β3-AR Agonist Research Articles

Adrenergic receptors signal to β‐arrestins to control the membrane trafficking of Monocarboxylate transporter 1 (MCT1) in glioblastoma

Monocarboxylate transporters (MCTs) are a family of 12‐pass transmembrane domain proteins that mediate the rapid symport of protons and monocarboxylates, including lactate, across the plasma membrane. MCT‐1, the primary MCT in glioblastoma cells, helps regulate mammalian metabolism and is thought to act as a scaffold for and facilitate the activation of pro‐migratory and pro‐invasive signaling pathways. Uncovering the cellular mechanisms governing MCT‐1 trafficking is crucial for the development of therapeutic treatments for MCT‐1 as an anticancer target. Here, we describe the role of signaling by β2‐adrenergic (β2AR) and α2‐adrenergic (α2AR) receptors in mediating MCT‐1 internalization into caveolae and sorting endosomes. Through MCT‐1 and Caveolin‐1 colocalization analysis, we show an increase in MCT‐1 internalization upon β2AR agonist stimulation and even greater increase in internalization upon simultaneous β2AR and α2AR agonist stimulation, which suggest that both receptors mediate this response in a synergistic manner. Furthermore, we demonstrate that siRNA depletion of endogenous β‐arrestin inhibits MCT‐1 internalization in cells when stimulated with a β2AR agonist, indicating that β2AR likely signals through β‐arrestin to mediate MCT‐1 internalization. We are currently investigating whether these results are recapitulated in single‐cell derived β‐arrestin knockout clones. Our current research also focuses on identifying which β‐arrestin‐dependent signaling pathways promote synergistic MCT‐1 internalization in response to adrenergic receptor activation.

CRISPR/Cas9‐mediated knockout of G proteins and β‐arrestins delineates their distinct but interdependent roles in dopamine D1 receptor signaling

The dopamine D1 receptor (D1R) is a G protein‐coupled neurotransmitter receptor which controls key brain functions including voluntary movement, memory, attention and reward. Selective agonist activation of the D1R may also provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. The D1R canonically activates the heterotrimeric G proteins Gαs, and possibly Gαolf, which activate adenylyl cyclase (AC) to increase cAMP/PKA signaling. The D1R also engages β‐arrestin proteins which may transduce β‐arrestin dependent signaling by scaffolding and activating kinases. However, the distinct roles of Gαs, Gαolf versus β‐arrestins 1 and 2 for D1R signaling remains largely undefined. Here we utilize CRISPR/Cas9 engineered HEK293 cells in which Gαs, Gαolf, β‐arrestin1 and β‐arrestin2 are stably knocked out (KO) to delineate D1R pharmacology and signaling mechanisms. Western blots confirmed a complete loss of Gαs, Gαolf, β‐arrestin1 or β‐arrestin2 proteins in KO cells versus parental wildtype cells. Dose responses with the D1R agonist SKF‐81297 or β2AR agonist isoproterenol induced robust cAMP production in wildtype cells that was eliminated in Gαs/olf KO cells. Re‐expression of Gαs or Gαolf in KO cells rescued D1R‐mediated cAMP signaling similar to wildtype cells. Notably, the D1R had significantly higher cAMP signaling when coupling via Gαs as compared to Gαolf, while the β2AR only coupled with Gαs to stimulate cAMP signaling. Western blotting indicated Gαs/olf KO completely blocked agonist‐induced CREB phosphorylation by both D1R and β2AR agonists, indicating a strict dependence on Gαs/olf and cAMP/PKA signaling to activate CREB. In major contrast, β‐arrestin1/2 KO significantly increased the intensity and duration of cAMP signaling and CREB phosphorylation by the β2AR. β‐arrestin1/2 KO also entirely blocked agonist‐induced D1R internalization and impaired D1R desensitization while Gαs/olf KO had no effect on D1R internalization. Key controls of re‐expressing Gαs, Gαolf, β‐arrestin1 or β‐arrestin2 in KO cells completely restored signaling or internalization similar to wildtype cells, indicating CRISPR/Cas9 targeting did not result in non‐specific or compensatory effects in the pathways under study. Notably, β‐arrestin1/2 KO also potentiated and prolonged D1R or β2AR‐induced ERK1/2 phosphorylation. D1R‐induced ERK1/2 phosphorylation in β‐arrestin1/2 KO cells was eliminated by the SRC inhibitor SU6656 or the MEK inhibitor U0126, suggesting that D1R signaling via a Gαs/SRC/MEK pathway is essential for ERK1/2 activation. Taken together, our studies indicate that Gαs is the dominant transducer for D1R cAMP, CREB and ERK1/2 signaling and that β‐arrestins provide distinctly interdependent roles with G proteins in modulating the duration and intensity of D1R and β2AR signaling cascades. Using CRISPR/Cas9 KO model cells, future studies will examine the distinct contributions of β‐arrestins and Gαs/olf for D1R pharmacology, which will be useful in defining and validating novel D1R biased agonists.Support or Funding InformationNIH DA047643 (JAA); Rising STAR award from the University of Texas System (JAA); Institutional funding from UTMB (JAA)

