• Fibrosis
• LIM kinases
• Myocardial infarction
• Wnt signaling

• Animals
• Male
• Mice
• Cell Proliferation
• Fibroblasts/metabolism
• Lim Kinases/metabolism
• Lim Kinases/genetics
• Mice, Inbred C57BL
• Mice, Knockout
• Myocardial Infarction/metabolism
• Myocardial Infarction/genetics
• Myocardial Infarction/pathology
• Myocardial Infarction/physiopathology
• Myocardium/pathology
• Myocardium/metabolism
• Myofibroblasts/metabolism
• Ventricular Remodeling
• Wnt Signaling Pathway

• Cell Movement
• Humans
• Infant, Newborn
• Keratinocytes/physiology
• Male
• Receptors, Adrenergic, alpha/metabolism
• Receptors, Adrenergic, alpha/physiology
• Receptors, Adrenergic, beta/metabolism
• Receptors, Adrenergic, beta/physiology
• Wound Healing

• ERK
• cardiac hypertrophy
• extracellular matrix
• heart

• G protein-coupled recpetor
• GPCR
• NTSR1
• PET
• addiction
• allosteric modulator
• cocaine
• dopamine
• methamphetamine
• neurotensin receptor 1
• positron emission tomography
• self-administration
• β-arrestin

• Allosteric Regulation/drug effects
• Allosteric Regulation/physiology
• Animals
• Behavior, Addictive/drug therapy
• Behavior, Addictive/metabolism
• Cell Line
• Female
• HEK293 Cells
• Humans
• Male
• Mice
• Mice, Inbred C57BL
• Models, Animal
• Receptors, G-Protein-Coupled/metabolism
• Receptors, Neurotensin/metabolism
• Signal Transduction/drug effects
• Signal Transduction/physiology
• Small Molecule Libraries/pharmacology
• beta-Arrestins/metabolism

• P30 DA029925 NIDA NIH HHS
• K08 HL125905 NHLBI NIH HHS
• R01 MH073853 NIMH NIH HHS
• P30 CA016086 NCI NIH HHS
• K01 AG041211 NIA NIH HHS
• R33 DA038019 NIDA NIH HHS
• R01 AG066184 NIA NIH HHS
• R00 DA048970 NIDA NIH HHS
• U24 CA220245 NCI NIH HHS
• K99 DA048970 NIDA NIH HHS
• R21 DA038019 NIDA NIH HHS
• R37 MH073853 NIMH NIH HHS
• F32 DA043931 NIDA NIH HHS

• 6-Octadecynoid acid
• 9-Octadecynoic acid
• Fatty acid receptor
• Insulin secretion

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of β-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate β₂AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated β₂AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant β₂AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant β₂AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which β-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

• cell biology
• hippocampus
• ion channel
• mouse
• rat
• signaling transduction

Cholinergic vagal nerves projecting from neurons in the brain stem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however, the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brain stem slices. Preganglionic NAm neurons were identified by ChAT-tdTomato fluorescence in young adult transgenic mice, and their cardiac projection was confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However, bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α₁-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, because postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current and hyperpolarization-activated current are involved in this excitation pattern, although not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons and that persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone.

NEW & NOTEWORTHY Cardiac preganglionic cholinergic neurons in the nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brain stem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

• cardiac vagal
• epinephrine
• nucleus ambiguus
• pacemaking
• parasympathetic

• JB6 P+ cells
• alprenolol
• biased β-blocker
• carcinogenesis
• β-blocker

To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide (LPS)-induced liver injury in vivo and in vitro.

Male β-arrestin 2^+/+ and β-arrestin 2^-/- C57BL/6J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.

Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines (including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 siRNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phospho-IκBα and phosho-p65, were upregulated.

β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathway-mediated inflammation.

• Lipopolysaccharide
• Liver injury
• β-arrestin 2
• TLR4/NF-κB signaling pathway
• Pro-inflammatory cytokines

• Animals
• Chemical and Drug Induced Liver Injury/etiology
• Chemical and Drug Induced Liver Injury/metabolism
• Chemical and Drug Induced Liver Injury/pathology
• Chemical and Drug Induced Liver Injury/prevention & control
• Cytokines/metabolism
• Disease Models, Animal
• Inflammation Mediators/metabolism
• Lipopolysaccharides
• Liver/metabolism
• Liver/pathology
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• NF-KappaB Inhibitor alpha/metabolism
• NF-kappa B/metabolism
• Phosphorylation
• RAW 264.7 Cells
• Signal Transduction
• Toll-Like Receptor 4/metabolism
• Transcription Factor RelA/metabolism
• beta-Arrestin 2/deficiency
• beta-Arrestin 2/genetics
• beta-Arrestin 2/metabolism

