β2AR Activation Research Articles

Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.

Sympathetic neurotransmitters promote the process of recellularization in decellularized liver matrix via activating the IL-6/Stat3 pathway

Recellularized liver, as an approach for hepatic tissue engineering, is an effective alternative to orthotopic liver transplantation for end-stage hepatic failure. When compared with normal liver, recellularized liver has a disparity in hepatocyte viability and function, owing to the difficulty of fully simulating the microenvironment of liver. Although the sympathetic nervous system (SNS) is considered an important constituent of liver function, few studies have examined the effect of the SNS on hepatic tissue engineering. It is imperative to explore the regulation of the SNS on a tissue-like configuration to obtain an intact recellularized liver with better hepatic function. We have observed that various subtypes of adrenergic receptors (ARs) are expressed on the hepatocyte membrane. Salbutamol, an agonist of β2-AR, promoted cell proliferation, albumin secretion and urea synthesis in the recellularized liver. Cytokines were screened in isoprenaline/salbutamol-treated recellularized liver, and the expression of IL-6 was significantly increased. Isoprenaline or salbutamol especially promoted the expression of Stat 3 and phosphorylated Stat 3, contributing to the activation of IL-6/Stat 3 signalling in promoting hepatocyte proliferation and recellularized liver function. This study suggests that activation of β2-AR accelerated hepatocyte proliferation and improved recellularized liver function by mediating the IL-6/Stat 3 signalling pathway, indicating that nervous system regulation may be an essential component contributing to the complexity of recellularized liver in tissue engineering.

β-Adrenergic Receptors Regulate the Acquisition and Consolidation Phases of Aversive Memory Formation Through Distinct, Temporally Regulated Signaling Pathways.

Memory formation requires the temporal coordination of molecular events and cellular processes following a learned event. During Pavlovian threat (fear) conditioning (PTC), sensory and neuromodulatory inputs converge on post-synaptic neurons within the lateral nucleus of the amygdala (LA). By activating an intracellular cascade of signaling molecules, these G-protein-coupled neuromodulatory receptors are capable of recruiting a diverse profile of plasticity-related proteins. Here we report that norepinephrine, through its actions on β-adrenergic receptors (βARs), modulates aversive memory formation following PTC through two molecularly and temporally distinct signaling mechanisms. Specifically, using behavioral pharmacology and biochemistry in adult rats, we determined that βAR activity during, but not after PTC training initiates the activation of two plasticity-related targets: AMPA receptors (AMPARs) for memory acquisition and short-term memory and extracellular regulated kinase (ERK) for consolidating the learned association into a long-term memory. These findings reveal that βAR activity during, but not following PTC sets in motion cascading molecular events for the acquisition (AMPARs) and subsequent consolidation (ERK) of learned associations.

Adaptive Activation of a Stress Response Pathway Improves Learning and Memory Through Gs and β-Arrestin-1–Regulated Lactate Metabolism

BackgroundStress is a conserved physiological response in mammals. Whereas moderate stress strengthens memory to improve reactions to previously experienced difficult situations, too much stress is harmful. MethodsWe used specific β-adrenergic agonists, as well as β2-adrenergic receptor (β2AR) and arrestin knockout models, to study the effects of adaptive β2AR activation on cognitive function using Morris water maze and object recognition experiments. We used molecular and cell biological approaches to elucidate the signaling subnetworks. ResultsWe observed that the duration of the adaptive β2AR activation determines its consequences on learning and memory. Short-term formoterol treatment, for 3 to 5 days, improved cognitive function; however, prolonged β2AR activation, for more than 6 days, produced harmful effects. We identified the activation of several signaling networks downstream of β2AR, as well as an essential role for arrestin and lactate metabolism in promoting cognitive ability. Whereas Gs–protein kinase A–cyclic adenosine monophosphate response element binding protein signaling modulated monocarboxylate transporter 1 expression, β-arrestin-1 controlled expression levels of monocarboxylate transporter 4 and lactate dehydrogenase A through the formation of a β-arrestin-1/phospho-mitogen-activated protein kinase/hypoxia-inducible factor-1α ternary complex to upregulate lactate metabolism in astrocyte-derived U251 cells. Conversely, long-term treatment with formoterol led to the desensitization of β2ARs, which was responsible for its decreased beneficial effects. ConclusionsOur results not only revealed that β-arrestin-1 regulated lactate metabolism to contribute to β2AR functions in improved memory formation, but also indicated that the appropriate management of one specific stress pathway, such as through the clinical drug formoterol, may exert beneficial effects on cognitive abilities.

