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A large number of β-adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of cardiovascular diseases and other diseases. Nonetheless, it remains unclear whether these commonly used β-AR drugs can activate downstream β- arrestin-biased signaling pathways. The objective of this study was to investigate β-arrestin2 recruitment effects of β-AR agonists and antagonists that were commonly used in clinical practice. We used TANGO (transcriptional activation following arrestin translocation) assay to detect the β-arrestin2 recruitment by β-AR ligands in HEK293 cell line (HTLA cells) stably transfected with tetracycline transactivator protein (tTA) dependent luciferase reporter and β-arrestin2-TEV fusion gene. Upon activation of β-AR by a β-AR ligand, β-arrestin2 was recruited to the C terminus of the receptor, followed by cleavage of the G protein-coupled receptors (GPCRs) fusion protein at the TEV protease-cleavage site. The cleavage resulted in the release of tTA, which, after being transported to the nucleus, activated transcription of the luciferase reporter gene. The results showed that β-AR non-selective agonists epinephrine, noradrenaline and isoprenaline all promoted β-arrestin2 recruitment at β1-AR and β2-AR. β1-AR selective agonists dobutamine and denopamine both promoted β-arrestin2 recruitment at β1-AR. β2-AR selective agonists procaterol and salbutamol promoted β-arrestin2 recruitment at β2-AR. β-AR non-selective antagonists alprenolol and pindolol promoted β-arrestin2 recruitment at β1-AR. β1-AR selective antagonists celiprolol and bevantolol showed β-arrestin2 recruitment at β1-AR. β2-AR selective antagonists butoxamine showed β-arrestin2 recruitment at β1-AR. These results provide some clues for the potential action of β-AR drugs, and lay a foundation for the screening of β-arrestin-biased β-AR ligands.

Background and Purpose: β-Adrenoceptor agonists relieve airflow obstruction by activating β 2 -adrenoceptors, which are G protein-coupled receptors (GPCRs) expressed on human airway smooth muscle (HASM) cells. The currently available β-adrenoceptor agonists are balanced agonists, however, and signal through both the stimulatory G protein (G s )- and β-arrestin-mediated pathways. While G s signalling is beneficial and promotes HASM relaxation, β-arrestin activation is associated with reduced G s efficacy. In this context, biased ligands that selectively promote β 2 -adrenoceptor coupling to G s signalling represent a promising strategy to treat asthma. Here, we examined several β-adrenoceptor agonists to identify G s -biased ligands devoid of β-arrestin-mediated effects., Experimental Approach: G s -biased ligands for the β 2 -adrenoceptor were identified by high-throughput screening and then evaluated for G s interaction, G i interaction, cAMP production, β-arrestin interaction, GPCR kinase (GRK) phosphorylation of the receptor, receptor trafficking, ERK activation, and functional desensitization of the β 2 -adrenoceptor., Key Results: We identified ractopamine, dobutamine, and higenamine as G s -biased agonists that activate the G s /cAMP pathway upon β 2 -adrenoceptor stimulation while showing minimal G i or β-arrestin interaction. Furthermore, these compounds did not induce any receptor trafficking and had reduced GRK5-mediated phosphorylation of the β 2 -adrenoceptor. Finally, we observed minimal physiological desensitization of the β 2 -adrenoceptor in primary HASM cells upon treatment with biased agonists., Conclusion and Implications: Our work demonstrates that G s -biased signalling through the β 2 -adrenoceptor may prove to be an effective strategy to promote HASM relaxation in the treatment of asthma. Such biased compounds may also be useful in identifying the molecular mechanisms that determine biased signalling and in design of safer drugs., (© 2022 British Pharmacological Society.)

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

The β 2 -adrenergic receptor (β 2 AR) is a member of the G protein-coupled receptor (GPCR) family that is an important drug target for asthma and COPD. Clinical studies coupled with biochemical data have identified a critical receptor variant, Thr164Ile, to have a reduced response to agonist-based therapy, although the molecular mechanism underlying this seemingly "non-deleterious" substitution is not clear. Here, we couple molecular dynamics simulations with network analysis and free-energy calculations to identify the molecular determinants underlying the differential drug response. We are able to identify hydration sites in the transmembrane domain that are essential to maintain the integrity of the binding site but are absent in the variant. The loss of these hydration sites in the variant correlates with perturbations in the intra-protein interaction network and rearrangements in the orthosteric ligand binding site. In conjunction, we observe an altered binding and reduced free energy of a series of agonists, in line with experimental trends. Our work identifies a functional allosteric pathway connected by specific hydration sites in β 2 AR that has not been reported before and provides insight into water-mediated networks in GPCRs in general. Overall, the work is one of the first step towards developing variant-specific potent and selective agonists., (Copyright © 2021 Elsevier B.V. All rights reserved.)

β-adrenergic receptor antagonists (β-blockers) are extensively used to improve cardiac performance in heart failure (HF), but the electrical improvements with these clinical treatments are not fully understood. The aim of this study was to analyze the electrophysiological effects of β-adrenergic system remodeling in heart failure with reduced ejection fraction and the underlying mechanisms. We used a combined mathematical model that integrated β-adrenergic signaling with electrophysiology and calcium cycling in human ventricular myocytes. HF remodeling, both in the electrophysiological and signaling systems, was introduced to quantitatively analyze changes in electrophysiological properties due to the stimulation of β-adrenergic receptors in failing myocytes. We found that the inotropic effect of β-adrenergic stimulation was reduced in HF due to the altered Ca 2+ dynamics resulting from the combination of structural, electrophysiological and signaling remodeling. Isolated cells showed proarrhythmic risk after sympathetic stimulation because early afterdepolarizations appeared, and the vulnerability was greater in failing myocytes. When analyzing coupled cells, β-adrenergic stimulation reduced transmural repolarization gradients between endocardium and epicardium in normal tissue, but was less effective at reducing these gradients after HF remodeling. The comparison of the selective activation of β-adrenergic isoforms revealed that the response to β 2 -adrenergic receptors stimulation was blunted in HF while β 1 -adrenergic receptors downstream effectors regulated most of the changes observed after sympathetic stimulation. In conclusion, this study was able to reproduce an altered β-adrenergic activity on failing myocytes and to explain the mechanisms involved. The derived predictions could help in the treatment of HF and guide in the design of future experiments., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Purpose: The physiological role of vascular β 3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β 3 -adrenoceptors are functionally effective after down-regulation of β 1 /β 2 -adrenoceptors. The functional interaction between the β 3 -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated., Experimental Approach: Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β 3 -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography., Key Results: All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β 3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β 3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β 1/2 -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β 3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β 3 -adrenoceptor agonists (in the presence of β 1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation., Conclusion and Implications: All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β 3 -adrenoceptor mediated vasodilation was only evident after blockade of β 1/2 -adrenoceptors. This suggests constitutive β 1/2 -adrenoceptor activity inhibits β 3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries., (© 2021 The British Pharmacological Society.)