Role of Distinct β2 Adrenergic Receptor Pathways in Mediating Inflammation

Chronic treatment of inflammatory diseases (i.e. asthma) with β2‐adrenergic receptor (β2AR) agonists can paradoxically lead to increased inflammation and poor therapeutic outcomes. The β2AR has at least two signaling pathways: the canonical Gs pathway and the β‐arrestin2 (βarr2) pathway. Both pathways are suggested to be involved in inflammatory disorders that are regulated by immunomodulatory cells, such as the airway epithelium. However, the inflammatory response of the epithelium to β2AR ligands that preferentially (relative to epinephrine) activate one of these pathways (biased signaling) remains unknown. Thus, we will use novel fusion proteins made of the β2AR receptor fused to Gαs (β2AR‐Gs) or to βarr2 (β2AR‐βarr2) to restrict signaling to a single pathway, to characterize the inflammatory profile of β2AR ligands, relative to their pharmacological properties (affinity, efficacy, and potency). Our central hypothesis is that by restricting signaling to a single conformational state of the receptor, we will be able to correlate the signaling of β2AR ligands with their pharmacological properties. Our long‐term goal is to understand how the distinct β2AR pathways mediate the inflammatory response in the epithelium. For pharmacological characterization, we used HEK293 cells. This cell line has been used to generate most of the previous data on β2AR biased signaling and allows for comparisons for experimental rigor. We stably transfected each fusion protein (β2AR‐Gs or β2AR‐βarr2) independently into HEK293 cells. Stably transfected HEK293 cells with wild‐type (WT) β2AR and untransfected HEK293 cells served as experimental controls. Using homologous time‐resolving fluorescence, we measured cAMP production and ERK phosphorylation as the surrogates for β2AR signaling via Gs and βarr2, respectively. For the Gs protein signaling pathway, all transfected cells showed increased constitutive activity compared to untransfected HEK293 cells. β2AR‐βarr2 transfected cells, however, showed the highest sensitivity to ICI 115,881 (ICI), a β2AR inverse agonist/antagonist for both pathways, at decreasing constitutive activity. Conversely, for the βarr2 signaling pathway, only β2AR‐βarr2 transfected cells showed constitutive activity. Furthermore, both signaling pathways (Gs or βarr2) could be modulated by isoproterenol (ISO), an agonist for both pathways. Thus, while the chimeric receptor is constitutively active, the fusion proteins are still able to respond to ligands, allowing us to explore the ‘absolute bias’ of β2AR ligands. Our data suggest ICI has a higher affinity for the inhibition of cAMP production by ISO in both the β2AR‐βarr2 and β2AR‐Gs fusion proteins (apparent pA2=10.38 and 10.16, respectively), compared to both control groups (WT β2AR pA2=9.42; untransfected HEK293 pA2=9.21). Furthermore, ISO showed a higher potency for the β2AR‐Gs fusion protein (pEC50=10.12) compared to cells expressing the β2AR‐βarr2 fusion protein (pEC50=8.98). We conclude that our fusion proteins can be used to generate a gain‐of‐function phenotype for studying our long‐term goals and can be useful tools to explore the signaling bias of β2AR ligands.