Vasopressin type1A receptor deletion enhances cardiac contractility, β-adrenergic receptor sensitivity and acute cardiac injury-induced dysfunction.

Vasopressin type1A receptor (V1AR) expression is elevated in chronic human heart failure (HF) and contributes to cardiac dysfunction in animal models, in part via reduced β-adrenergic receptor (βAR) responsiveness. Although cardiac V1AR overexpression and V1AR stimulation are each sufficient to decrease βAR activity, it is unknown whether V1AR inhibition conversely augments βAR responsiveness. Further, although V1AR has been shown to contribute to chronic progression of HF, its impact on cardiac function following acute ischaemic injury has not been reported. Using V1AR knockout (V1AR KO) mice we assessed the impact of V1AR deletion on cardiac contractility at baseline and following ischaemic injury, βAR sensitivity and cardiomyocyte responsiveness to βAR stimulation. Strikingly, baseline cardiac contractility was enhanced in V1AR KO mice and they experienced a greater loss in contractile function than control mice following acute ischaemic injury, although the absolute levels of cardiac dysfunction and survival rates did not differ. Enhanced cardiac contractility in V1AR KO mice was associated with augmented β-blocker sensitivity, suggesting increased basal βAR activity, and indeed levels of left ventricular cAMP, as well as phospholamban (PLB) and cardiac troponin I (cTnI) phosphorylation were elevated compared with control mice. At the cellular level, myocytes isolated from V1AR KO mice demonstrated increased responsiveness to βAR stimulation consistent with the finding that acute pharmacological V1AR inhibition enhanced βAR-mediated contractility in control myocytes. Therefore, although V1AR deletion does not protect the heart from the rapid development of cardiac dysfunction following acute ischaemic injury, its effects on βAR activity suggest that acute V1AR inhibition could be utilized to promote myocyte contractile performance.

Antiadrenergic autoimmunity in postural tachycardia syndrome.

AimsPostural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and β1/2-adrenergic receptors (β1/2AR).Methods and resultsImmunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and β1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and β1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, β1AR and β2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and β1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the β1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated β1AR and α1AR activity but not with β2AR activity.ConclusionThese data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and β1AR responsiveness are important in the pathophysiology of postural tachycardia.

Cardiac physiologic regulation of sub-type specific adrenergic receptors in transgenic mice overexpressing β1- and β2-adrenergic receptors.

ObjectiveCombination of β1-adrenergic receptor (AR) blockade and β2-AR activation might be a potential novel therapy for treating heart failure. However, use of β-AR agonists and/or antagonists in the clinical setting is controversial because of the lack of information on cardiac inotropic or chronotropic regulation by AR signaling.MethodsIn this study, we performed hemodynamic evaluation by examining force frequency response (FFR), Frank-Starling relationship, and response to a non-selective β-AR agonist (isoproterenol) in hearts isolated from 6-month-old transgenic (TG) mice overexpressing β1- and β2-ARs (β1- and β2-AR TG mice, respectively).ResultsCardiac physiologic consequences of β1- and β2-AR overexpression resulted in similar maximal response to isoproterenol and faster temporary decline of positive inotropic response in β2-AR TG mice. β1-AR TG mice showed a pronounced negative limb of FFR, whereas β2-AR TG mice showed high stimulation frequencies with low contractile depression during FFR. In contrast, Frank-Starling relationship was equally enhanced in both β1- and β2-AR TG mice.ConclusionHemodynamic evaluation performed in the present showed a difference in β1- and β2-AR signaling, which may be due to the difference in the desensitization of β1- and β2-ARs.

