Adrenergic Receptor Signaling Pathway Research Articles

Exploring the potential pharmacological mechanism of aripiprazole against hyperprolactinemia based on network pharmacology and molecular docking

The current primary therapeutic approach for schizophrenia is antipsychotic medication, and antipsychotic-induced hyperprolactinemia occurs in 40–80% of patients with schizophrenia. Aripiprazole, an atypical antipsychotic belonging to the quinolinone derivative class, can reduce the likelihood of developing hyperprolactinemia, but the pharmacological mechanisms of this reduction are unknown. This study aimed to explore the molecular mechanism of action of aripiprazole in treating hyperprolactinemia based on network pharmacology and molecular docking techniques. This study identified a total of 151 potential targets for aripiprazole from the DrugBank, TCMSP, BATMAN-TCM, TargetNet, and SwissTargetPrediction databases. Additionally, 71 hyperprolactinemia targets were obtained from the PharmGKB, DrugBank, TTD, GeneCards, OMIM, and DisGENET databases. Utilizing Venny 2.1.0 software, an intersection of 27 genes was identified between aripiprazole and hyperprolactinemia. To construct a common target protein–protein interaction (PPI) network, the common targets obtained from both sources were input into the STRING database. The resulting PPI network was then imported into Cytoscape 3.7.2 software, which identified eight core targets associated with aripiprazole’s treatment of hyperprolactinemia. Subsequently, a PPI network was established for these targets. Enrichment analysis of the key targets was conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes in the DAVID database. Additionally, molecular docking verification of the interaction between aripiprazole and the core targets was performed using AutoDock Vina software. Aripiprazole’s intervention in hyperprolactinemia primarily targets the following core proteins: Solute Carrier Family 6 Member 3 (SLC6A3), monoamine oxidase (MAO-B), Dopamine D2 receptor (DRD2), 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A), 5-hydroxytryptamine (serotonin) receptor 2C (HTR2C), cytochrome P450 2D6 (CYP2D6), Dopamine D1 receptor (DRD1), Dopamine D4 receptor (DRD4). These targets are predominantly involved in biological processes such as the adenylate cyclase-activating adrenergic receptor signaling pathway, G-protein coupled receptor signaling pathway coupled to cyclic nucleotide second messenger, phospholipase C-activating G-protein coupled receptor signaling pathway, chemical synaptic transmission, and response to xenobiotic stimulus. Primary enrichment occurs in signaling pathways such as the neuroactive ligand-receptor interaction and serotonergic synapse pathways. Molecular docking results demonstrate a favorable affinity between aripiprazole and the core target proteins MAO-B, DRD2, SLC6A3, HTR2C, HTR2A, CYP2D6, DRD4, and DRD1. Network pharmacology predicted potential targets and signaling pathways for aripiprazole’s intervention in hyperprolactinemia, offering theoretical support and a reference basis for optimizing clinical strategies and drug development involving aripiprazole.

An Ethanol Extract of Perilla frutescens Leaves Suppresses Adrenergic Agonist-Induced Metastatic Ability of Cancer Cells by Inhibiting Src-Mediated EMT.

Previous studies have indicated that the adrenergic receptor signaling pathway plays a fundamental role in chronic stress-induced cancer metastasis. In this study, we investigated whether an ethanol extract of Perilla frutescens leaves (EPF) traditionally used to treat stress-related symptoms by moving Qi could regulate the adrenergic agonist-induced metastatic ability of cancer cells. Our results show that adrenergic agonists including norepinephrine (NE), epinephrine (E), and isoproterenol (ISO) increased migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocellular carcinoma cells. However, such increases were completely abrogated by EPF treatment. E/NE induced downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Slug. Such effects were clearly reversed by pretreatment with EPF, suggesting that the antimetastatic activity of EPF could be related to epithelial-mesenchymal transition (EMT) regulation. EPF suppressed E/NE-stimulated Src phosphorylation. Inhibition of Src kinase activity with dasatinib completely suppressed the E/NE-induced EMT process. Transfecting MDA-MB-231 cells with constitutively activated Src (SrcY527F) diminished the antimigration effect of EPF. Taken together, our results demonstrate that EPF can suppress the adrenergic agonist-promoted metastatic ability of cancer cells by inhibiting Src-mediated EMT. This study provides basic evidence supporting the probable use of EPF to prevent metastasis in cancer patients, especially those under chronic stress.