SPARC: Atomistic Modeling of Autonomic Stimulation for a Multi‐Scale Neurocardiovascular Simulator.

Heart rhythm and vascular tone are modulated by stimulation of the autonomic nervous system, and dysregulation of sympathetic or parasympathetic stimuli is associated with multiple cardiovascular disorders such as arrhythmias and hypertension. At the molecular level, such neuromodulation is established by stimulating myocyte G protein‐coupled receptor (GPCR) mediated signal transduction pathways with neurotransmitters like norepinephrine or acetylcholine. In this study, funded by NIH Common Fund program “Stimulating Peripheral Activity to Relieve Conditions” (SPARC), we used atomistic modeling to identify structural, energetic, and kinetic determinants of several molecular processes crucial for cardiovascular neuromodulation such as neurotransmitter interactions with GPCR / G protein complexes at the neuroeffector junction, as well as G protein interactions with adenylyl cyclase (AC), a key enzyme for downstream subcellular signaling. To determine these parameters, we developed atomistic models of human β1‐ and β2‐adrenergic receptors (β1AR and β2AR) as well as muscarinic acetylcholine receptor M2 (M2R) in different conformational states with and without bound Gs or Gi protein using available structures and Rosetta structural modeling. These models were validated via molecular docking of βAR agonists norepinephrine and isoproterenol and MR agonists acetylcholine and carbachol to compare with experimental crystal structures. Model structural stabilities were assessed via microsecond‐long all‐atom molecular dynamics (MD) simulations with and without bound ligand. Enhanced sampling atomistic MD simulations were used to estimate state‐dependent receptor ‐ ligand binding affinities and association / dissociation rates, which were compared with available experimental values. Also, coarse‐grained and Brownian dynamics implicit‐solvent techniques were utilized to assess G protein interactions with AC and GPCRs. This information will be used to inform functional kinetic models of autonomic control of myocyte subcellular signaling, a crucial component of our predictive multi‐scale neurocardiovascular simulator.Support or Funding InformationNIH SPARC 1OT2OD026580 (C.E.C., I.V.), UC Davis Pharmacology T32GM099608 (J.R.D.D.), AHA career development award 19CDA34770101 (I.V.), NIH NHLBI 5R01HL128537 (C.E.C., V.Y.Y.) and 5U01HL126273 (C.E.C., V.Y.Y.). Computer time allocations: Anton 2 PSCA18077P (I.V., C.E.C., K.R.D.), XSEDE MCB170095 (I.V., K.R.D.), NCSA Blue Waters Broadening Participation Allocation (C.E.C., I.V., K.R.D.).

Beta Adrenoceptor Ligands for the Treatment of Group 3 Pulmonary Hypertension and Cor Pulmonale: A Novel Therapeutic Target?

Pulmonary hypertension is a common complication of idiopathic pulmonary fibrosis (IPF), which leads to cor pulmonale and right-heart failure. β-blockers have been used extensively for the treatment of left heart disease, yet their use for right-heart failure is controversial. Recent studies have shown that β-blockers may be beneficial for the treatment of pulmonary arterial hypertension. However, whether β-receptors ligands are effective in PH and cor pulmonale associated with lung fibrosis has not been evaluated. Here we tested the effects of β adrenergic receptors (βAR) ligands in an experimental model of lung fibrosis and PH where fibrotic injury and cardiovascular complications co-exist. Using lung tissues isolated from explanted lungs from patients with IPF (with and without a diagnosis of PH), we determined βAR gene expression levels. Next, the capacity of either carvedilol (a βAR blocker) or clenbuterol (a β2AR agonist) to improve bleomycin (BLM)-induced alterations in cardiovascular physiology or lung function were assessed. These experiments were performed using lung function and transthoracic Echocardiography (Fig 1). After completion of these physiological readouts, the lungs were excised for assessment of fibrotic injury by RT-PCR, immunoblots and histology. Our experiments demonstrate reduced expression levels of the β2AR, but not β1AR or β3AR in IPF lung vs controls. We also report a significant correlation between elevated mPAP and reduced β2AR expression. Remarkably, our results using carvedilol or clenbuterol revealed that these agents improved right-heart function without impacting lung function. Taken together, these results demonstrate that β-receptor ligands are able to treat PH and cor pulmonale associated with lung fibrosis without altering fibrotic matrix deposition. These results are significant since PH is the single most significant predictor of mortality in IPF where limited therapies exist.