• Apoptosis
• Arrestin
• FSHR
• GPCR
• LHCGR
• hGL5

• Apoptosis
• Caspase 3/metabolism
• Cell Line
• Cell Proliferation/drug effects
• Cell Survival
• Cyclic AMP/metabolism
• Cyclic AMP-Dependent Protein Kinases/metabolism
• Female
• Gene Silencing
• Granulosa Cells/cytology
• Granulosa Cells/metabolism
• Humans
• Intracellular Space/metabolism
• Progesterone/biosynthesis
• Receptors, FSH/metabolism
• Receptors, Gonadotropin/metabolism
• Receptors, LH
• Signal Transduction
• beta-Arrestins/metabolism

The mu opioid receptor is a G-protein coupled receptor able to signal through the Gα_i/o class of G-protein and β-arrestin pathways, stimulating down-stream effector pathways. Signaling bias occurs when different receptor agonists lead to different signaling outcomes. Traditionally these have been studied using end-point assays. Real-time cellular analysis platforms allow for the analysis of the holistic effects of receptor activation as an integrated output. While this allows for different ligands to be compared rapidly, the cellular mechanisms underlying the signal are not well described. Using an impedance based system, the impedance responses for two opioid ligands, morphine and DAMGO were examined.

The impedance responses for these two agonists, while showing similar features, were distinct from each other. Some of the mechanisms underlying the mu opioid receptor coupled impedance changes were investigated. It was found that the response is a result of discrete cellular processes, including G-protein signaling and protein kinase phosphorylation.

•

An impedance assay was used to capture label-free real-time data for two opioids.

• CHO, Chinese hamster ovary
• CI, cell index
• DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin
• FCS, foetal calf serum
• G-protein
• GPCR
• GPCR, G-protein coupled receptor
• Impedance
• Label-free
• MOR, mu opioid receptor
• Opioid
• RTCA, real-time cellular analysis
• Real-time cellular analysis

• FSH
• Granulosa cells
• LH
• LHCGR
• hCG

• Adult
• Apoptosis/drug effects
• Cells, Cultured
• Chorionic Gonadotropin/pharmacology
• Cyclic AMP/metabolism
• Cyclic AMP Response Element-Binding Protein/metabolism
• Drug Synergism
• Female
• Follicle Stimulating Hormone/pharmacology
• Humans
• In Vitro Techniques
• Luteal Cells/cytology
• Luteal Cells/drug effects
• Luteinizing Hormone/pharmacology
• Signal Transduction/drug effects
• Steroids/metabolism

Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

• Animals
• Apoptosis
• Arrestins/physiology
• Cell Movement
• Cells, Cultured
• Gene Expression Regulation
• Mice
• Mice, Knockout
• Phosphorylation
• Protein Binding
• Protein Transport
• RNA, Messenger/genetics
• Real-Time Polymerase Chain Reaction
• Receptors, Formyl Peptide/genetics
• Receptors, Formyl Peptide/metabolism
• Reverse Transcriptase Polymerase Chain Reaction
• Signal Transduction
• src-Family Kinases/genetics
• src-Family Kinases/metabolism

• P30 CA118100 NCI NIH HHS
• R01 CA163890 NCI NIH HHS
• CA163890 NCI NIH HHS
• R01 GM068901 NIGMS NIH HHS
• GM68901 NIGMS NIH HHS
• BC030217 NCI NIH HHS

• Cardiac tissue-remodeling
• Cyr61
• Gα(q)/Gα(11)-signaling
• Matricellular proteins

• Animals
• Cardiomegaly/genetics
• Cardiomegaly/metabolism
• Cardiomegaly/pathology
• GTP-Binding Protein alpha Subunits, Gq-G11/deficiency
• GTP-Binding Protein alpha Subunits, Gq-G11/genetics
• Heart Rate/physiology
• Male
• Mice
• Mice, Knockout
• Mice, Transgenic
• Myocytes, Cardiac/physiology
• Signal Transduction/physiology
• Stroke Volume/physiology
• Ventricular Remodeling/physiology

Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). The EPIC-BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration-dependent negative DMR deflection that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Subtype-selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR-mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and –independent pathways.

• cardiac fibroblast
• dynamic mass redistribution
• epidermal growth factor receptor
• fluorescence resonance energy transfer
• β-adrenergic receptor

• 5-bromo-2′-deoxyuridine
• BMP
• BrdU
• CCK2R
• G-17
• G-protein−coupled receptor
• GAS(−/−)
• GFP
• GPCR
• Id4
• PCR
• Progastrin
• WT
• amidated gastrin 17
• bone morphogenetic protein
• cholecystokinin-2 receptor
• gastrin knockout
• green fluorescent protein
• hGAS
• human progastrin
• inhibitor of DNA binding 4
• mRNA
• messenger RNA
• polymerase chain reaction
• wild-type