G protein-coupled receptor (GPCR) is activated by extracellular signals. After their function at plasma membrane, GPCRs are internalized to be desensitized, while emerging evidence suggests that some GPCRs maintain their activity even after internalization. The endosomal trafficking pathway of a prototypic GPCR, β adrenergic receptor 2 (B2AR), is in the range of several hours, however, spatiotemporal B2AR activity during this long-term endosomal trafficking pathway has not been characterized yet. Here, we analyze an agonist-induced real-time B2AR activity and its downstream function at the level of individual vesicles, utilizing a fluorescence resonance energy transfer (FRET)-based B2AR biosensor and cAMP reporters tethered at different trafficking stages of endosomes. Our results report that the internalized B2ARs sustain the activity and maintain the production of cAMP for several hours during the endosomal trafficking pathway. Temporal kinetics of B2AR activity is mathematically well explained by our active-vesicle population model modified from the Ricker model. Therefore, our GPCR monitoring system and a new kinetics model can be applied to understand the spatiotemporal GPCR activity and its downstream function during the endosomal trafficking pathway.

In the context of pharmacology teaching, hands-on activities constitute an essential complement to theoretical lectures. Frequently, these activities consist in exposing fresh animal tissues or even living animals to selected drugs and qualitatively or quantitatively evaluating functional responses. However, technological advancements in pharmacological research and the growing concerns for animal experimentation support the need for innovative and flexible in vitro assays adapted for teaching purposes. We herein report the implementation of a luciferase complementation assay (LCA) enabling to dynamically monitor β-arrestin2 recruitment at the β 2 adrenergic receptor in the framework of pharmacological training at the faculty of Pharmacy and Biomedical Sciences. The assay allowed students to quantitatively characterize the competitive antagonism of propranolol, and to calculate pEC 50 , pK B , and pA 2 values after a guided data analysis session. Moreover, the newly implemented workshop delivered highly reproducible results and were generally appreciated by students. As such, we report that the luciferase complementation-based assay proved to be a straightforward, robust, and cost-effective alternative to experiments performed on animal tissues, constituting a useful and flexible tool to enhance and update current hands-on training in the context of pharmacological teaching., (© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Higenamine was included in the World Anti-Doping Agency (WADA) Prohibited Substances and Methods List as a β 2 -adrenoceptor agonist in 2017, thereby resulting in its prohibition both in and out of competition. The present mini review describes the physiology and pharmacology of adrenoceptors, summarizes the literature addressing the mechanism of action of higenamine and extends these findings with previously unpublished in silico and in vitro work. Studies conducted in isolated in vitro systems, whole-animal preparations and a small number of clinical studies suggest that higenamine acts in part as a β 2 -adrenoceptor agonist. In silico predictive tools indicated that higenamine and possibly a metabolite have a high probability of interacting with the β 2 -receptor as an agonist. Stable expression of human β 2 -receptors in Chinese hamster ovary (CHO) cells to measure agonist activity not only confirmed the activity of higenamine at β 2 but also closely agreed with the in silico prediction of potency for this compound. These data confirm and extend literature findings supporting the inclusion of higenamine in the Prohibited List., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Rationale: β 1 ARs (β 1 -adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β 1 AR in cardiac contractility remains to be elucidated., Objective: Test localization and function of intracellular β 1 AR on cardiac contractility., Methods and Results: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β 1 ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca 2+ -ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility., Conclusions: Functional β 1 ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β 1 ARs requires catecholamine transport via OCT3.

Background: Considerable evidence suggests that the sympathetic nervous system, mainly via adrenergic signaling, contributes to prostate cancer (PCa) progression. However, the underlying molecular mechanisms remain unknown., Methods: The expression level of β 2 -adrenergic receptor (β 2 -AR) in tissue microarray was evaluated by immunohistochemistry. The effects of isoproterenol (ISO) or Sonic Hedgehog (Shh) signaling inhibitor on tumor growth were analyzed in proliferation and colony formation assays. The apoptosis of cells was analyzed by flow cytometry. Small hairpin RNA-based knockdown of β 2 -AR or Gli1 was validated by Western blot analysis and real-time PCR. Effects of β 2 -AR on prostate carcinogenesis in vivo were observed in a mouse xenograft model. The expression levels of the indicated proteins in xenograft tissues were evaluated by immunohistochemistry. Expression levels of Shh signaling components and downstream proteins were assessed by immunoblotting., Results: We determined that β 2 -AR was expressed at significantly higher levels in carcinoma than in normal prostate tissues. β 2 -AR signaling also played an essential role in sustaining PCa cell proliferation in vivo and in vitro. We also found that inhibition of Shh signaling or knockdown of Gli1 expression significantly restrained ISO-induced cell proliferation in vitro. ISO alleviated the apoptosis induced by suppressing or knocking down of Gli1. The β 2 -AR agonist ISO upregulated the transcription and protein expression of target genes of Shh signaling, including c-Myc, Cyclin D1, and VEGFA. Conversely, knocking down β 2 -AR markedly suppressed the expression of Shh components in vivo and in vitro. In Gli1 knockdown cells, ISO failed to increase the expression of target genes of Shh signaling., Conclusions: In this study, we uncovered an important role of β 2 -AR signaling in regulating the Shh pathway activity in PCa tumorigenesis and provide further insight into the mechanism of the involvement of the Hh signaling pathway. Furthermore, given the efficacy of β 2 -adrenergic modulation on PCa, our study might also add evidence for potential therapeutic options of β-blockers for PCa., (© 2020 The Authors. The Prostate published by Wiley Periodicals LLC.)

The bladder wall is constantly subjected to intravesical pressure during the filling and voiding cycles. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) under elevated intravesical pressure contributes to pathological changes in the bladder. To investigate the changes in human urothelial cells (HUCs) under elevated intravesical pressure, this study analyzed the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs exposed to pathological hydrostatic pressure (HP) (70 cm H 2 O) for 6 hours. Quantitative polymerase chain reaction, Western blot analysis, and cell fluorescence staining were used to explore the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs after agonist and/or antagonist treatment. The expression levels of β 2 - and β 3 -adrenoceptor, MMP1, and MMP2 were greatly downregulated, while the expression of TIMP1 was greatly upregulated. Formoterol and BRL 37344, which are agonists of β 2 - and β 3 -adrenoceptor, respectively, significantly increased MMP1 and MMP2 expression under 70 cm H 2 O. As a classic downstream pathway of β 2 - and β 3 -adrenoceptor, protein kinase A (PKA) signaling inhibited MMP1 and MMP2 expression by regulating cAMP response element binding protein (CREB) activity. MMP1 and MMP2 expression in HUCs under 70 cm H 2 O was modified by β 2 - and β 3 -adrenoceptor via the PKA/CREB pathway. This outcome suggests that MMPs likely participate in the pathological effects of elevated intravesical pressure. The underlying mechanism of β 2 - and β 3 -adrenoceptor in elevated intravesical pressure was also revealed; this mechanism constitutes a new potential therapeutic target for partial bladder outlet obstruction., (© 2020 Wiley Periodicals, Inc.)