Upregulations of α1 adrenergic receptors and noradrenaline synthases in the medial prefrontal cortex are associated with emotional and cognitive dysregulation induced by post-weaning social isolation in male rats

Early-life social isolation induces emotional and cognitive dysregulation, such as increased aggression and anxiety, and decreases neuron excitability in the medial prefrontal cortex (mPFC). The noradrenergic system in the mPFC regulates emotion and cognitive function via α1 or α2A adrenergic receptors, depending on noradrenaline levels. However, social isolation-induced changes in the mPFC noradrenergic system have not been reported. Here, male Wistar rats received post-weaning social isolation for nine consecutive weeks and were administered behavioral tests (novel object recognition, elevated plus maze, aggression, and forced swimming, sequentially). Protein expression levels in the mPFC noradrenergic system (α1 and α2A adrenergic receptors, tyrosine hydroxylase, and dopamine-β-hydroxylase used as indices of noradrenaline synthesis and release) were examined through western blotting. Social isolation caused cognitive dysfunction, anxiety-like behavior, and aggression, but not behavioral despair. Socially-isolated rats exhibited increased protein levels of the α1 adrenergic receptor, tyrosine hydroxylase, and dopamine-β-hydroxylase in the mPFC; there was no significant difference between the groups in the α2A adrenergic receptor expression levels. Preferential activation of the α1 adrenergic receptor caused by high noradrenaline concentration in the mPFC may be involved in social isolation-induced emotional and cognitive regulation impairments. Targeting the α1 adrenergic receptor signaling pathway is a potential therapeutic strategy for psychiatric disorders with symptomatic features such as emotional and cognitive dysregulation.

Potato protease inhibitor II prevents obesity by inducing browning of white adipose tissue in mice via β3 adrenergic receptor signaling pathway.

There are currently few effective and safe pharmacologic means for inducing beige adipogenesis in humans. This study highlights the role of potato protease inhibitor II (PPI II) in regulating the browning of adipose tissue. The in vitro results showed that PPI II increased the expression of the uncoupling protein 1 (UCP1) protein and gene and beige-specific genes, including Cd137, Cited1, Tbx1, and Tmem26 in vitro. PPI II treatment for three months in diet-induced obesity mice increased the levels of the UCP1 protein in white adipose tissue, causing elevated energy expenditure, thus preventing obesity and improving glucose tolerance. Mechanistic studies further revealed that PPI II regulated the abundance and activity of β3 adrenergic receptor (β3 -AR) in white adipocytes. Chemical-inhibition experiments revealed the crucial role of β3 -AR-dependent protein kinase A (PKA)-p38 kinase (p38)/extracellular signal-related kinase1/2 (ERK1/2) signaling in PPI II-mediated browning program of white adipose tissues. In summary, our findings highlight the role of PPI II in beige adipocyte differentiation and thermogenesis and provide new insights into its use in preventing obesity.

Insulin acts as an atypical KCNQ1/KCNE1‐current activator and reverses long QT in insulin‐resistant aged rats by accelerating the ventricular action potential repolarization through affecting the β3‐adrenergic receptor signaling pathway

Insufficient-heart function is associated with myocardial insulin resistance in the elderly, particularlyassociated with long-QT, in a dependency on dysfunctional KCNQ1/KCNE1-channels. So, we aimed to examine the contribution of alterations in KCNQ1/KCNE1-current (IKs ) to the aging-related remodeling of the heart as well as the role of insulin treatment on IKs in the aged rats. Prolonged late-phase action potential (AP) repolarization of ventricular cardiomyocytes from insulin-resistant 24-month-old rats was significantly reversed by in vitro treatment of insulin or PKG inhibitor (in vivo, as well) via recovery in depressed IKs . Although the protein level of either KCNQ1 or KCNE1 in cardiomyocytes was not affected with aging, PKG level was significantly increased in those cells. The inhibited IKs in β3 -ARs-stimulated cells could be reversed with a PKG inhibitor, indicating the correlation between PKG-activation and β3 -ARs activation. Furthermore, in vivo treatment of aged rats, characterized by β3 -ARs activation, with either insulin or a PKG inhibitor for 2 weeks provided significant recoveries in IKs , prolonged late phases of APs, prolonged QT-intervals, and low heart rates without no effect on insulin resistance. In vivo insulin treatment provided also significant recovery in increased PKG and decreased PIP2 level, without the insulin effect on the KCNQ1 level in β3 -ARs overexpressed cells. The inhibition of IKs in aged-rat cardiomyocytes seems to be associated with activated β3 -ARs dependent remodeling in the interaction between KCNQ1 and KCNE1. Significant recoveries in ventricular-repolarization of insulin-treated aged cardiomyocytes via recovery in IKs strongly emphasize two important issues: (1) IKs can be a novel target in aging-associated remodeling in the heart and insulin may be a cardioprotective agent in the maintenance of normal heart function during the aging process. (2) This study is one of the first to demonstrate insulin'sbenefits on long-QT in insulin-resistant aged rats by accelerating the ventricular AP repolarization through reversing the depressed IKs via affecting the β3 -ARs signaling pathway and particularly affecting activated PKG.

