Intracellular cAMP Concentration Research Articles

Expression of FSHR in chondrocytes and the effect of FSH on chondrocytes

ObjectiveChondrocytes express many kinds of hormone receptors. The function of Follicle stimulating hormone (FSH) in the ovary is mediated by FSH receptor (FSHR). FSH receptor (FSHR) is found in many non-ovarian tissues, however it has been unclear if chondrocytes express FSHR. The purpose of this study is to determine it. MethodsMouse primary chondrocytes and human articular cartilage tissues were examined. The expression and sequence of FSHR mRNA by reverse transcription polymerase chain reaction (RT-PCR) and sequenced, respectively, and its protein expression was tested using western blotting and location was observed under immunofluorescence microscopy. Ovarian tissue was as a positive control. After FSH stimulated mouse chondrocytes, intracellular cAMP levels were assessed by ELISA, and gene expression relative to Mouse WNT Signaling Pathway was tested by RT2 Profiler PCR Arrays. ResultsFSHR was detected at the transcriptional level and confirmed to have the same sequence as that of ovary-derived mRNA of FSHR. FSHR proteins presented at the same line as the positive proteins of ovary, in mouse chondrocytes and human cartilage tissue, respectively. FSHR proteins were located at the cell membrane. Intracellular cAMP contents were significantly elevated up to 7-fold in mouse chondrocytes by forskolin (100 mM) (P < 0.001); however, different doses of FSH did not change the cAMP contents in mouse primary chondrocytes. RT2 Profiler PCR Arrays demonstrated that FSH could cause changes in gene expression among the 84 preordained genes, such as Fosl1, Rhou, and Dkk1, in mouse chondrocytes relative to the control. ConclusionMouse chondrocytes and human articular cartilage express functional FSHR. Moreover, FSH can act on chondrocytes and cause genetic changes.

PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana.

The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i) whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

ID: 1039 The antidepressant fluoxetine inhibits adenylate cyclase stimulation by FSH or Forskolin in the COV434 human ovarian granulosa tumor cell line

Fluoxetine (Prozac), a selective Serotonin Reuptake Inhibitor antidepressant, exhibits other mechanisms of action in various cell types and has been shown to induce cell death in cancer cells, paving the way for its potential use in cancer therapy. The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis, and human granulosa cells are essential for scientific research to improve the understanding of these two processes. However, little is known about fundamental signaling pathways in human granulosa cells. In this study, we investigated the dynamics of intracellular cyclic adenosine monophosphate AMP, a conserved signaling messenger that can regulate virtually every physiological process. We show that incubating COV434 human ovarian granulosa cells with fluoxetine induces a decrease in intracellular cAMP response to Follicle-stimulating hormone (FSH) and forskolin (FSK). In order to study the intracellular cAMP kinetic responses of COV434 cells to FSH or FSK, we used COV434 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be monitored, by using oxiluciferin luminescence produced from catalyzed luciferin oxidation. Our data show that fluoxetine induces an increase in the extracellular Ca2+ entry and reduces ATP concentration as well as cell viability. Targeting these signaling pathways with fluoxetine could permit to get better knowledge in the molecular mechanisms involved in ovarian follicular development

High extracellular Ca2+ enhances the adipocyte accumulation of bone marrow stromal cells through a decrease in cAMP

Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca2+]o enhances adipocyte accumulation.We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca2+ and Sr2+) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca2+]o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1μM forskolin and 200nM IBMX) and a cAMP analog (10μM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca2+]o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca2+]o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25–40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP.

Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice

Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.