S-Palmitoylation of a Novel Site in the β2-Adrenergic Receptor Associated with a Novel Intracellular Itinerary

We report here that a population of human β2-adrenergic receptors (β2AR), a canonical G protein-coupled receptor, traffics along a previously undescribed intracellular itinerary via the Golgi complex that is associated with the sequential S-palmitoylation and depalmitoylation of a previously undescribed site of modification, Cys-265 within the third intracellular loop. Basal S-palmitoylation of Cys-265 is negligible, but agonist-induced β2AR activation results in enhanced S-palmitoylation, which requires phosphorylation by the cAMP-dependent protein kinase of Ser-261/Ser-262. Agonist-induced turnover of palmitate occurs predominantly on Cys-265. Cys-265 S-palmitoylation is mediated by the Golgi-resident palmitoyl transferases zDHHC9/14/18 and is followed by depalmitoylation by the plasma membrane-localized acyl-protein thioesterase APT1. Inhibition of depalmitoylation reveals that S-palmitoylation of Cys-265 may stabilize the receptor at the plasma membrane. In addition, β2AR S-palmitoylated at Cys-265 are selectively preserved under a sustained adrenergic stimulation, which results in the down-regulation and degradation of βAR. Cys-265 is not conserved in β1AR, and S-palmitoylation of Cys-265 may thus be associated with functional differences between β2AR and β1AR, including relative resistance of β2AR to down-regulation in multiple pathophysiologies. Trafficking via the Golgi complex may underlie new roles in G protein-coupled receptor biology.

β2-Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function

Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of β2-adrenergic receptors (β2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by ∼5-fold when β2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected β2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca2+]i with β2AR co-transfection, ∼53% decrease in [Ca2+]i signaling with shRNA knockdown of β2AR in H292 cells, and ∼60% loss of [Ca2+]i responsiveness in βAR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with β2AR activation rapidly uncoupling TAS2R14 function (∼65% desensitization). Cross-talk was independent of β2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged β-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by ∼50% with β-agonist co-treatment. Thus the β2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by β-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function.

Regulation of Brown and White Adipocyte Transcriptome by the Transcriptional Coactivator NT-PGC-1α.

The β3-adrenergic receptor (AR) signaling pathway is a major component of adaptive thermogenesis in brown and white adipose tissue during cold acclimation. The β3-AR signaling highly induces the expression of transcriptional coactivator PGC-1α and its splice variant N-terminal (NT)-PGC-1α, which in turn activate the transcription program of adaptive thermogenesis by co-activating a number of transcription factors. We previously reported that NT-PGC-1α is able to increase mitochondrial number and activity in cultured brown adipocytes by promoting the expression of mitochondrial and thermogenic genes. In the present study, we performed genome-wide profiling of NT-PGC-1α-responsive genes in brown adipocytes to identify genes potentially regulated by NT-PGC-1α. Canonical pathway analysis revealed that a number of genes upregulated by NT-PGC-1α are highly enriched in mitochondrial pathways including fatty acid transport and β-oxidation, TCA cycle and electron transport system, thus reinforcing the crucial role of NT-PGC-1α in the enhancement of mitochondrial function. Moreover, canonical pathway analysis of NT-PGC-1α-responsive genes identified several metabolic pathways including glycolysis and fatty acid synthesis. In order to validate the identified genes in vivo, we utilized the FL-PGC-1α-/- mouse that is deficient in full-length PGC-1α (FL-PGC-1α) but expresses a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α254). The β3-AR-induced increase of NT-PGC-1α254 in FL-PGC-1α-/- brown and white adipose tissue was closely associated with elevated expression of genes involved in thermogenesis, mitochondrial oxidative metabolism, glycolysis and fatty acid synthesis. Increased adipose tissue thermogenesis by β3-AR activation resulted in attenuation of adipose tissue expansion in FL-PGC-1α-/- adipose tissue under the high-fat diet condition. Together, the data strengthen our previous findings that NT-PGC-1α regulates mitochondrial genes involved in thermogenesis and oxidative metabolism in brown and white adipocytes and further suggest that NT-PGC-1α regulates a broad spectrum of genes to meet cellular needs for adaptive thermogenesis.