Regulation of the β-Adrenergic Receptor Signaling Pathway in Sustained Ligand-Activated Preconditioning.

Sustained ligand-activated preconditioning (SLP), induced with chronic opioid receptor (OR) agonism, enhances tolerance to ischemia/reperfusion injury in young and aged hearts. Underlying mechanisms remain ill-defined, although early data implicate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) during the induction phase, and β 2-adrenoceptor (β 2-AR), Gs alpha subunit (Gα s), and protein kinase A (PKA) involvement in subsequent cardioprotection. Here, we tested for induction of a protective β 2-AR/Gα s/PKA signaling axis with SLP to ascertain whether signaling changes were PI3K-dependent (by sustained cotreatment with wortmannin), and whether the downstream PKA target Rho kinase (ROCK) participates in subsequent cardioprotection (by acute treatment with fasudil). A protected phenotype was evident after 5 days of OR agonism (using morphine) in association with increased membrane versus reduced cytosolic levels of total and phosphorylated β 2-ARs; increased membrane and cytosolic expression of 52 and 46 kDa Gα s isoforms, respectively; and increased phosphorylation of PKA and Akt. Nonetheless, functional sensitivities of β 2-ARs and adenylyl cyclase were unchanged based on concentration-response analyses for formoterol, fenoterol, and 6-[3-(dimethylamino)propionyl]-forskolin. Protection with SLP was not modified by ROCK inhibition, and changes in β 2-AR, Gα s, and PKA expression appeared insensitive to PI3K inhibition, although 5 days of wortmannin alone exerted unexpected effects on signaling (also increasing membrane β 2-AR and PKA expression/phosphorylation and Gα s levels). In summary, sustained OR agonism upregulates cardiac membrane β 2-AR expression and phosphorylation in association with increased Gα s subtype levels and PKA phosphorylation. While Akt phosphorylation was evident, PI3K activity appears nonessential to OR upregulation of the β 2-AR signal axis. This opioidergic remodeling of β 2-AR signaling may explain β 2-AR, Gα s, and PKA dependence of SLP protection.

Chronic Testosterone Increases Impulsivity and Influences the Transcriptional Activity of the Alpha-2A Adrenergic Receptor Signaling Pathway in Rat Brain

Testosterone is an anabolic androgenic steroid hormone involved in brain development, reproduction, and social behavior. Several studies have shown that testosterone can cause impulsivity in humans, which in turn, is linked with mood-related psychiatric disorders and higher risk of death by suicide. The mechanisms by which testosterone abuse influences impulsivity are unclear. The present study aims to understand how testosterone influences impulsivity in a rodent model both at behavioral and molecular levels. In this study, rats were either only gonadectomized or gonadectomized and injected with supraphysiological doses of testosterone. Their relative impulsivity levels were assessed using the go/no-go task. Serum level of testosterone was measured using ELISA. Transcript levels of alpha-2A adrenergic receptor (Adra2a), G proteins (stimulatory subunit-Gαs [Gnas], inhibitory subunit-Giα [Gnai1 and Gnai2]), and catalytic and regulatory subunits of protein kinase A (PKA) were examined using quantitative PCR (qPCR) in brain areas associated with limbic system (prefrontal cortex (PFC), hippocampus, and amygdala). The testosterone-treated (T) group showed significantly higher level of serum testosterone and displayed a lower go/no-go ratio, indicating greater impulsivity compared to the gonadectomized (GDX) group. The transcript levels Adra2a and Gαs genes and PKA subunits encoded by Prkar1a, Prkar1b, Prkar2a, and Prkaca genes were significantly upregulated in PFC of testosterone treated rats. The expression levels of these genes were not significantly altered in hippocampus. On the other hand, amygdala showed changes only in Gnas and Prkar2a. These results suggest that chronic testosterone influences impulsivity possibly via hyperactive alpha-2A adrenergic receptor-PKA signaling axis, specifically in the PFC.