Background and Purpose: In cardiovascular diseases, cardiac fibroblasts (CFs) participate in the myocardial inflammation by producing pro-inflammatory cytokines, worsening the prognosis. β2-adrenergic receptor (AR) and β3AR are expressed in CFs, and β-adrenergic stimulation promotes CFs to produce pro-inflammatory cytokines. However, the mechanism of the expression of pro-inflammatory cytokines in response to β-adrenergic stimulation remains to be fully elucidated., Experimental Approach: CFs were isolated from adult wild-type or AT-rich interactive domain-containing protein 5A (Arid5a) knockout mice. The expression of mRNA was measured by real-time RT-PCR. Interleukin (IL)-6 protein was measured by ELISA. The activity of nuclear factor-κB (NF-κB) and cyclic AMP (cAMP) response element binding protein (CREB) was assessed by ELISA-like assay or Western blotting., Key Results: The β-adrenergic stimulation remarkably induced IL-6 mRNA and protein through β2AR in CFs. The activation of adenylate cyclase and the enhancement of intracellular cAMP resulted in the upregulation of IL-6 mRNA expression. The induction of IL-6 transcript by β2AR signaling was independent of NF-κB. Concomitant with IL-6, the expression of Arid5a, an IL-6 mRNA stabilizing factor, was enhanced by β2-adrenergic stimulation and by cAMP increase. Importantly, β2AR signaling-mediated IL-6 induction was suppressed in Arid5a knockout CFs. Finally, β2AR stimulation phosphorylated CREB via PKA pathway, and the activation of CREB was essential for the induction of Arid5a and IL-6 mRNA., Conclusion and Implications: β2-adrenergic stimulation post-transcriptionally upregulates the expression of IL-6 by the induction of Arid5a through cAMP/PKA/CREB pathway in adult CFs. β2AR/Arid5a/IL-6 axis could be a therapeutic target against cardiac inflammation., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

A series of β 2 -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 β 2 -adrenoceptor and β 1 -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 β 2 -adrenoceptor. One of the compounds, (R)-18c, possessed a strong β 2 -adrenoceptor agonistic effect with an EC 50 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 β 2 -AR agonists., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of the present study was to reveal the effect of hyperlipidemia on β2- and β3-adrenergic signaling in late pregnant rat uterus. Hyperlipidemia was induced in female Wistar rats by feeding a high-fat high-cholesterol diet for 8 weeks before and after mating upto the 21st day of gestation. The effect of hyperlipidemia on β-adrenergic signaling was studied with the help of tension experiments, real-time PCR and cAMP ELISA in 21-day pregnant rat uterus. In tension experiments, hyperlipidemia neither altered the spontaneous contractility nor the oxytocin-induced contractions. However, it decreased the -logEC50 values of β2-adrenoceptor agonist, salbutamol and β3-adrenoceptor agonist, BRL37344. It also decreased the efficacy of adenylyl cyclase activator, forskolin. Further, there was a significant decrease in salbutamol and BRL37344-stimulated cAMP content in uterine tissues. However, there was no alteration in mRNA expressions of β2-adrenoceptor (Adrb2), β3-adrenoceptor (Adrb3) and Gs protein (Gnas) though there was a significant increase in the mRNA expression of Gi protein (Gnai). In conclusion, reduced cAMP content after beta-adrenergic receptor stimulation, which correlates with an increase in Gnai mRNA, may explain the mechanism of the impairment of uterine β-adrenergic signaling in hyperlipidemic pregnant rats. The clinical implication of the present study may relate to reduced myometrial relaxant response to β-adrenergic agonists in high fat-induced uterine dysfunction.

New Findings: What is the central question of this study? Are there specific contributions of β 1 - and β 2 -adrenoceptor subtypes to the impaired β-adrenoceptor responsiveness of the type 2 diabetic heart? What is the main finding and its importance? In hearts isolated from the Zucker diabetic fatty rat model of type 2 diabetes, we showed that the β 1 -adrenoceptors are the main subtype to regulate heart rate, contraction and relaxation. Notably, the β 2 -adrenoceptor subtype actions seem to support function in the diabetic heart indirectly., Abstract: Impaired β-adrenoceptor (β-AR) responsiveness causes cardiac vulnerability in patients with type 2 diabetes, but the independent contributions of β 1 - and β 2 -AR subtypes to β-AR-associated cardiac dysfunction in diabetes are unknown. Our aim was to determine the specific β 1 - and β 2 -AR responsiveness of heart rate (HR), contraction and relaxation in the diabetic heart. Isolated Langendorff-perfused hearts of Zucker type 2 diabetic fatty (ZDF) rats were stimulated with the β-AR agonist isoprenaline (1 × 10 -11 to 3 × 10 -8  mol l -1 ) with or without the selective β 1 -AR antagonist CGP20712A (3 × 10 -8  mol l -1 ) or the β 2 -AR antagonist ICI-118,551 (5 × 10 -8  mol l -1 ), and HR, contraction and relaxation were measured. Diabetic hearts showed lower basal HR (non-diabetic 216 ± 17 beats min -1 versus diabetic 151 ± 23 beats min -1 , P < 0.05). However, the β-AR-induced increase in HR was similar and was completely blocked by the β 1 -AR antagonist, but not by the β 2 -AR antagonist. The β-AR-induced increase in contraction and acceleration of relaxation was impaired in diabetic hearts, completely blocked by the β 1 -AR antagonist and partly impaired by the β 2 -AR antagonist. Western blots revealed 41% higher phosphorylation levels of AMP kinase (AMPK), a key regulator of cardiac energy metabolism, in diabetic hearts (non-diabetic 1.62 ± 0.19 a.u. versus diabetic 2.30 ± 0.25 a.u., P < 0.05). In conclusion, the β 1 -AR is the main subtype regulating chronotropic, inotropic and lusitropic β-AR responses in the healthy heart and the type 2 diabetic heart. The β 2 -AR subtype indirectly supports the β 1 -AR functional response in the diabetic heart. This suggests that β 2 -ARs could be an indirect target to improve the function of the heart in type 2 diabetes., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Background: Ischemic stroke provokes a neuroinflammatory response and simultaneously promotes release of epinephrine and norepinephrine by the sympathetic nervous system. This increased sympathetic outflow can act on β2-adrenergic receptors expressed by immune cells such as brain-resident microglia and monocyte-derived macrophages (MDMs), but the effect on post-stroke neuroinflammation is unknown. Thus, we investigated how changes in β2-adrenergic signaling after stroke onset influence the microglia/MDM stroke response, and the specific importance of microglia/MDM β2-adrenergic receptors to post-stroke neuroinflammation., Methods: To investigate the effects of β2-adrenergic receptor manipulation on post-stroke neuroinflammation, we administered the β2-adrenergic receptor agonist clenbuterol to mice 3 h after the onset of photothrombotic stroke. We immunostained to quantify microglia/MDM numbers and proliferation and to assess morphology and activation 3 days later. We assessed stroke outcomes by measuring infarct volume and functional motor recovery and analyzed gene expression levels of neuroinflammatory molecules. Finally, we evaluated changes in cytokine expression and microglia/MDM response in brains of mice with selective knockout of the β2-adrenergic receptor from microglia and monocyte-lineage cells., Results: We report that clenbuterol treatment after stroke onset causes enlarged microglia/MDMs and impairs their proliferation, resulting in reduced numbers of these cells in the peri-infarct cortex by 1.7-fold at 3 days after stroke. These changes in microglia/MDMs were associated with increased infarct volume in clenbuterol-treated animals. In mice that had the β2-adrenergic receptor specifically knocked out of microglia/MDMs, there was no change in morphology or numbers of these cells after stroke. However, knockdown of β2-adrenergic receptors in microglia and MDMs resulted in increased expression of TNFα and IL-10 in peri-infarct tissue, while stimulation of β2-adrenergic receptors with clenbuterol had the opposite effect, suppressing TNFα and IL-10 expression., Conclusions: We identified β2-adrenergic receptor signaling as an important regulator of the neuroimmune response after ischemic stroke. Increased β2-adrenergic signaling after stroke onset generally suppressed the microglia/MDM response, reducing upregulation of both pro- and anti-inflammatory cytokines, and increasing stroke size. In contrast, diminished β2-adrenergic signaling in microglia/MDMs augmented both pro- and anti-inflammatory cytokine expression after stroke. The β2-adrenergic receptor may therefore present a therapeutic target for improving the post-stroke neuroinflammatory and repair process.