Abstract 460: β 1 -Adrenergic Receptor O-glycosylation Regulates Receptor N-terminal Cleavage and Signaling Responses in Cardiomyocytes

β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Recent efforts to structurally characterize β1ARs have focused on the ligand binding sites in transmembrane helices and effector docking sites in the intracellular loops and C-terminus. The notion that the β1AR extracellular N-terminus (which is a substrate for both N- and O-linked glycosylation and a target for proteolytic cleavage) functions as a structural determinant of β1AR activation has never been considered. This study takes advantage of site-directed mutagenesis and expression studies in Chinese Hamster ovary cells that harbor a reversible glycosylation defect to map β1ARs O-glycosylation sites to Ser37/Ser41 and show that O-glycosylation at these sites prevents β1AR N-terminal cleavage. We then used an adenoviral overexpression approach to show that both full-length fully-glycosylated β1ARs (β1AR-FL) and N-terminally truncated glycosylation-defective β1ARs (β1AR-Δ52) increase cAMP accumulation and activate the ERK-MAPK signaling cascade in cardiomyocytes. However, isoproterenol-dependent cAMP accumulation is markedly enhanced, and ERK activation is markedly attenuated, in cardiomyocytes that express β1AR-Δ52, compared with β1AR-FL cultures. The conclusion that β1ARs are stabilized in a conformation that is biased toward the cAMP pathway as a result of N-terminal truncation gains further support from additional studies showing that agonistic β1AR autoantibodies induce a more rapid and robust increase in membrane PKA activity in cells that express glycosylation-defective β1ARs, compared to cells that express full-length glycosylated β1ARs. These results identify a novel role for O-glycosylation as a structural determinant of β1AR responsiveness in the heart.

Monoterpene phenolic compound thymol promotes browning of 3T3-L1 adipocytes

Appearance of brown-like adipocytes within white adipose tissue depots (browning) is associated with improved metabolic phenotypes, and thus a wide variety of dietary agents that contribute to browning of white adipocytes are being studied. The aim of this study was to assess the browning effect of thymol, a dietary monoterpene phenolic compound, in 3T3-L1 white adipocytes. Thymol-induced fat browning was investigated by determining expression levels of brown fat-specific genes and proteins by real-time RT-PCR and immunoblot analysis, respectively. Moreover, the molecular mechanism underlying the fat-browning effect of thymol was investigated by determining expression levels of key players responsible for browning in the presence of kinase inhibitors. Thymol promoted mitochondrial biogenesis and enhanced expression of a core set of brown fat-specific markers as well as increased protein levels of PPARγ, PPARδ, pAMPK, pACC, HSL, PLIN, CPT1, ACO, PGC-1α, and UCP1, suggesting its possible role in browning of white adipocytes, augmentation of lipolysis, fat oxidation, and thermogenesis, and reduction of lipogenesis. Increased expression of UCP1 and other brown fat-specific markers by thymol was tightly coordinated with activation of β3-AR as well as AMPK, PKA, and p38 MAPK. Our findings suggest that 3T3-L1 is a potential cell model for screening browning agents. Thymol plays multiple modulatory roles in the form of inducing the brown-like phenotype as well as enhancing lipid metabolism. Thus, thymol may be explored as a potentially promising food additive for prevention of obesity.