Abstract 5583: Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells

Abstract Extracellular adenosine can be generated from ATP and/or ADP through the concerted action of the ectoenzymes CD39 and CD73. Adenosine can bind different type-1 purinergic receptors eliciting potent cytoprotective and immunosuppressive effects. We previously focused on chronic lymphocytic leukemia (CLL), a disease characterized by the progressive expansion of a mature population of B lymphocytes, showing that a subset of patients with a CD73+ clone possess a poor prognosis. This patient subset can actively generate adenosine, which favors leukemic cell survival, synthesis of immunosuppressive cytokines, while inhibiting T lymphocyte proliferation. ADA2 is a human enzyme that catalyzes the conversion of adenosine into inosine. We hypothesized that depletion of high adenosine levels from the tumor microenvironment through the administration of exogenous ADA2 could be therapeutically relevant to limit tumor protection and immunosuppression. Several engineered PEG-ADA2 variants possessed improved enzyme activity and pharmacokinetics compared to wild-type ADA2. Inhibition of adenosine receptor activation was evaluated by measuring intracellular cAMP concentrations in T lymphocytes purified from healthy donors. Adenosine depletion was observed with increasing concentrations of PEG-ADA2-R222Q/S265N and significantly reduced cAMP levels. PEG-ADA2 variants were then tested on CD73+ CLL lymphocytes to determine their effects on extracellular adenosine and inosine. Treatment of leukemic B cells with PEG-ADA2-R222Q/S265N and PEG-ADA2-R222Q/S265N/K374D resulted in complete removal of high levels of adenosine, as measured by HPLC. Conversely, PEG-ADA2-E182T, which possesses significantly attenuated catalytic activity, was less effective in reducing adenosine. The functional effects of adenosine depletion on CLL cell survival were analyzed following treatment with the chemotherapeutic, DNA-damaging agent etoposide that robustly induces apoptosis within 16 hours. Addition of exogenous adenosine to cultures of purified CD73+ CLL cells significantly rescued cells from etoposide-induced apoptosis. However, when these primary leukemic cells were pretreated with PEG-ADA2-R222Q/S265N or PEG-ADA2-R222Q/S265N/K374D, the cell viability rate was significantly decreased, abrogating the cytoprotective effects of adenosine. On the contrary, PEG-ADA2-E182T had a minimal effect, suggesting enzymatic depletion of adenosine is critical to observe these effects. Similar effects were observed by the PEG-ADA2 variants on a CLL cell line expressing CD73+. These preliminary results suggest that enzymatic depletion of extracellular adenosine following treatment with PEG-ADA2 is a relevant approach to counteract the cytoprotective effects of adenosine, warranting further development of PEG-ADA2 as a possible approach to treat CLL. Citation Format: Sara Serra, Cinzia Bologna, Luz Londono, Lin Wang, Michael Shepard, Sanna Rosengren, Christopher Thanos, Silvia Deaglio. Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2017-5583

Erratum to: Dynamic monitoring of Gi/o-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors.

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Forster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.

Identification of the fatty acid activation site on human ClC-2.

Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl- channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET691, of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP]i), EP2, or EP4 receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl- current activation (increasing EC50s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA655/RGEA691) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl- currents without increasing [cAMP]i, while PGE2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP2 or EP4 receptors. L-161,982, a supposed EP4-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl- currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i, and EP2 or EP4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.

Dynamic monitoring of Gi/o-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.

CAMP: a multifaceted modulator of immune synapse assembly and T cell activation

T Lymphocyte activation involves a substantial reorganization of the membranous and intracellular compartments. Signaling complexes assemble and dismantle in a highly ordered fashion in both compartments and orchestrate the activation of T cells with high sensitivity and specificity. TCR ligation leads to a short burst of cAMP production, which is centrally required for T cell activation; however, sustained elevations in intracellular cAMP concentrations are immunosuppressive. Emerging evidence of the existence of local cAMP pools gleaned from studies on other cell types suggests that cAMP compartmentalization may account, in part, for these opposing effects. Whereas cAMP compartmentalization has been identified as a central factor in the control of the cAMP-dependent processes in other cell types, this has, as yet, not been addressed in T lymphocytes. In this review, we discuss the role of cAMP in T cell activation and differentiation, with an emphasis on the effects mediated by the cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC)1, and on the regulatory proteins that may control the generation of local cAMP pools in T cells. We also present an overview of the available tools to image cAMP production at the subcellular level and discuss how bacterial adenylate cyclase (AC) toxins that are known to generate local cAMP pools can be exploited to address the role of cAMP compartmentalization in T cell activation.