Trauma exposure interacts with the genetic risk of bipolar disorder in alcohol misuse of US soldiers.

To investigate whether trauma exposure moderates the genetic correlation between substance use disorders and psychiatric disorders, we tested whether trauma exposure modifies the association of genetic risks for mental disorders with alcohol misuse and nicotine dependence (ND) symptoms. High-resolution polygenic risk scores (PRSs) were calculated for 10 732 US Army soldiers (8346 trauma-exposed and 2386 trauma-unexposed) based on genome-wide association studies of bipolar disorder (BD), major depressive disorder, and schizophrenia. The main finding was a significant BD PRS-by-trauma interaction with respect to alcohol misuse (P = 6.07 × 10-3 ). We observed a positive correlation between BD PRS and alcohol misuse in trauma-exposed soldiers (r = 0.029, P = 7.5 × 10-3 ) and a negative correlation in trauma-unexposed soldiers (r = -0.071, P = 5.61 × 10-4 ). Consistent (nominally significant) result with concordant effect, directions were observed in the schizophrenia PRS-by-trauma interaction analysis. The variants included in the BD PRS-by-trauma interaction showed significant enrichments for gene ontologies related to high voltage-gated calcium channel activity (GO:0008331, P = 1.51 × 10-5 ; GO:1990454, P = 4.49 × 10-6 ; GO:0030315, P = 2.07 × 10-6 ) and for Beta1/Beta2 adrenergic receptor signaling pathways (P = 2.61 × 10-4 ). These results indicate that the genetic overlap between alcohol misuse and BD is significantly moderated by trauma exposure. This provides molecular insight into the complex mechanisms that link substance abuse, psychiatric disorders, and trauma exposure.

0509 : Beta 2 adrenegic receptor expression and activation of endogenous progenitor cells

Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction. The beta adrenergic pathway has been proposed to induce proliferation and migration of progenitor cells. However the mechanisms have not yet been clarified. The mechanism underlying beta adrenergic signaling on endogenous c-kit+/CD45 – cardiac cells was investigated by inducing myocardial infarction in adult mice. Hearts were dissociated and flow cytometry analysis demonstrated that one week after ligation, the percentage of c-kit+/ CD45 – cells expressing beta 1 or beta 2 adrenergic receptor was significantly increased (88.1±3% and 106.8±36.5% increase compared to sham respectively). Flow cytometry studies on cultured cardiac c-kit+/CD45 – cells confirmed increased beta 1 and 2 adrenergic receptor expression in response to stress conditions, specifically hypoxia (5%) or serum starvation. Interestingly, stress conditions altered localization of the beta 2 adrenergic receptor by increasing membrane expression. The beta 2 adrenergic receptor signaling pathway was stimulated in adult sham mice with the agonist fenoterol (0.25 mg/kg/day) administered in drinking water. Seven days after treatment the mice and non-treated controls were sacrificed and progenitor cells were measured by flow cytometry in the heart and blood. Fenoterol increased the proliferation and percentage of c-kit+/CD45 – cells in the heart (123.3±86.2% and 70.9±44.6% increase compared to control respectively). Fenoterol treatment also elevated levels of circulating endothelial progenitor cells (158.5±87.9% compared to control) and c-kit/CD45 – cells (70.6±33.5% increase) in the peripheral blood. Beta adrenergic receptor expression is increased after coronary ligation in vivo and in stress conditions in vitro . A beta 2 adrenergic receptor agonist may be used to improve endogenous cardiac repair through the activation of progenitor cells. The author hereby declares no conflict of interest

Role of β2 adrenergic receptor signaling pathway in hepatocellular carcinoma

Role of β2 adrenergic receptor signaling pathway in hepatocellular carcinoma

Role of adrenergic receptor signaling pathway in colorectal cancer

Role of adrenergic receptor signaling pathway in colorectal cancer

Abstract 628: Early Life Stress Exacerbates The Adrenergic Receptor Subtypes Mrna Expression In The Kidney