Aims: Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium-potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure., Methods and Results: A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects., Conclusions: NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Primary and secondary lymphoid organs are heavily innervated by the autonomic nervous system. Norepinephrine, the primary neurotransmitter secreted by post-ganglionic sympathetic neurons, binds to and activates β-adrenergic receptors expressed on the surface of immune cells and regulates the functions of these cells. While it is known that both activated and memory CD8 + T-cells primarily express the β2-adrenergic receptor (β2-AR) and that signaling through this receptor can inhibit CD8 + T-cell effector function, the mechanism(s) underlying this suppression is not understood. Under normal activation conditions, T-cells increase glucose uptake and undergo metabolic reprogramming. In this study, we show that treatment of murine CD8 + T-cells with the pan β-AR agonist isoproterenol (ISO) was associated with a reduced expression of glucose transporter 1 following activation, as well as decreased glucose uptake and glycolysis compared to CD8 + T-cells activated in the absence of ISO. The effect of ISO was specifically dependent upon β2-AR, since it was not seen in adrb2 -/- CD8 + T-cells and was blocked by the β-AR antagonist propranolol. In addition, we found that mitochondrial function in CD8 + T-cells was also impaired by β2-AR signaling. This study demonstrates that one mechanism by which β2-AR signaling can inhibit CD8 + T-cell activation is by suppressing the required metabolic reprogramming events which accompany activation of these immune cells and thus reveals a new mechanism by which adrenergic stress can suppress the effector activity of immune cells.

Label-free cell phenotypic assays were performed to establish a β 2 -adrenoceptor (β 2 -AR) target model in A431 cells and a β 1 -AR target model in transfected HEK293-β 1 cells, using known β 2 -AR and β 1 -AR agonists and antagonists. A list of natural compounds was screened on the target models, among which seven new compounds were found to be antagonistically active against β 2 -AR. After receptor specificity evaluations on hydroxyl carboxylic acid receptor-2 (ΗΧΑ-2), histamine receptor (H 1 R), and β 1 -adrenoceptor (β 1 -AR), six out of the seven compounds, including nuciferine, epiberberine, harmaline, harmine, palmatine, and columbamine, exhibited specific antagonistic activity against β 2 -AR. Epiberberine and palmatine showed the strongest antagonistic activities against β 2 -AR with IC 50 values of 2.3 ± 0.2 μM and 2.6 ± 0.3 μM, respectively. Docking palmatine to the crystal structure of human β 2 -AR (PDB 5X7D) suggested that the ligand forms a hydrogen bond with N312 and hydrophobic interaction with several amino acid residues in the binding pocket, such as D113 and V114. The kinetic binding profile of palmatine was further investigated using co-stimulation assays. Results suggested that palmatine was a competitive antagonist for β 2 -AR. The six novel β 2 -AR antagonists provide a promising chemical starting point for identification and optimization of drugs used for treating hypertension, glaucoma, and infantile hemangiomas. This study also lays the foundation for the in-depth investigation of biochemical mechanisms and pharmacological properties of natural compounds.

Myocardial PDE2 activity increases in terminal human heart failure and after isoprenaline infusion in rat heart. PDE2 inhibitors do not potentiate the murine sinoatrial tachycardia produced by noradrenaline. We investigated whether isoprenaline infusion induces PDE2 to decrease the chronotropic and inotropic effects of catecholamines in rat heart. Sprague-Dawley rats were infused with isoprenaline (2.4 mg kg -1  day -1 ) for 3 days. We used spontaneously beating right atria, paced right ventricular strips and left ventricular papillary muscles. The effects of the PDE2 inhibitors EHNA (10 μM) and Bay 60-7550 (0.1-1 μM) were investigated on the cardiostimulation produced by noradrenaline (ICI118551 50 nM present to block β 2 -adrenoceptors) and adrenaline (CGP20712A 300 nM present to block β 1 -adrenoceptors). Hydrolysis of cAMP by PDE2 was measured by radioenzyme assay. Bay 60-7550 but not EHNA increased sinoatrial beating. A stable tachycardia elicited by noradrenaline (10 nM) or adrenaline (1 μM) was not increased by the PDE2 inhibitors. Isoprenaline infusion increased the hydrolytic PDE2 activity threefold in left ventricle, reduced the chronotropic and inotropic effects and potency of noradrenaline and abolished the effects of adrenaline. The potency of the catecholamines was not increased by the PDE2 inhibitors. Neither EHNA nor Bay 60-7550 potentiated the effects of the catecholamines. Rat PDE2 decreased basal sinoatrial beating but did not reduce the sinoatrial tachycardia or increases of ventricular force mediated through β 1 - and β 2 -adrenoceptors. The β-adrenoceptor desensitization induced by the isoprenaline infusion was not reversed by the PDE2 inhibitors despite the increased hydrolysis of cAMP by PDE2.

