-Jun N-terminal Kinase and Tropomyosin related Kinase receptors, on the increase in intracellular calcium levels, on polypeptide release and on the endogenous Brain Derived Neurotrophic Factor (BDNF). We also observed that treatment with IL-4 enhances c-AMP response element binding and Mitogen Activated Protein Kinase phosphorylation and increases the expression of BDNF. Concerning the IL-4 expression our data show an increase in IL-4 levels during post natal development. Taken together our results demonstrate that the trophic effect of IL-4 on retinal ganglion cells of newborn rats is mediated by cAMP pathway and BDNF release.

The function of Ca(2+) and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca(2+) and reactivated with MgATP(2-) (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13-27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca(2+) (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875-1883), beat with three-dimensional waves if they were reactivated with low MgATP(2-). At a high MgATP(2-), almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca(2+). These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP(2-).