Maternal separation (MS) in rodents is a model of human chronic behavioral stress during early life. Previously we have shown that renal sympathetic system is sensitized in adult MS rats. Accordingly, we found that reduced glomerular filtration rate in intact MS rats was normalized after bilateral renal denervation (DnX). The aims of this study were to test the mRNA expression of adrenergic receptor (AR) subtypes in intact and DnX renal cortex and renal vessels. Also, to test the levels of mediators related to the alpha (1,4,5-trisphosphate, IP3) and beta (adenosine-3′,5′-cyclic monophosphate, cAMP) AR signaling pathways. MS was performed in WKY by separating approx half of the male pups from the dam 3 hr a day from days 2-14 of life. Renal tissue from 12 weeks old rats was harvested and snapped frozen. RT-PCR was performed using primers for ARs and IP3 receptor 1 and 2 and mRNA levels. cAMP was determined in renal cortex homogenates by ELISA. IP3 receptor mRNA expression was not different between MS and control rats in intact cortex, Dnx cortex or kidney vessels. cAMP was increased in intact renal cortex of MS rats compared to control (2.37±0.23 vs. 1.55±0.0.24 pmol/mg protein, respectively, p<0.05). Relative mRNA expression (2 - ΔΔ ct ) of α1a, α1b, α1d and α2b were not different in intact cortex from control and MS rats, while Dnx did not have any significant effect. However, α2c AR was increased in intact renal cortex from MS rats compared to control rats (4.5±1.2 vs. 1.8±0.3 2 - ΔΔ ct , p<0.05). DnX significantly reduced α2c mRNA expression in renal cortex from MS and control rats (0.9±0.2 and 0.5±0.1 2 - ΔΔ ct , p<0.05 vs. intact cortex). Relative mRNA expression of α1a, α1b, α1d, α2b and α2c were significantly increased in renal vessels tissue from MS rats compared to control rats (p<0.05). We conclude that enhanced renal mRNA expression of ARs, especially α2c, may be a key mechanisms contributing to the lower glomerular filtration capacity in MS rats. Distinctive pharmacodynamic properties of α2c ARs suggest that they may be attractive targets for therapeutic intervention in case of exposition to early life stress.

Modulation of Purinoceptor mediated Ca2+ signals by β‐adrenergic receptor activation in Salivary Glands

Crosstalk between Ca2+ signaling pathways has been demonstrated to facilitate secretory activity. However it is not known whether such a crosstalk exists between adrenergic receptor signaling pathway and a non‐cholinergic non‐adrenergic (NANC) pathway (purinergic pathway). In this study we use enzymatically isolated mouse parotid acini along with live cell Ca2+ imaging and pharmacological manipulations to assess crosstalk between these two pathways.We showed that [Ca2+]i rises induced by selective activation of P2X4R and P2X7R were potentiated three to eightfold following treatment with the cAMP elevating compounds forskolin, 8‐cpt‐cAMP or 3‐isobutyl‐1‐methylxanthine. Importantly, rises in cAMP levels evoked by β‐adrenergic receptor activation using isoprenalin also resulted in a marked enhancement of P2X‐mediated [Ca2+]i signals.Potentiation of P2X4R‐mediated [Ca2+]i signals following β‐adrenergic receptor activation was likely mediated by PKA because protein kinase inhibitor application in the presence of forskolin or isoprenalin abolished the enhancement of evoked [Ca2+]i signals. Interestingly, application of 2‐Aminoethoxydiphenyl borate during P2X7R stimulation also abolished the enhancement of [Ca2+]i rises following isoprenalin treatment.The results from this study reveal a crosstalk between purinergic and β‐adrenergic signaling pathways and may provide novel targets for construction of therapies for salivary hypofunction patients.