Cardiovascular disease development has been associated with sex differences, suggesting that sex hormones are implicated in vascular function and development of hypertension. Vascular tone comparison at different stages of rat growth represents a good model to study testosterone-related vascular response. We explored the role of testosterone in modulation of age-dependent impaired β-adrenergic vasodilation. The 3-week-old male Sprague-Dawley rats were sorted in 3-week-old rats without any manipulation and 3-week-old rats treated with testosterone. The 9-week-old rats were randomly grouped into 9-week-old rats without any manipulation (sham), 9-week-old rats that underwent gonadectomy (9-week-old castrated), and 9-week-old castrated treated with testosterone replacement therapy (9-week-old castrated + testosterone). Vascular relaxation was evaluated in aortic rings. β-adrenergic receptor protein expression, cyclic adenosine monophosphate production, testosterone levels, and adenylyl cyclase (AC) gene expression were assessed. Testosterone levels were low in 3-week-old and 9-week-old castrated rats compared with 9-week-old sham rats. Testosterone replacement raised these levels in 3-week-old and 9-week-old castrated rats similar to those of 9-week-old sham rats. SQ22536, the AC inhibitor, prevented isoproterenol-induced relaxation in aortic rings from 3-week-old and 9-week-old castrated rats. The β-adrenergic receptor protein expression was similar in all experimental groups. AC mRNA and protein expression and cyclic adenosine monophosphate levels were elevated in 3-week-old and 9-week-old castrated rats compared with 3-week-old + testosterone, 9-week-old sham, and 9-week-old castrated + testosterone rats. In conclusion, we demonstrated that age maturation was associated with vascular relaxation impairment. Variations in testosterone levels and reduced AC expression may be responsible for this altered vascular function.

Object We aimed to identify the β-adrenoceptor (β-AR) subtypes involved in isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle using pharmacological and biochemical approaches. Methods Longitudinal smooth muscle was prepared from the male guinea pig ascending colon and contracted with histamine prior to comparing the relaxant responses to three catecholamines (isoprenaline, adrenaline, and noradrenaline). The inhibitory effects of subtype-selective β-AR antagonists on isoprenaline-induced relaxation were then investigated. Results The relaxant potencies of the catecholamines were ranked as: isoprenaline > noradrenaline ≈ adrenaline, whereas the rank order was isoprenaline > noradrenaline > adrenaline in the presence of propranolol (a non-selective β-AR antagonist; 3 × 10 -7 M). Atenolol (a selective β 1 -AR antagonist; 3 × 10 -7 -10 -6  M) acted as a competitive antagonist of isoprenaline-induced relaxation, and the pA 2 value was calculated to be 6.49 (95% confidence interval: 6.34-6.83). The relaxation to isoprenaline was not affected by ICI-118,551 (a selective β 2 -AR antagonist) at 10 -9 -10 -8  M, but was competitively antagonized by 10 -7 -3 × 10 -7  M, with a pA 2 value of 7.41 (95% confidence interval: 7.18-8.02). In the presence of propranolol (3 × 10 -7 M), the relaxant effect of isoprenaline was competitively antagonized by bupranolol (a non-selective β-AR antagonist), with a pA 2 value of 5.90 (95% confidence interval: 5.73-6.35). Conclusion These findings indicated that the β-AR subtypes involved in isoprenaline-induced relaxation of colonic longitudinal guinea pig muscles are β 1 -AR and β 3 -AR.

Object We identified the β-adrenoceptor (β-AR) subtypes responsible for the relaxant responses to adrenaline (AD) and noradrenaline (NA) in the rat thoracic aorta and examined the role of cAMP which is involved in these relaxant responses. Methods The effects of β-AR antagonists or the adenylyl cyclase inhibitor SQ 22,536 on AD- and NA-induced relaxant responses in phenylephrine-induced contraction and increases in cAMP levels were examined in isolated, endothelium-denuded rat thoracic aorta segments. Results AD-induced relaxation was completely suppressed by propranolol (10 -7  M) or by ICI-118,551 (10 -8  M) plus atenolol (10 -6 M), and was also very strongly inhibited by ICI-118,551 (10 -8  M) alone. AD (10 -5  M) increased tissue cAMP levels by approximately 1.9-fold compared with that in non-stimulated aortic tissue, but did not significantly increase cAMP levels in the presence of ICI-118,551 (10 -8  M) or SQ 22,536 (10 -4 M). AD-induced relaxation was strongly suppressed by SQ 22,536 (10 -4 M). NA-induced relaxation was almost completely suppressed by atenolol (10 -6  M) plus ICI-118,551 (10 -8  M) although it was hardly affected by ICI-118,551 (10 -8  M) alone. NA (10 -5  M) increased tissue cAMP levels by approximately 2.2-fold compared with that in non-stimulated aortic tissue, but did not significantly increase cAMP levels in the presence of atenolol (10 -6  M) or SQ 22,536 (10 -4 M). NA-induced relaxation was strongly suppressed by SQ 22,536 (10 -4 M). Conclusion In rat thoracic aorta, AD- and NA-induced relaxations, which are both strongly dependent on increased tissue cAMP levels, are mainly mediated through β 2 - and β 1 -adrenoceptors respectively.

Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the β 2 -adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or βarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.

Visualization of the G-protein coupled receptor (GPCR) is of great importance for studying its function in a native cell. We have synthesized a series of red-emitting fluorescent probes targeting β-adrenergic receptor (βAR) that are compatible with confocal and Stimulated Emission Depletion (STED) microscopy as well as with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay in living cells. The probe based on the agonist BI-167107 and fluorescent dye KK114 demonstrates nanomolar binding affinity and up to nine-fold β 2 AR selectivity over β 1 AR. Carazolol-derived probes are fluorogenic and allow no-wash imaging experiments. STED microscopy of β 2 ARs stained at the native expression level on pancreatic CAPAN cells provides two-fold improvement in lateral optical resolution over confocal mode and reveals the formation of receptor microdomains. These probes retain their functional (agonist or antagonist) properties, allowing simultaneous modulation of cyclic adenosine monophosphate (cAMP) levels and receptor internalization as well as imaging receptor localization.