Aminophylline Effect on Renal Ischemia-Reperfusion Injury in Mice

BackgroundAminophylline increases the intracellular concentration of cAMP and exerts an anti-inflammatory effect. The aim of this study was to investigate the effect of aminophylline on renal ischemia-reperfusion (I/R) injury in mice. MethodsThirty C57BL/6 mice were divided into 3 groups. In the sham group (group S, n = 10), only right nephrectomy was performed. In the control group (group C, n = 10), after right nephrectomy, the mice were subjected to 30 minutes of left renal ischemia. In the aminophylline group (group A, n = 10), an intraperitoneal injection of aminophylline (5 mg/kg) was performed before renal ischemia. Twenty-four hours after reperfusion, the mice were euthanized, and plasma and kidney samples were obtained to analyze the serum creatinine, renal histology, and expression levels of nuclear factor-kappa B (NF-kB) and pro-inflammatory cytokines. ResultsThe serum creatinine concentration in group C was markedly elevated at 24 hours after reperfusion. Aminophylline treatment significantly reduced serum creatinine, compared with group C. Aminophylline also reduced the histological evidence of renal damage. The expression levels of NF-kB, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and intercellular adhesion molecule-1 (ICAM-1) mRNA were significantly increased in group C (P < .001). Group A showed lower expression of NF-kB, TNF-α, MCP-1, MIP-2, and ICAM-1 mRNA than group C (P < .01). ConclusionsAminophylline treatment improved the renal function and indexes of renal inflammation, which suggests that it provided reno-protection against renal I/R injury.

Examining the Role of Phosphorylation on Interactions between the Cardiac Potassium Channel Alpha-Subunits hERG and KVLQT1

KvLQT1 and hERG are the voltage-gated K+ channel α-subunits of the channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and cell culture have motivated our investigations into interactions between hERG and KvLQT1. These data suggest that a dynamic physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which HERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is thought to play a regulatory role in this process: modifying the phosphorylation state of each the proteins has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP concentration. Through classic biochemical assays and quantitative FRET approaches, we aim to characterize the effects of phosphorylation in regulating interactions between KvLQT1 and hERG in cellular model systems. We have developed ion channel fusions to fluorescent proteins, which include hERG and KvLQT1 phosphonull and phosphomimetic mutants. We hypothesize that phosphorylation abrogates protein-protein interactions, as suggested by findings that increased cAMP levels leads to decreased hERG-KvLQT1 interaction. This work potentially furthers our understanding of hERG-KvLQT1 interactions and may elucidate mechanisms that underlie many types of arrhythmia as well as characterize novel interactions between two distinct potassium channel families.

Naringenin Regulates CFTR Activation and Expression in Airway Epithelial Cells

Background/Aims: Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin. Methods: Vectorial Cl^- transport was determined by measuring short-circuit current (I_SC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR. Results: Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in I_SC. The current was suppressed when exposed to Cl^–-free solution or by bumetanide, BaCl₂, and DPC but not by DIDS and IBMX. Forskolin-induced I_SC increase and CFTRinh-172/MDL-12330A-induced I_SC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression. Conclusion: These results demonstrate that naringenin has the ability to stimulate Cl^- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum.