Design of the β3-Adrenergic Agonist Treatment in Chronic Pulmonary Hypertension Secondary to Heart Failure Trial

Combined pre-and post-capillary hypertension (CpcPH) is a relatively common complication of heart failure (HF) associated with a poor prognosis. Currently, there is no specific therapy approved for this entity. Recently, treatment with beta-3 adrenergic receptor (β3AR) agonists was able to improve pulmonary hemodynamics and right ventricular (RV) performance in a translational, large animal model of chronic PH. The authors present the design of a phase II randomized clinical trial that tests the benefits of mirabegron (a clinically available β3AR agonist) in patients with CpcPH due to HF. The effect of β3AR treatment will be evaluated on pulmonary hemodynamics, as well as clinical, biochemical, and advanced cardiac imaging parameters. (Beta3 Agonist Treatment in Chronic Pulmonary Hypertension Secondary to HeartFailure [SPHERE-HF]; NCT02775539).

Psychological stress enhances tumor growth and diminishes radiation response in preclinical model of lung cancer

Background and purposePatients with life-threatening illnesses, such as cancer, experience emotional distress. This study was to investigate the molecular and cellular mechanisms of relevant psychological stressor on tumor growth and therapeutic resistance. Materials and MethodsStress was induced in C57BL/6J mice bearing LLC lung tumors by exposure to a conspecific mice receiving inescapable foot shocks. Mice were irradiated at 7 Gy for 3 consecutive days. Behaviors were monitored by open field test (OFT), elevated plus maze (EPM), sucrose preference test (SPT), and learned helplessness (LH) test. Protein expression in tissues and cultured cells were measured by Western blot. ResultsThis study in animals showed that observing a conspecific mouse receiving foot shocks induced depression like behaviors with increased plasma corticosterone and adrenaline levels which increased tumor growth and radioresistance. Stress increased Wnt1, Drosha, and vimentin expression and decreased E-cadherin expression in tumor tissues. The combination of stress and irradiation enhanced radioresistance along with the increase in vimentin expression. The in vitro study showed that a β2-adrenergic receptor (β2-AR) agonist blocked irradiation-induced cell apoptosis and decreased cell viability, while silencing β2-AR expression reduced the protective effects of β2-AR agonist. β2-AR agonist obviously increased Wnt1 and Drosha expression in LLC-1 cells. ConclusionPsychological stress increased tumor growth and enhanced radioresistance associated with the activation of epithelial-mesenchymal transition by stress hormone-stimulated adrenergic receptors.

Functions of beta2-adrenergic receptor in human periodontal ligament cells.

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor.

G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although β3-adrenergic receptor (β3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce β3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of β3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human β3AR presented a short-term desensitization of cAMP response stimulated by the β3AR agonist, BRL37344, and not by forskolin. We found that β3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased β3AR desensitization. Consistently, stimulation of β3AR increased interaction between GRK2 and Gαs subunit. Furthermore, in rat cardiomyocytes endogenously expressing β3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the β3AR in heart function and disease.

β2-adrenergic signals downregulate the innate immune response and reduce host resistance to viral infection.