Regular exercise training is known to affect the action potential duration (APD) and improve heart function, but involvement of β-adrenergic receptor (β-AR) subtypes and/or the ATP-sensitive K + (K ATP ) channel is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to voluntary wheel-running or control groups; they were anesthetized after 6-8 wk of training, and myocytes were isolated. Exercise training significantly increased APD of apex and base myocytes at 1 Hz and decreased APD at 10 Hz. Ca 2+ transient durations reflected the changes in APD, while Ca 2+ transient amplitudes were unaffected by wheel running. The nonselective β-AR agonist isoproterenol shortened the myocyte APD, an effect reduced by wheel running. The isoproterenol-induced shortening of APD was largely reversed by the selective β 1 -AR blocker atenolol, but not the β 2 -AR blocker ICI 118,551, providing evidence that wheel running reduced the sensitivity of the β 1 -AR. At 10 Hz, the K ATP channel inhibitor glibenclamide prolonged the myocyte APD more in exercise-trained than control rats, implicating a role for this channel in the exercise-induced APD shortening at 10 Hz. A novel finding of this work was the dual importance of altered β 1 -AR responsiveness and K ATP channel function in the training-induced regulation of APD. Of physiological importance to the beating heart, the reduced response to adrenergic agonists would enhance cardiac contractility at resting rates, where sympathetic drive is low, by prolonging APD and Ca 2+ influx; during exercise, an increase in K ATP channel activity would shorten APD and, thus, protect the heart against Ca 2+ overload or inadequate filling. NEW & NOTEWORTHY Our data demonstrated that regular exercise prolonged the action potential and Ca 2+ transient durations in myocytes isolated from apex and base regions at 1-Hz and shortened both at 10-Hz stimulation. Novel findings were that wheel running shifted the β-adrenergic receptor agonist dose-response curve rightward compared with controls by reducing β 1 -adrenergic receptor responsiveness and that, at the high activation rate, myocytes from trained animals showed higher K ATP channel function., (Copyright © 2017 the American Physiological Society.)

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs., Enzymes: Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96)., (© 2017 Federation of European Biochemical Societies.)

Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β 2 AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca 2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β 2 AR activation leads to robust Ca 2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP 3 ) receptors. This pathway did not involve cAMP, Gα s , or Gα i or the participation of the other members of the canonical β 2 AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca 2+ mobilization by β 2 AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Exposure to environmental polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B(a)P), has been linked to several health-threatening risks. PAHs were also shown to hinder adrenergic receptor (ADR) responses. As we previously demonstrated that B(a)P can directly interact with the β2ADR, we investigated here whether B(a)P could decrease β2ADR responsiveness by triggering receptor desensitization phenomena. We firstly showed that exposure to B(a)P reduced β2ADR-mediated epinephrine-induced induction of NR4A gene mRNAs and of intracellular cAMP. Analysis of β2ADR protein expression demonstrated that B(a)P rapidly decreased membrane expression of β2ADR with a subsequent degradation of receptor protein. B(a)P exposure concomitantly rapidly increased the β2ADR mRNA levels. The use of the β-blockers, propranolol and ICI 118.551, demonstrated the involvement of β2ADR itself in this increase. However, sustained exposure to B(a)P induced a diminution of β2ADR mRNA steady-state as a result of the acceleration of its degradation. Together, these results show that, beside the well-known activation of the aryl hydrocarbon receptor, PAH deleterious effects may involve the dysfunction of adrenergic responses through, in part, the desensitization of β2ADR. This may be taken in consideration when β2-agonists/antagonists are administered in patients exposed to important concentrations of PAHs, e.g. in cigarette smokers.

The L-type Ca 2+ channel Ca v 1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser 1928 in Ca v 1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser 1928 is not required for regulation of cardiac Ca v 1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Ca v 1.2 and enhancement of its activity by the β 2 -adrenergic receptor (β 2 AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Ca v 1.2 by PKA in cardiomyocytes and hippocampal neurons., (Copyright © 2017, American Association for the Advancement of Science.)

Study Question: Does chronic stress in mice accelerate the development of endometriosis, and, if so, through what mechanism?, Summary Answer: Exposure to chronic stress accelerates the development of endometriosis and exacerbates the endometriosis-associated generalized hyperalgesia, most likely through activation of the adrenoceptor β2 (ADRB2) and cAMP responsive element-binding protein (CREB)., What Is Known Already: Women with endometriosis tend to have higher levels of psychological stress, which is known to impact negatively on health in general and to promote tumor growth and metastasis in particular. Exposure to chronic stress before and after the induction of endometriosis is reported to increase lesion sizes in rodents, but it is unclear whether adrenoceptors are involved or not in the stress-promoted development of endometriosis., Study Design, Size, Duration: Three independent, prospective, randomized mouse experimentations. A total of 184 virgin female Balb/C mice were used., Participants/materials, Setting, Methods: In Experiment 1, the mice were randomly divided into four groups: the control group, which received no stress; the before, after and both groups, which received immobilization stress before, after and both before and after the induction of endometriosis, respectively. In Experiment 2, mice were randomly divided into four groups one day after the induction of endometriosis: phosphate buffer saline (PBS) and propranolol (PROP) groups, which received the mini-pump containing, respectively, PBS only and propranolol (a non-selective ADRB antagonist) but no stress, STR+PROP and STR+PBS groups, which received stress and the mini-pump containing, respectively, propranolol and PBS. The immobilization stress started after the insertion of mini-pumps. In Experiment 3, mice were induced with endometriosis. Three days after the induction, they were randomly divided into four groups: control, ADRAa, ADRB2a, and ADRBa, which received the mini-pump containing solution only, metaraminol (a non-specific α adrenoceptor agonist), tebutaline (a specific ADRB2 agonist), or isoproterenol (a non-specific ADRB agonist), respectively. In all three experiments, the bodyweight and hotplate latency were evaluated before sacrifice 14 days after the induction. In all experimentations, the lesion weight was evaluated and the harvested ectopic endometrial tissue samples were subjected to immunohistochemistry analysis of vascular endothelial growth factor (VEGF), CD31-positive microvessels, proliferating cell nuclear antigen (PCNA), phosphorylated CREB, ADRB1, ADRB2, ADRB3, adrenergic receptor α1 (ADRA1) and ADRA2., Main Results and the Role of Chance: Exposure to chronic stress accelerated the development of endometriosis and exacerbated the endometriosis-associated generalized hyperalgesia. This promotional effect is likely to be mediated through the systemic activation of the sympatho-adreno-medullary (SAM) axis, which results in subsequent release of catecholamines. The surging catecholamines may activate ADRB2 and CREB, yielding increased angiogenesis and cellular proliferation in ectopic endometrium in mice with induced endometriosis. In addition, β adrenergic receptor blockade completely abolished the promotional effect of chronic stress, likely through suppression of ADRB2 and CREB activation, thus suppressing angiogenesis and proliferation. Moreover, a non-specific adrenergic β agonist and a specific adrenergic β2 agonist, but not non-specific adrenergic α agonist, acted similarly to chronic stress, accelerating the development of endometriosis and exacerbating the generalized hyperalgesia in mice with pre-existing endometriosis., Large Scale Data: NA., Limitations, Reasons for Caution: This study is limited by the use of immunohistochemistry analyses only and the lack of molecular data., Wider Implications of the Findings: The present study provides the experimental evidence that chronic stress can promote the development of endometriosis through the activation of ADRB2. Given ADRB2 is also expressed in human endometriosis and appears to be functional, and in light of recent awareness that adrenergic signaling plays critical roles in tumorigenesis, it is likely that adrenergic signaling may play important roles in the development of endometriosis and is potentially a target for intervention., Study Funding/competing Interests: This research was supported in part by grants (81270676,  81471434 and 81530040  to S.W.G.;  81370695 and 81671436  to X.S.L.) from the National Natural Science Foundation of China, and grant (2013ZYJB0019 to X.S.L.) from Shanghai Municipal Commission of Health and Family Planning. None of the authors has anything to disclose., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