β1 Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β(1) isoform in energy homeostasis. First, the 30 min i.v. infusion of norepinephrine (NE) or the β(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β(1) gene (KO of β(1) adrenergic receptor (β(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, β(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Abstract 8142: By Virtue of Its Interaction with the β 2 -adrenergic Receptor, CXCR4 Plays a Role in the Regulation of β 2 -adrenergic Receptor Signaling Pathway Affecting Calcium and Contractility in Cardiac Myocytes

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α (SDF-1α) and its G-protein coupled receptor, CXCR4. SDF-1 α /CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective β-adrenergic receptor (βAR) agonist, isoproterenol. As a result of the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. We hypothesis that β 2 adrenergic receptor in the heart is negatively modulated by interactions with CXCR4 chemokine receptor. For this we have used biophysical techniques such as bioluminescence resonance energy transfer assay and biochemical techniques such as immunoprecipitation and signaling assays to identify the CXCR4-β2AR interaction and determine the physiological significance of this interaction. Our studies demonstrate activation of CXCR4 by SDF-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR-dependent. In conclusion, we demonstrated a physical interaction between CXCR4 and β2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal switching from Gαs to Gαi-mediated pathway and suggests the interaction is a novel mechanism for regulating cardiac myocyte calcium handling and subsequent contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4- β2AR complex could represent a hitherto unknown target for therapeutic intervention.

A Systems Approach to the Biology of Mood Disorders through Network Analysis of Candidate Genes

Meta analysis of association data of mood disorders has shown evidence for the role of particular genes in genetic risk. Integration of association data from meta analysis with differential expression data in brains of mood disorder patients could heighten the level of support for specific genes. To identify molecular mechanisms that may be disrupted in disease, a systems approach that involves analysis of biological networks created by these selected genes was employed.Interaction networks of hierarchical groupings of selected genes were generated using the Michigan Molecular Interactions (MiMI) software. Large networks were deconvoluted into subclusters of core complexes by using a community clustering program, GLay. Network nodes were functionally annotated in DAVID Bioinformatics Resource to identify enriched pathways and functional clusters. MAPK and beta adrenergic receptor signaling pathways were significantly enriched in the ANK3 and CACNA1C network. The PBRM1 network bolstered the enrichment of chromatin remodeling and transcription regulation functional clusters. Lowering the stringency for inclusion of other genes in network seeds increased network complexity and expanded the recruitment of enriched pathways to include signaling by neurotransmitter and hormone receptors, neurotrophin, ErbB and the cell cycle. We present a strategy to interrogate mechanisms in the cellular system that might be perturbed in disease. Network analysis of meta analysis- generated candidate genes that exhibited differential expression in mood disorder brains identified signaling pathways and functional clusters that may be involved in genetic risk for mood disorders.

S-Nitrosylation of Rap1 and Relationship to Rap1 Activity and Disease Status In SCD

AbstractAbstract 2662Red cell adhesion appears to be a critical contributor to vaso-occlusion in sickle cell disease (SCD). We have been interested in intra-erythrocytic signaling events that can affect sickle red cell (SS RBC) adhesion and thereby stimulate vaso-occlusion. One such agonist-inducible signaling pathway is that of the β2 adrenergic receptor. β2 adrenergic receptor signaling pathways, involving both protein kinase A and the small guanosine triphosphatase Rap1, have been shown to affect multiple RBC adhesion receptors. Therefore, we sought to determine whether specific post-translational modifications of RBC membrane and intracellular proteins were associated with the signaling pathways affecting adhesion. Protein S-nitrosylation, a post-translational modification of cysteine thiol by nitric oxide (NO), is one of several post-translational protein modifications that can regulate diverse cell signaling pathways. Given the many reports of abnormal NO biology in SCD, we investigated how S-nitrosylation of erythrocyte proteins affects molecules involved in regulation of SS RBC adhesion. First, we identified RBC membrane proteins that underwent S-nitrosylation in RBCs from healthy donors (n=2) and SCD patients (n=6; three patients treated with hydroxyurea [HU] and three without HU treatment). Highly purified RBC membrane ghosts were pretreated with or without 0.05 mM CysNO (to promote S-nitrosocysteine formation) for 10 min, and S-nitrosylated proteins were isolated using the resin-assisted capture (SNO-RAC) method (Forrester et al. 2009). Captured S-nitrosylated proteins were visualized by SDS-PAGE, and protein identification was accomplished via an in-situ digestion of the bead-bound proteins, release of the residual bead-bound peptides, and analysis using nanoscale capillary liquid chromatography coupled to high resolution, accurate mass tandem mass spectrometry (LC/MS/MS). A number of proteins underwent S-nitrosylation in both normal and SS RBCs. Among these, MS analysis indicated that Rap1, which has previously been shown to affect SS RBC adhesion to laminin, underwent S-nitrosylation much more extensively in SS than in normal RBCs. These data were confirmed by SNO-RAC followed by immunoblotting. Using a RalGDS Ras-binding domain pull-down assay, we further quantitated the amount of detectable activated Rap1 in highly purified preparations of normal and SS RBCs (lacking either contaminating leukocytes or platelets), before and after RBC loading with NO. Active Rap1 was undetectable or minimally present in RBCs from healthy donors (n=6) but easily detectable in all SS RBCs (n=4). Loading of RBCs with NO in these same samples led to an increased amount of detectable activated Rap1 in both normal and SS RBCs. Finally, we found that patients admitted for pain episodes (n=3) had higher levels of Rap1 nitrosylation and activity than patients in steady state (n=5). In summary, we have found that Rap1, a protein that acts downstream of adrenergic signaling in RBCs, undergoes S-nitrosylation and that this modification is associated with increased Rap1 activity. Most importantly, SS RBCs contain significantly more S-nitrosylated Rap1 and activated Rap1 than do normal RBCs, and this difference in content of active Rap1 is more pronounced in patients with ongoing vaso-occlusion. Disclosures:Telen:GlycoMimetics: Consultancy.