In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the β2-adrenergic receptor (β2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a β2-AR agonist were more susceptible to MCMV infection. By contrast, β2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the β2-AR signaling could be used to increase resistance to infectious diseases.

Atomistic Modeling of Neuro-cardiovascular Coupling Modulation

Cardiac heart rhythm and vascular tone are tightly controlled by sympathetic and parasympathetic autonomic nervous systems and their dysregulation is associated with multiple cardiovascular disorders such as arrythmias and hypertension. At the molecular level such control is established through a complex set of events including synaptic neurotransmitter release, binding to myocyte adrenergic or cholinergic G-protein coupled receptors (GPCRs), subcellular signal transduction through G-proteins, neurotransmitter - GPCR unbinding, reuptake or degradation. In this study, funded by NIH Common Fund program “Stimulating Peripheral Activity to Relieve Conditions” (SPARC), we used atomistic modeling to identify structural, energetic and kinetic determinants of several molecular processes crucial for cardiovascular neuromodulation such as neurotransmitter interactions with GPCR / G-protein complex at neuroeffector junction as well as G-protein interaction with adenylyl cyclase (AC), a key enzyme for downstream subcellular signaling. To this end, we developed atomistic models of human β-adrenergic receptor (βAR) / Gs and muscarinic receptor (MR) / Gi GPCR / G-protein complexes in different conformational states using available structures and Rosetta structural modeling. We tested these models via molecular docking of βAR agonists norepinephrine and isoproterenol and MR agonists acetylcholine and carbachol. Moreover, receptor model structural stabilities were assessed via microsecond-long all-atom molecular dynamics (MD) simulations with and without ligand bound. Enhanced sampling atomistic MD runs were used to estimate receptor - ligand binding affinities and association / dissociation rates. Martini coarse-grained and Brownian dynamics implicit-solvent techniques were utilized to assess G-protein interactions with AC and GPCR as well. This information will be used to inform functional kinetic models of autonomic control of myocyte subcellular signaling, a crucial component of our predictive multi-scale neurocardiovascular simulator.

GRK2-Mediated Crosstalk Between β-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein βarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. β-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the βAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the βAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol’s effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the β-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Spatial Distribution of (R)-salbutamol in Rat Brain Following Nasal and Intravenous Administration Using DESI-MS.

Recent studies have shown that β2-Adrenoreceptor is a regulator of the a-synuclein gene driving risk of Parkinson’s disease. The β2-AR agonist (R)-salbutamol, eutomer of rac-salbutamol, may hold therapeutic potential for Parkinson’s disease (PD) following nasal administration. In this study, we use desorption electrospray ionization mass spectrometry (DESI-MS) to analyze spatial distribution of (R)-salbutamol in rat brain following nasal and intravenous administration. Here, we report that (R)-salbutamol efficiently deliver to the brain and had more drug dosage exposure in rat’s brain through nasal route administration than that of intravenous route administration. In conclusion, administering (R)-salbutamol through nasal route of administration may hold advantages in improving spatial distribution and increased exposure of drug in brain.

β3-Adrenergic receptor blockade reduces mortality in endotoxin-induced heart failure by suppressing induced nitric oxide synthase and saving cardiac metabolism.