β-Adrenergic signaling can regulate macrophage involvement in several diseases and often produces anti-inflammatory properties in macrophages, which are similar to M2 properties in a dichotomous M1 vs. M2 macrophage taxonomy. However, it is not clear that β-adrenergic-stimulated macrophages may be classified strictly as M2. In this in vitro study, we utilized recently published criteria and transcriptome-wide bioinformatics methods to map the relative polarity of murine β-adrenergic-stimulated macrophages within a wider M1-M2 spectrum. Results show that β-adrenergic-stimulated macrophages did not fit entirely into any one pre-defined category of the M1-M2 spectrum but did express genes that are representative of some M2 side categories. Moreover, transcript origin analysis of genome-wide transcriptional profiles located β-adrenergic-stimulated macrophages firmly on the M2 side of the M1-M2 spectrum and found active suppression of M1 side gene transcripts. The signal transduction pathways involved were mapped through blocking experiments and bioinformatics analysis of transcription factor binding motifs. M2-promoting effects were mediated specifically through β2-adrenergic receptors and were associated with CREB, C/EBPβ, and ATF transcription factor pathways but not with established M1-M2 STAT pathways. Thus, β-adrenergic-signaling induces a macrophage transcriptome that locates on the M2 side of the M1-M2 spectrum but likely accomplishes this effect through a signaling pathway that is atypical for M2-spectrum macrophages., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is characterized by inflammation of the synovium, which leads to the progressive destruction of cartilage and bone. Adrenoreceptor (AR) signaling may play an important role in modulating dendritic cell (DC), which may be involved in the pathogenesis of RA. We examined the effect of the β-AR agonist isoprenaline (ISO) on DC function, the impact of the β2-AR agonist salbutamol on adjuvant-induced arthritic (AA) rats, and changes in β2-AR signaling in DCs during the course of AA. ISO inhibited the expression of the surface molecules CD86 and MHC-II, inhibited the stimulation of T lymphocyte proliferation by DC and TNF-α secretion, and promoted DC antigen uptake and IL-10 secretion. The effects of ISO on MHC-II expression, DC stimulation of T lymphocyte proliferation, and DC antigen uptake were mediated by β2-AR. Treatment with salbutamol ameliorated the severity of AA and histopathology of the joints and inhibited proliferation of thymus lymphocytes and FLS in vivo. β2-AR signaling was weaker in AA rats compared to the control. Elevated GRK2 and decreased β2-AR expression in DC cytomembranes were observed in AA and may have decreased the anti-inflammatory effect of β2-AR signaling. Decreased β2-AR signaling may be relevant to the exacerbation of arthritis inflammation.

Chronic stress is widely considered to trigger or enhance itch, especially for pruritic dermatitis. However, the molecular mechanisms linking chronic stress and itch are still unknown. The present study aimed to elucidate the role of adrenergic signaling in itch hypersensitivity following heterotypic chronic intermittent stress (HIS) in rats. HIS significantly increased hindlimb scratching, but not forepaw swiping, induced by intradermal injection of 5-hydroxytryptamine (5-HT) in the rat cheek. Coadministration of stress mediators such as norepinephrine or epinephrine dose-dependently increased both 5-HT-induced hindlimb scratching and 5-HT-induced forepaw swiping. HIS-induced itch hypersensitivity was attenuated by blockade of sympathetic signaling through guanethidine treatment, and systemic administration of the β-adrenoceptor antagonist propranolol and the β2-adrenoceptor antagonist butoxamine, but not on treatment with an α-adrenoceptor antagonist phentolamine and a β1-adrenoceptor antagonist atenolol. Moreover, HIS selectively increased the expression of β2-adrenoceptors and proinflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nerve growth factor (NGF)] in rat skin. The β-blockers propranolol and butoxamine abolished the upregulation of proinflammatory factors. The β2-adrenoceptor agonist terbutaline was sufficient to enhance the skin expression of TNF-α and IL-1β and to increase 5-HT-induced scratching in naive rats. Pretreatment with TNF-α could increase 5-HT-induced scratching. Together, these results demonstrate that β2-adrenoceptors mediate itch hypersensitivity following chronic stress by inducing proinflammatory factors, such as TNF-α, in the skin.

Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.

Background and Purpose: The early management of the cardiovascular dysfunction of septic shock is critical as it is associated with a poor outcome. Although the use of catecholamines is a common therapy in this syndrome, no data are available on the involvement of β-adrenoceptor (β-AR) subtypes and only few studies report an alteration of β-adrenergic-induced vasodilation in septic shock. The purpose of the study was to evaluate vascular β1, β2 and β3-AR expression and function in an endotoxemic rat model., Experimental Approach: Endotoxemia was induced in rats by intravenous injection of lipopolysaccharide (LPS). β1, β2 and β3-AR mRNA expression was evaluated by RT-PCR in aorta and vascular β1, β2 and β3-AR responses were determined on conducting (aorta) and/or resistance (mesenteric and renal) arteries by constructing relaxation curves in response to different β-AR agonists., Results: The maximal effect of isoproterenol decreased by 31 to 61% in the three vascular beds of LPS-treated rats compared to controls. In aortas from LPS-treated rats, β1 and β3-AR mRNA expression was decreased and associated to a reduced β1 and β3-induced vasodilation. Conversely, albeit β2-AR mRNA was unchanged, the maximal β2-AR-induced vasodilation increased by 49% in aortas from LPS-treated rats compared to controls. This increase was not affected by endothelium removal but was abolished in the presence of a β2-AR antagonist or an adenylate cyclase inhibitor., Conclusions: In endotoxemia, β2-AR vasodilation was increased by a potential recruitment of β2-AR located on smooth muscle cells. This study suggests that vascular β2-AR should be a putative new therapeutic target in septic shock., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context., Methods: β2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization., Results: Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization., Conclusion: Amphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2., General Significance: Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2's kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hypoglycemic effect is an efficient means to modulate elevated blood glucose levels in patients with diabetes. We found that the extract of lotus plumule (the germ of Nelumbo nucifera Gaertn. seed) showed potent glucose uptake enhancement activity against L6 myotubes, which results in a hypoglycemic effect. This activity was further investigated, and an active constituent was identified as a single bioactive compound, higenamine 4'-O-β-d-glucoside. Mechanistic studies employing phosphatidylinositol 3-kinase (PI3K) inhibitor, AMP-activated protein kinase (AMPK) inhibitor, or adrenergic receptor antagonist showed that the compound induced its activity through β2-adrenergic receptor. Patients with type II diabetes mellitus frequently develop insulin resistance. Owing to the differences between the mechanism of action of insulin and of the isolated compound, the compound or lotus plumule itself may have the possibility of modulating blood glucose levels in insulin-resistant patients effectively., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Salmeterol is a long-acting β2-adrenergic receptor (β2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the β2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-β2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to β2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events., (U.S. Government work not protected by U.S. copyright.)