Abstract 4662: Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis

Abstract Therapeutic strategies to target EGFR tyrosine kinase inhibitor (TKI) resistance mediated by mechanisms other than T790M are a major clinical challenge. Studies have implicated IL-6 as a mediator of EGFR TKI resistance, and IL-6 is known to be regulated by adrenergic receptors (AR) in some cancers. We investigated whether adrenergic pathways can promote T790M-independent EGFR TKI resistance in preclinical models and in clinical studies. We found that β2-AR was highly expressed in our panel of 119 cell lines and in NSCLC clinical specimens. Activation of β-ARs by stress hormones such as norepinephrine (NE) induced a dramatic rise in IL-6, and this occurred through β2-ARs. β-AR inhibitors (i.e. propranolol), but not α-AR inhibitors, blocked IL-6 induction. Analysis of downstream signaling pathways revealed that β2-ARs induced IL-6 expression through activation of adenylyl cyclase, p90RSK and CREB. To identify novel signaling pathways modulated by ARs, we stimulated NSCLC cell lines with NE and analyzed protein lysates by RPPA to detect expression and activation of >100 proteins. β-AR signaling inactivated the tumor suppressor LKB1 through phosphorylation of S428 and subsequently increased mTOR activity. LKB1 inactivation was critical for IL-6 induction. This finding is important as LKB1 loss is known to be a driver of NSCLC resistance and metastasis. Moreover, we found that β2-AR activation promoted EGFR TKI resistance in cell lines and in mouse models of EGFR mutant NSCLC. The effect of β-AR on EGFR TKI resistance was blocked by the addition of the beta blocker propranolol or IL-6 antibodies, in vitro and in vivo. Consistent with our preclinical studies, in the phase III ZEST clinical study testing erlotinib vs vandetanib, we found that high plasma levels of IL-6 was associated with a worse PFS and OS in the erlotinib arm. In addition, we found that circulating levels of IL-6 were significantly lower in NSCLC patients incidentally receiving beta blockers in the BATTLE trial. Finally, we analyzed the influence of incidental beta blocker use in the LUX-Lung3 study testing afatinib vs chemotherapy in EGFR mutant NSCLC patients. In patients not receiving beta blockers, the median PFS was 11.1 and 6.9 months for afatinib and chemotherapy, respectively, and afatinib improved PFS with a hazard ratio (HR) of 0.60. In patients receiving beta blockers, the median PFS was 13.6 and 2.5 for afatinib and chemotherapy, respectively, and afatinib improved PFS with a HR of 0.25. In conclusion, our preclinical and clinical data provide evidence that β2-AR activation can upregulate IL-6 in EGFR mutant+ NSCLC, modulate the LKB1/AMPK/mTOR axis, and promote EGFR TKI resistance. Moreover, EGFR mutant+ patients using beta blockers had greater relative PFS benefit from afatinib vs chemotherapy compared with those not using beta blockers, supporting future clinical testing of EGFR TKIs in combination with beta blockers. Citation Format: Monique B. Nilsson, Huiying Sun, Lixia Diao, Pan Tong, Youhong Fan, Hai Tran, Diane Liu, Guillermo Armaiz Pena, Jing Wang, Phil Rowe, Alan Webster, Jack Lee, Daniel Gomez, Waun Ki Hong, Ignacio Wistuba, Anil Sood, John Heymach. Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4662.

Leukocyte-Expressed β2-Adrenergic Receptors Are Essential for Survival After Acute Myocardial Injury.

Immune cell-mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through β-adrenergic receptors (βARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. Wild-type (WT) mice were irradiated followed by isoform-specific βAR knockout (βARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. β2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. β2ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via β-arrestin-dependent β2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to β2AR activity, and spleens from human tissue donors treated with β-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of β2AR in β2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. Immune cell-expressed β2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

Effect and potential mechanism of β3-adrenoceptor activation on fibrosis in cardiac fibroblast cell

To observe the effect of β3 adrenergic receptor (β3-AR) on fibrosis in cardiac fibroblasts(CFBs) and explore the related mechanisms. Neonatal CFBs were divided into negative control group (N-CFC): CFBs without any intervention; group treated with β3 adrenergic receptor agonist (AngⅡ-CFC-β3-AR BRL): CFBs treated with 10(-6) mol/L angiotensin Ⅱ(AngⅡ), 1 hour later treated with 10(-5) mol/L β3 adrenergic receptor agonist (β3-AR BRL37344); group treated with β3 adrenergic receptor antagonist (AngⅡ-CFC-β3-AR SR): CFBs treated with 10(-6) mol/L AngⅡ, 1 hour later treated with 10(-5) mol/L β3 adrenergic receptor antagonist (β3-AR SR59230A); and positive control group (AngⅡ-CFC): CFBs treated with 10(-6) mol/L AngⅡonly. Proliferation of CFBs was detected by the method of WST-1. Protein expression of β3-AR, transforming growth factor β1 receptor (TGF-β1-R), transforming growth factor β1(TGF-β1), Smad-2, phospho-Smad-2 (p-Smad-2), collagen-Ⅰ (COL-Ⅰ) and collagen-Ⅲ(COL-Ⅲ) was determined by Western blot assay. (1) The proliferation of CFBs was the highest in AngⅡ-CFC-β3-AR BRL, followed by AngⅡ-CFC-β3-AR SR and AngⅡ-CFC group (all P<0.05 vs. N-CFC group). (2) The protein expression level of β3-AR, TGF-β1-R, TGF-β1 and p-Smad-2 was in the same order as proliferation of CFBs. (3) The expression level of COL-Ⅰ and COL-Ⅲ protein was also in the same order as proliferation of CFBs. Activation of β3-AR may promote fibrosis of CFBs through the TGF-β/Smad signaling pathway and thus aggravate myocardial remodeling.