The β3-adrenergic receptor (β3AR) is related to myocardial fatty acid metabolism and its expression has been implicated in heart failure. In this study, we investigated the role of β3AR in sepsis-related myocardial dysfunction using lipopolysaccharide (LPS)-induced endotoxemia as a model of cardiac dysfunction. We placed mice into three treatment groups and treated each with intraperitoneal injections of the β3AR agonist CL316243 (CL group), the β3AR antagonist SR59230A (SR group), or normal saline (NS group). Survival rates were significantly improved in the SR group compared with the other treatment groups. Echocardiography analyses revealed cardiac dysfunction within 6-12 h of LPS injections, but the outcome was significantly better for the SR group. Myocardial ATP was preserved in the SR group but was decreased in the CL-treated mice. Additionally, quantitative PCR analysis revealed that expression levels of genes associated with fatty acid oxidation and glucose metabolism were significantly higher in the SR group. Furthermore, the expression levels of mitochondrial membrane protein complexes were preserved in the SR group. Electron microscope studies showed significant accumulation of lipid droplets in the CL group. Moreover, inducible nitric oxide synthase (iNOS) protein expression and nitric oxide were significantly reduced in the SR group. The in vitro study demonstrated that β3AR has an independent iNOS pathway that does not go through the nuclear factor-κB pathway. These results suggest that blockading β3AR improves impaired energy metabolism in myocardial tissues by suppressing iNOS expression and recovers cardiac function in animals with endotoxin-induced heart failure.NEW & NOTEWORTHY Nitric oxide production through stimulation of β3-adrenergic receptor (β3AR) may improve cardiac function in cases of chronic heart failure. We demonstrated that the blockade of β3AR improved mortality and cardiac function in endotoxin-induced heart failure. We also determined that LPS-induced inducible nitric oxide synthase has a pathway that is independent of nuclear factor-κB, which worsened cardiac metabolism and mortality in the acute phase of sepsis. Treatment with the β3AR antagonist had a favorable effect. Thus, the blockade of β3AR could offer a novel treatment for sepsis-related heart failure.

Exacerbation of ozone-induced pulmonary and systemic effects by \u03b22-adrenergic and/or glucocorticoid receptor\xa0agonist/s

Agonists of β2 adrenergic receptors (β2AR) and glucocorticoid receptors (GR) are prescribed to treat pulmonary diseases. Since ozone effects are mediated through the activation of AR and GR, we hypothesized that the treatment of rats with relevant therapeutic doses of long acting β2AR agonist (LABA; clenbuterol; CLEN) and/or GR agonist (dexamethasone; DEX) would exacerbate ozone-induced pulmonary and systemic changes. In the first study, male 12-week-old Wistar-Kyoto rats were injected intraperitoneally with vehicle (saline), CLEN (0.004 or 0.02 mg/kg), or DEX (0.02 or 0.1 mg/kg). Since dual therapy is commonly used, in the second study, rats received either saline or combined CLEN + DEX (each at 0.005 or 0.02 mg/kg) one day prior to and on both days of exposure (air or 0.8ppm ozone, 4 hr/day x 2-days). In air-exposed rats CLEN, DEX or CLEN + DEX did not induce lung injury or inflammation, however DEX and CLEN + DEX decreased circulating lymphocytes, spleen and thymus weights, increased free fatty acids (FFA) and produced hyperglycemia and glucose intolerance. Ozone exposure of vehicle-treated rats increased bronchoalveolar lavage fluid protein, albumin, neutrophils, IL-6 and TNF-α. Ozone decreased circulating lymphocytes, increased FFA, and induced hypeerglycemia and glucose intolerance. Drug treatment did not reverse ozone-induced ventillatory changes, however, lung effects (protein and albumin leakage, inflammation, and IL-6 increase) were exacerbated by CLEN and CLEN + DEX pre-treatment in a dose-dependent manner (CLEN > CLEN + DEX). Systemic effects induced by DEX and CLEN + DEX but not CLEN in air-exposed rats were analogous to and more pronounced than those induced by ozone. These data suggest that adverse air pollution effects might be exacerbated in people receiving LABA or LABA plus glucocorticoids.