M64 - Genome-Wide Association Analyses Reveal Snp-By-Consumption Interaction Effects On ALT And AST Levels In Alcohol Dependent Patients

Background Heavy alcohol use is causally associated with liver dysfunction. Prior genome wide association studies (GWAS) identified single nucleotide polymorphisms (SNPs) associated with liver cirrhosis or elevation of liver enzymes (including aspartate aminotransferase or AST and alanine aminotransferase or ALT) in alcohol dependent subjects. Yet, it is possible that effects of SNPs on liver function may depend on the extent of recent alcohol exposure, which was not investigated in these studies. To address this gap in our knowledge, we investigated SNP-by-consumption interaction effects on the levels of three liver enzymes (AST, ALT and gamma-glutamyl transferase or GGT) commonly used in clinical practice to assess liver function in alcohol dependent subjects Methods Self-reported alcohol consumption during previous 30 days (measured by Timeline Follow back), DNA and plasma samples for measurement of AST, ALT and GGT levels were collected from 480 alcohol dependent subjects treated in community-based treatment programs. Genotyping was conducted using Illumina HumanCore array. Multivariable linear regression models were used to assess the effects of SNP-by-consumption interaction on ALT, AST and GGT levels. Results Genome-wide significant evidence of consumption-interaction effects on AST (p Discussion Genomic location of our top association findings indicates their potential involvement in the essential cellular function, which may also be relevant for alcohol-related liver damage. Specifically, EXOSC10 gene is a putative catalytic component of the RNA exosome complex involved in proper maturation of stable RNA species such as rRNA, snRNA and snoRNA as well as elimination of RNA processing by-products. Moreover, the top SNP in ALT analysis (EXOSC10 rs2258621) might be a blood eQTL for MTOR, which is a serine/threonine protein kinase, belonging to a family of kinases mediating cellular responses to stresses such as DNA damage and nutrient deprivation. Similarly, GALNT2 encodes protein involved in metabolism and O-linked glycosylation pathways, while PDE9A encodes protein involved in signal transduction by regulating the intracellular concentration of cAMP and cGMP. Replication in the independent sample as well as investigation of the functional mechanisms behind these associations needs to follow.

Functional expression of G protein-coupled receptor 30 in immature rat epididymal epithelium.

The aim of this study is to investigate the functional role of G protein-coupled receptor 30 (GPR30) in the epididymis. We found that GPR30 is expressed in the epithelium of the immature rat epididymis and is involved in chloride secretion into the caudal epididymis lumen. The short-circuit current (Isc) experiments showed that in primary cultured caudal epididymis epithelium, activation of GPR30 by its specific agonist G1 induced a mono-phasic current increase, and G15, the specific antagonist of GPR30, could completely inhibit the current induced by G1. The G1-induced Isc was largely blocked by application of the non-specific chloride channel inhibitor diphenylamine-dicarboxylic acid (DPC), or by the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 , suggesting that the current was mainly mediated through CFTR. In addition, after stimulating GPR30 by G1, the intracellular concentration of cAMP in the epithelium was significantly increased, indicating that the cAMP signal pathway is involved and could be responsible for the CFTR activation. Finally, to further investigate the function of GPR30 in vivo, G15 was administrated into rats subcutaneously. The osmotic pressure of the micro perfusion solution from epididymis was measured and the sperms were collected. Results showed that there was an osmotic pressure increase of the perfusion solution from G15 treated rats. When the GPR30 was inhibited by G15 endogenously, the motility of sperms decreased. Our data demonstrated that GPR30 is involved in the formation of caudal epididymis fluid micro-environment thus affecting sperm motility.

AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity

The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs).Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP]i). Type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP]i. The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient.The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine.

Enhancing cAMP Levels as Strategy for the Treatment of Neuropsychiatric Disorders.

Phosphodiesterases (PDEs) have gained attention as potential pharmacological targets for neuropsychiatric diseases due to their ability to hydrolyze the second messengers, cGMP and/or cAMP. It is well-known the relationship between cAMP signaling and inflammation. Since neuroinflammation is considered to play an important role in the pathology of brain disorders, inhibition of PDEs highly expressed in brain has emerged as an innovative strategy for the treatment of these pathologies. PDE4, 7, 8 and 10 inhibitors are presented here as promising drug candidates to overcome the partial efficacy and adverse effects of the current therapy for neuropsychiatric illness such as depression, cognitive impairments or schizophrenia. Enhancing the intracellular concentration of cAMP underlies the effects of PDE inhibitors in the above mentioned disorders.