Activation of β-adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume.

Brain extracellular space (ECS) is an interconnected channel that allows diffusion-mediated transport of signaling molecules, metabolites, and drugs. We tested the hypothesis that β-adrenergic receptor (βAR) activation impacts extracellular diffusion-mediated transport of molecules through alterations in the morphology of astrocytes. Two structural parameters of ECS-volume fraction and tortuosity-govern extracellular diffusion. Volume fraction (α) is the volume of ECS relative to the total tissue volume. Tortuosity (λ) is a measure of the hindrance that molecules experience in the ECS, compared to a free medium. The real-time iontophoretic (RTI) method revealed that treatment of acutely prepared visual cortical slices of adult female rats with a βAR agonist, DL-isoproterenol (ISO), decreases α significantly, from 0.22 ± 0.03 (mean ± SD) for controls without agonist to 0.18 ± 0.03 with ISO, without altering λ (control: 1.64 ± 0.04; ISO: 1.63 ± 0.04). Electron microscopy revealed that the ISO treatment significantly increased the cytoplasmic area of astrocytic distal endings per unit area of neuropil by 54%. These findings show that norepinephrine decreases α, in part, through an increase in astrocytic volume following βAR activation. Norepinephrine is recognized to be released within the brain during the awake state and increase neurons' signal-to-noise ratio through modulation of neurons' biophysical properties. Our findings uncover a new mechanism for noradrenergic modulation of neuronal signals. Through astrocytic activation leading to a reduction of α, noradrenergic modulation increases extracellular concentration of neurotransmitters and neuromodulators, thereby facilitating neuronal interactions, especially during wakefulness. Synapse 70:307-316, 2016. © 2016 Wiley Periodicals, Inc.

Persistent Catechol-O-methyltransferase-dependent Pain Is Initiated by Peripheral β-Adrenergic Receptors.

Patients with chronic pain disorders exhibit increased levels of catecholamines alongside diminished activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines. The authors found that acute pharmacologic inhibition of COMT in rodents produces hypersensitivity to mechanical and thermal stimuli via β-adrenergic receptor (βAR) activation. The contribution of distinct βAR populations to the development of persistent pain linked to abnormalities in catecholamine signaling requires further investigation. Here, the authors sought to determine the contribution of peripheral, spinal, and supraspinal βARs to persistent COMT-dependent pain. They implanted osmotic pumps to deliver the COMT inhibitor OR486 (Tocris, USA) for 2 weeks. Behavioral responses to mechanical and thermal stimuli were evaluated before and every other day after pump implantation. The site of action was evaluated in adrenalectomized rats receiving sustained OR486 or in intact rats receiving sustained βAR antagonists peripherally, spinally, or supraspinally alongside OR486. The authors found that male (N = 6) and female (N = 6) rats receiving sustained OR486 exhibited decreased paw withdrawal thresholds (control 5.74 ± 0.24 vs. OR486 1.54 ± 0.08, mean ± SEM) and increased paw withdrawal frequency to mechanical stimuli (control 4.80 ± 0.22 vs. OR486 8.10 ± 0.13) and decreased paw withdrawal latency to thermal heat (control 9.69 ± 0.23 vs. OR486 5.91 ± 0.11). In contrast, adrenalectomized rats (N = 12) failed to develop OR486-induced hypersensitivity. Furthermore, peripheral (N = 9), but not spinal (N = 4) or supraspinal (N = 4), administration of the nonselective βAR antagonist propranolol, the β2AR antagonist ICI-118,511, or the β3AR antagonist SR59230A blocked the development of OR486-induced hypersensitivity. Peripheral adrenergic input is necessary for the development of persistent COMT-dependent pain, and peripherally-acting βAR antagonists may benefit chronic pain patients.