Stimulation of α_1aAdrenergic Receptors (ARs) is known to have anti-proliferative and hypertrophic effects; however, some studies also suggests this receptor can increase cell proliferation. Surprisingly, we find the α_1aAR expressed in rat-1 fibroblasts can produce either phenotype, depending exclusively on agonist concentration. Stimulation of the α_1aAR by high dose phenylephrine (>10⁻⁷ M) induces an antiproliferative, hypertrophic response accompanied by robust and extended p38 activation. Inhibition of p38 with SB203580 prevented the antiproliferative response, while inhibition of Erk or Jnk had no effect. In stark contrast, stimulation of the α_1aAR with low dose phenylephrine (∼10⁻⁸ M) induced an Erk-dependent increase in cellular proliferation. Agonist-induced Erk phosphorylation was preceded by rapid FGFR and EGFR transactivation; however, only EGFR inhibition blocked Erk activation and proliferation. The general matrix metalloprotease inhibitor, GM6001, blocked agonist induced Erk activation within seconds, strongly suggesting EGFR activation involved extracellular triple membrane pass signaling. Erk activation required little Ca²⁺ release and was blocked by PLCβ or PKC inhibition but not by intracellular Ca²⁺ chelation, suggesting Ca²⁺ independent activation of novel PKC isoforms. In contrast, Ca²⁺ release was essential for PI3K/Akt activation, which was acutely maximal at non-proliferative doses of agonist. Remarkably, our data suggests EGFR transactivation leading to Erk induced proliferation has the lowest activation threshold of any α_1aAR response. The ability of α_1aARs to induce proliferation are discussed in light of evidence suggesting antagonistic growth responses reflect native α_1aAR function.

• Adrenergic alpha-Agonists/pharmacology
• Animals
• Blotting, Western
• Cell Line
• Cell Proliferation/drug effects
• Dose-Response Relationship, Drug
• Enzyme Activation
• Imidazoles/pharmacology
• Phosphorylation
• Protein Kinases/metabolism
• Pyridines/pharmacology
• Rats
• Receptors, Adrenergic, alpha-1/drug effects

• R37 HL049103 NHLBI NIH HHS
• R37 HL49103 NHLBI NIH HHS
• 5R37HL049103-19 NHLBI NIH HHS

• Blotting, Western
• Colonic Neoplasms/metabolism
• Colonic Neoplasms/pathology
• Electrophoresis, Gel, Two-Dimensional
• HT29 Cells
• Humans
• Membrane Proteins/genetics
• Membrane Proteins/metabolism
• Membrane Proteins/physiology
• Models, Biological
• Proteome/analysis
• Proteome/metabolism
• Proteomics/methods
• Signal Transduction/physiology
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• Transfection
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/metabolism
• Tumor Suppressor Proteins/physiology

The serotonin (5-HT) 2A and 2C receptors (5-HT_2AR and 5-HT_2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Ca_i⁺⁺) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.

We optimized the Ca_i⁺⁺ assay for stable cell lines expressing either 5-HT_2AR or 5-HT_2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT_2AR- and 5-HT_2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT_2AR-expressing cells showed significant release of Ca_i⁺⁺ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT_2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.

These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT_2AR and 5-HT_2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.

• β-arrestin
• β-adrenergic receptor
• angiotensin ii type 1a receptor
• g protein coupled receptor kinase
• extracellular-signal receptor kinase
• epidermal growth factor receptor transactivation
• signaling

• Animals
• Arrestins/chemistry
• Arrestins/metabolism
• Heart Failure/drug therapy
• Heart Failure/metabolism
• Humans
• Molecular Targeted Therapy
• Myocardium/metabolism
• Protein Conformation
• Protein Transport
• Proteolysis
• Receptors, Adrenergic, beta-1/metabolism
• Receptors, Angiotensin/metabolism
• Receptors, G-Protein-Coupled/chemistry
• Receptors, G-Protein-Coupled/metabolism
• Signal Transduction
• beta-Arrestins

• P01 HL075443 NHLBI NIH HHS
• R01 HL056687 NHLBI NIH HHS
• HL-56687 NHLBI NIH HHS
• HL-075443 NHLBI NIH HHS

• gpcr
• g protein
• β-arrestin
• cardiac contractility
• hypertrophy

• Arrestins/metabolism
• Heart/physiology
• Humans
• Receptors, G-Protein-Coupled/metabolism
• Signal Transduction/physiology
• beta-Arrestins

• R01 HL105414 NHLBI NIH HHS

• Angiotensin II/pharmacology
• GTP-Binding Protein alpha Subunits, Gq-G11/metabolism
• Gene Expression Regulation/drug effects
• HEK293 Cells
• Humans
• Polymerase Chain Reaction
• Receptor, Angiotensin, Type 1/genetics
• Receptor, Angiotensin, Type 1/metabolism
• Receptors, Adrenergic, beta-2/metabolism
• Reproducibility of Results
• Response Elements/genetics
• Signal Transduction/genetics
• Time Factors
• Transcription Factors/genetics
• Transcription Factors/metabolism
• Transcription, Genetic/drug effects

• Animals
• Heart/physiology
• Humans
• MAP Kinase Signaling System/physiology
• Mitogen-Activated Protein Kinases/physiology
• Myocardium/metabolism

• R01 HL098954 NHLBI NIH HHS
• P01 HL080111 NHLBI NIH HHS
• R01 HL103205 NHLBI NIH HHS
• R01 HL070079 NHLBI NIH HHS
• R01 HL062311 NHLBI NIH HHS