Characterization of bovine neutrophil β2‐adrenergic receptor function

This study compares bovine leukocyte beta-adrenergic receptor densities to that of the rat, demonstrates for the first time a functional beta(2)-adrenergic receptor signaling pathway in steer neutrophils, and investigates the effect of an inflammatory stimulus on that signaling pathway. The beta(1)-/beta(2)-adrenergic antagonist ([3H])CGP-12177 demonstrated that rat lymphocyte specific binding-site density was highest, followed by steer and dairy cow lymphocytes, and lastly steer and dairy cow neutrophils. The beta(2)-adrenergic agonist terbutaline stimulated steer neutrophil adenosine 3,5-cyclic monophosphate (cAMP) production, an effect increased by inclusion of > or = 1 x 10(-8) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Both terbutaline and the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) independently decreased steer neutrophil superoxide anion production in a concentration-dependent manner, with 1 x 10(-4) M IBMX enhancing both the potency and efficacy of the terbutaline effect (up to 74% reduction in superoxide anion production). Superoxide anion production was also reduced by the synthetic cAMP analog 8-bromo-cAMP, which increased the potency of the IBMX effect on superoxide anion production. Taken together, these data demonstrate the presence of a beta(2)-adrenergic receptor signaling pathway in bovine neutrophils much like that described in other animal species, as well as the potential for an inflammatory stimulus to alter its function.

Noradrenaline activation of neurotrophic pathways protects against neuronal amyloid toxicity

Degeneration of locus coeruleus (LC) noradrenergic forebrain projection neurons is an early feature of Alzheimer's disease. The physiological consequences of this phenomenon are unclear, but observations correlating LC neuron loss with increased Alzheimer's disease pathology in LC projection sites suggest that noradrenaline (NA) is neuroprotective. To investigate this hypothesis, we determined that NA protected both hNT human neuronal cultures and rat primary hippocampal neurons from amyloid-beta (Abeta(1-42) and Abeta(25-35)) toxicity. The noradrenergic co-transmitter galanin was also effective at preventing Abeta-induced cell death. NA inhibited Abeta(25-35)-mediated increases in intracellular reactive oxygen species, mitochondrial membrane depolarization, and caspase activation in hNT neurons. NA exerted its neuroprotective effects in these cells by stimulating canonical beta(1) and beta(2) adrenergic receptor signaling pathways involving the activation of cAMP response element binding protein and the induction of endogenous nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Treatment with functional blocking antibodies for either NGF or BDNF blocked NA's protective actions against Abeta(1-42) and Abeta(25-35) toxicity in primary hippocampal and hNT neurons, respectively. Taken together, these data suggest that the neuroprotective effects of noradrenergic LC afferents result from stimulating neurotrophic NGF and BDNF autocrine or paracrine loops via beta adrenoceptor activation of the cAMP response element binding protein pathway.

The 27-kDa Heat Shock Protein Confers Cytoprotective Effects through a β2-Adrenergic Receptor Agonist-Initiated Complex with β-Arrestin

Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical beta adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with beta-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this beta-arrestin/Hsp27 complex in response to the selective beta adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the beta2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both beta-arrestin isoforms to confer this cytoprotective consequence of beta adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and beta-arrestin to form a previously unknown antiapoptotic "signalosome."