β3 adrenergic receptor antagonist SR59230A exerts beneficial effects on right ventricular performance in monocrotaline-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease with a high mortality rate. Previous studies have revealed the important function of the β3 adrenergic receptor (β3-AR) in cardiovascular diseases, and the potential beneficial effects of numerous β3-AR agonists on pulmonary vasodilation. Conversely, a number of studies have proposed that the antagonism of β3-AR may prevent heart failure. The present study aimed to investigate the functional involvement of β3-AR and the effects of the β3-AR antagonist, SR59230A, in PAH and subsequent heart failure. A rat PAH model was established by the subcutaneous injection of monocrotaline (MCT), and the rats were randomly assigned to groups receiving four weeks of SR59230A treatment or the vehicle control. SR59230A treatment significantly improved right ventricular function in PAH in vivo compared with the vehicle control (P<0.001). Additionally, the expression level of β3-AR was significantly upregulated in the lung and heart tissues of PAH rats compared with the sham group (P<0.01), and SR59230A treatment inhibited this increase in the lung (P<0.05), but not the heart. Specifically, SR59230A suppressed the elevated expression of endothelial nitric oxide and alleviated inflammatory infiltration to the lung under PAH conditions. These results are, to the best of our knowledge, the first to reveal that SR59230A exerts beneficial effects on right ventricular performance in rats with MCT-induced PAH. Furthermore, blocking β3-AR with SR59230A may alleviate the structural changes and inflammatory infiltration to the lung as a result of reduced oxidative stress.

β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P&lt; 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P&lt;0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

Design, synthesis and biological evaluation of 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one derivatives as potent β2-adrenoceptor agonists

A series of β2-adrenoceptor agonists with an 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one moiety is presented. The stimulatory effects of the compounds on human β2-adrenoceptor and β1-adrenoceptor were characterized by a cell-based assay. Their smooth muscle relaxant activities were tested on isolated guinea pig trachea. Most of the compounds were found to be potent and selective agonists of the β2-adrenoceptor. One of the compounds, (R)-18c, possessed a strong β2-adrenoceptor agonistic effect with an EC50 value of 24 pM. It produced a full and potent airway smooth muscle relaxant effect same as olodaterol. Its onset of action was 3.5 min and its duration of action was more than 12 h in an in vitro guinea pig trachea model of bronchodilation. These results suggest that (R)-18c is a potential candidate for long-acting β2-AR agonists.

β2 Adrenergic Regulation of the Phagocytic and Microbicide Capacity of Macrophages from Obese and Lean Mice: Effects of Exercise.

Macrophages are crucial in the inflammation associated with obesity. Exercise is the main non-pharmacological strategy against obesity, not only for improving metabolic impairment, but also because of its anti-inflammatory effects, particularly those mediated by β2 adrenergic receptors (β2-AR). Nevertheless, these anti-inflammatory effects could immunocompromise the innate response against pathogen challenge. Thus, the objective of this work was to evaluate the effect of obesity, and of exercise in this condition, on the β2 adrenergic regulation of the innate function of macrophages. High fat diet-induced obese C57BL/6J mice were used to evaluate the effects of acute and regular exercise on the phagocytic and microbicide capacities of peritoneal macrophages. Selective β2-AR agonist terbutaline (1 µM) decreased the phagocytic and microbicide activities of macrophages from control lean and obese sedentary animals. While acute exercise did not modify the inhibitory capacity of terbutaline, regular exercise abolished this inhibitory effect. These effects cannot be explained only by changes in the surface expression of β2-AR. In conclusion, (1) obesity does not alter the β2-AR-mediated decrease of the innate response of macrophages and (2) regular exercise can revert the inhibitory effect of terbutaline on the phagocytic activity of macrophages, although obesity seems to hinder this immunophysiological adaptation.

Beta-3 Receptor Agonists

Beta-3 adrenergic receptors (β3-AR) have a more widespread tissue distribution in the human body as compared to beta1- and beta2 (β1/2)-adrenergic receptors, including in the bladder, brain, adipose tissue and cardiovascular system. Thus, β3- AR are potential drug targets for a wide range of therapeutic areas, both cardiovascular and non-cardiovascular including overactive bladder (OAB), depression, metabolic syndrome, obesity and heart failure (HF). β3-AR agonists that are selective to the β3-AR include CL 316,243, amibegron (SR58611A), mirabegron (YM-178) and vibegron (RVT-901). However, in HF, study results regarding a possible inotropic effect of β3-AR agonists remain equivocal and some authors report a negative inotropic effect in HF and β3-AR antagonists are also under study.