Angiotensin II counteracts the effects of cAMP/PKA on NHE3 activity and phosphorylation in proximal tubule cells.

Binding of angiotensin II (ANG II) to the AT1 receptor (AT1R) in the proximal tubule stimulates Na+/H+ exchanger isoform 3 (NHE3) activity through multiple signaling pathways. However, the effects of ANG II/AT1R-induced inihibitory G protein (Gi) activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that ANG II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, ANG II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), ANG II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of ANG II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in ANG II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of ANG II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that ANG II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by ANG II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.

SiRNA-mediated silencing of phosphodiesterase 4B expression affects the production of cytokines in endotoxin-stimulated primary cultured microglia

Phosphodiesterase 4 (PDE4) has four subtypes: PDE4A, PDE4B, PDE4C and PDE4D. The expression of PDE4 subtypes in microglial cells and the specific contribution of each subtype to inflammation remain unclear. In this study, the expression of PDE4 subtypes in primary microglial cells was assayed. Primary microglial cells were then transfected with specific small interfering RNA (siRNA) against each PDE4 subtype. PDE4 subtype A-D knockdown was confirmed by quantitative polymerase chain reaction. Secreted cytokines in the supernatant and intracellular cyclic adenosine monophosphate (cAMP) levels of transfected cells were measured. The effect of PDE4B siRNA on the activation of extracellular regulated protein kinase (ERK) induced by lipopolysaccharide (LPS) in microglia was further tested by western blotting. Results showed that the primary microglial cells expressed all four types of PDE4s at the protein level. Transfection with the four siRNAs inhibited PDE4 subtype A-D mRNA expression, respectively. In primary microglial cells, treatment with PDE4B siRNA significantly inhibited the expression of tumor necrosis factor-α and interleukin (IL)-1β, and enhanced the expression of cAMP, while siRNAs to other subtypes had no significant effects. However, none of the four siRNAs had any significant effect on the expression of IL-10. Furthermore, in the PDE4B group, the level of phosphorylated ERK was reduced. Among the four PDE4 subtypes, PDE4B plays an important role in regulating inflammatory responses in microglia, potentially through initially regulating the intracellular cAMP concentration.

Distinct transcriptional regulation of the two Escherichia coli transhydrogenases PntAB and UdhA.

Transhydrogenases catalyse interconversion of the redox cofactors NADH and NADPH, thereby conveying metabolic flexibility to balance catabolic NADPH formation with anabolic or stress-based consumption of NADPH. Escherichia coli is one of the very few microbes that possesses two isoforms: the membrane-bound, proton-translocating transhydrogenase PntAB and the cytosolic, energy-independent transhydrogenase UdhA. Despite their physiological relevance, we have only fragmented information on their regulation and the signals coordinating their counteracting activities. Here we investigated PntAB and UdhA regulation by studying transcriptional responses to environmental and genetic perturbations. By testing pntAB and udhA GFP reporter constructs in the background of WT E. coli and 62 transcription factor mutants during growth on different carbon sources, we show distinct transcriptional regulation of the two transhydrogenase promoters. Surprisingly, transhydrogenase regulation was independent of the actual catabolic overproduction or underproduction of NADPH but responded to nutrient levels and growth rate in a fashion that matches the cellular need for the redox cofactors NADPH and/or NADH. Specifically, the identified transcription factors Lrp, ArgP and Crp link transhydrogenase expression to particular amino acids and intracellular concentrations of cAMP. The overall identified set of regulators establishes a primarily biosynthetic role for PntAB and link UdhA to respiration.