Monoterpene limonene induces brown fat-like phenotype in 3T3-L1 white adipocytes

AimsSeveral dietary compounds that are able to induce the brown fat-like phenotype in white adipocytes have been considered for treatment of obesity due to their ability to increase energy expenditure. Here, we report that limonene induces the brown fat-like phenotype in 3T3-L1 adipocytes by increasing expression of brown adipocyte-specific genes and proteins. Main methodsLimonene-induced browning in white adipocytes was investigated by determining expression levels of brown fat-specific genes and proteins by real-time RT-PCR, immunoblot analysis, and immunocytochemical staining. Key findingsLimonene enhanced mitochondrial biogenesis, as evidenced by increased mitochondrial content and immunofluorescent intensity. Limonene also significantly elevated protein levels of HSL, PLIN, p-AMPK, p-ACC, ACO, COX4, CPT1, and CYT C, suggesting its possible role in enhancement of lipolysis and lipid catabolism. Increased expression of PRDM16, UCP1, C/EBPβ, and other brown fat-specific markers by limonene was possibly mediated by activation of β3-adnergenic receptor (β3-AR), as inhibition of β3-AR inhibited up-regulation of brown fat-specific markers. Similarly, limonene-mediated activation of ERK and up-regulation of key brown adipocyte specific markers were eliminated by treatment with ERK antagonist. SignificanceTaken together, these results suggest that limonene induces browning of 3T3-L1 adipocytes via activation of β3-AR and the ERK signaling pathway. In conclusion, our findings suggest that limonene plays a dual modulatory role in induction of the brown adipocyte-like phenotype as well as promotion of lipid metabolism and thus may have potential therapeutic implications for treatment of obesity.

β2-adrenergic receptor signaling promotes pancreatic ductal adenocarcinoma (PDAC) progression through facilitating PCBP2-dependent c-myc expression

The β2-adrenergic receptor (β2-AR) plays a crucial role in pancreatic ductal adenocarcinoma (PDAC) progression. In this report, we identified poly(rC)-binding protein 2 (PCBP2) as a novel binding partner for β2-AR using immunoprecipitation-mass spectrometry (IP-MS) approach. The association between β2-AR and PCBP2 was verified using reciprocal immunoprecipitation. Importantly, we found significant interaction and co-localization of the two proteins in the presence of β2-AR agonist in Panc-1 and Bxpc3 PDAC cells. β2-AR-induced recruitment of PCBP2 led to augmented protein level of c-myc in PDAC cells, likely as a result of enhanced internal ribosome entry segment (IRES)-mediated translation of c-myc. The activation of β2-AR accelerated cell proliferation and colony formation, while knockdown of PCBP2 or c-myc restrained the effect. Furthermore, overexpression of PCBP2 was observed in human PDAC cell lines and tissue specimens compared to the normal pancreatic ductal epithelial cells and the non-cancerous tissues respectively. Overexpression of β2-AR and PCBP2 was associated with advanced tumor stage and significantly worsened prognosis in patients with PDAC. Our results elucidate a new molecular mechanism by which β2-AR signaling facilitates PDAC progression through triggering PCBP2-dependent c-myc expression.

Norepinephrine Controls Effector T Cell Differentiation through β2-Adrenergic Receptor-Mediated Inhibition of NF-κB and AP-1 in Dendritic Cells.

Despite accumulating evidence indicating that neurotransmitters released by the sympathetic nervous system can modulate the activity of innate immune cells, we still know very little about how norepinephrine impacts signaling pathways in dendritic cells (DC) and the consequence of that in DC-driven T cell differentiation. In this article, we demonstrate that β2-adrenergic receptor (β2AR) activation in LPS-stimulated DC does not impair their ability to promote T cell proliferation; however, it diminishes IL-12p70 secretion, leading to a shift in the IL-12p70/IL-23 ratio. Although β2AR stimulation in DC induces protein kinase A-dependent cAMP-responsive element-binding protein phosphorylation, the effect of changing the profile of cytokines produced upon LPS challenge occurs in a protein kinase A-independent manner and, rather, is associated with inhibition of the NF-κB and AP-1 signaling pathways. Moreover, as a consequence of the inverted IL-12p70/IL-23 ratio following β2AR stimulation, LPS-stimulated DC promoted the generation of CD4(+) T cells that, upon TCR engagement, produced lower amounts of IFN-γ and higher levels of IL-17. These findings provide new insights into molecular and cellular mechanisms by which β2AR stimulation in murine DC can influence the generation of adaptive immune responses and may explain some aspects of how sympathetic nervous system activity can modulate immune function.