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated β2 adrenergic receptors (β2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated β2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a β2AR-dependent manner. Upon β2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated β2AR complex, and facilitates trafficking of the ubiquitinated β2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the β2AR to autophagosomes while promoting plasma membrane recycling of internalized β2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cancer research relies heavily on murine models for evaluating the anti-tumour efficacy of therapies. Here we show that the sensitivity of several pancreatic tumour models to cytotoxic therapies is significantly increased when mice are housed at a thermoneutral ambient temperature of 30 °C compared with the standard temperature of 22 °C. Further, we find that baseline levels of norepinephrine as well as the levels of several anti-apoptotic molecules are elevated in tumours from mice housed at 22 °C. The sensitivity of tumours to cytotoxic therapies is also enhanced by administering a β-adrenergic receptor antagonist to mice housed at 22 °C. These data demonstrate that standard housing causes a degree of cold stress sufficient to impact the signalling pathways related to tumour-cell survival and affect the outcome of pre-clinical experiments. Furthermore, these data highlight the significant role of host physiological factors in regulating the sensitivity of tumours to therapy.

β2-AR activation increases the risk of sudden cardiac death (SCD) in heart failure (HF) patients. Non-selective β-AR blockers have greater benefits on survival than selective β1-AR blockers in chronic HF patients, indicating that β2-AR activation contributes to SCD in HF. This study investigated the role of β2-AR activation on repolarization and ventricular arrhythmia (VA) in the experimental HF model. The guinea pig HF was induced by descending aortic banding. The effective refractoriness period (ERP), corrected QT (QTc) and the incidence of VA were examined using Langendorff and programmed electrical stimulation. Ikr and APD were recorded by the whole cell patch clamp. Selective β2-AR agonist salbutamol significantly increased the incidence of VA, prolonged QTc and shortened ERP. These effects could be prevented by the selective β2-AR antagonist, ICI118551. Salbutamol prolonged APD90 and reduced Ikr in guinea pig HF myocytes. The antagonists of cAMP (Rp-cAMP) and PKA (KT5720) attenuated Ikr inhibition and APD prolongation induced by salbutamol. However, the antagonists of Gi protein (PTX) and PDE III (amrinone) showed opposite effects. This study indicates that β2-AR activation increases the incidence of VA in the experimental HF model via activation of Gs/cAMP/PKA and/or inhibition of Gi/PDE pathways.

G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the β2-adrenergic receptor (β2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of β2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the β2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

How cell fate (survival or death) is determined and whether such determination depends on the strength of stimulation has remained unclear. In this study, we discover that the cell fate of cardiomyocytes switches from survival to death with the increase of β-adrenergic receptor (β-AR) stimulation. Mathematical simulations combined with biochemical experimentation of β-AR signalling pathways show that the gradual increment of isoproterenol (a non-selective β1/β2-AR agonist) induces the switching response of Bcl-2 expression from the initial increase followed by a decrease below its basal level. The ERK1/2 and ICER-mediated feed-forward loop is the hidden design principle underlying such cell fate switching characteristics. Moreover, we find that β1-blocker treatment increases the survival effect of β-AR stimuli through the regulation of Bcl-2 expression leading to the resistance to cell death, providing new insight into the mechanism of therapeutic effects. Our systems analysis further suggests a novel potential therapeutic strategy for heart disease.

Cardiac disorders are the main cause of mortality in autosomal-dominant polycystic kidney disease (ADPKD). However, how mutated polycystins predispose patients with ADPKD to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased levels of polycystin 2 (PC2), a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2(+/-)) undergo cardiac remodeling as a result of changes in calcium handling, separate from renal complications. We found that Pkd2(+/-) cardiomyocytes have altered calcium handling, independent of desensitized calcium-contraction coupling. Paradoxically, in Pkd2(+/-) mice, protein kinase A (PKA) phosphorylation of phospholamban (PLB) was decreased, whereas PKA phosphorylation of troponin I was increased, explaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2(+/-) mice have increased left ventricular ejection fraction after stimulation with isoproterenol (ISO), a β-adrenergic receptor (βAR) agonist. Blockers of βAR-1 and βAR-2 inhibited the ISO response in Pkd2(+/-) mice, suggesting that the dephosphorylated state of PLB is primarily by βAR-2 signaling. Importantly, the Pkd2(+/-) mice were normotensive and had no evidence of renal cysts. Our results showed that decreased PC2 levels shifted the βAR pathway balance and changed expression of calcium handling proteins, which resulted in altered cardiac contractility. We propose that PC2 levels in the heart may directly contribute to cardiac remodeling in patients with ADPKD in the absence of renal dysfunction.

The sympathetic nervous system (SNS) regulates bone resorption through β-2 adrenergic receptor (Adrb2). In orthodontic tooth movement (OTM), mechanical force induces and regulates alveolar bone remodeling. Compressive force-associated osteoclast differentiation and alveolar bone resorption are the rate-limiting steps of tooth movement. However, whether mechanical force can activate Adrb2 and thus contribute to OTM remains unknown. In this study, orthodontic nickel-titanium springs were applied to the upper first molars of rats and Adrb1/2(-/-) mice to confirm the role of SNS and Adrb2 in OTM. The results showed that blockage of SNS activity in the jawbones of rats by means of superior cervical ganglion ectomy reduced OTM distance from 860 to 540 μm after 14 d of force application. In addition, the injection of nonselective Adrb2 agonist isoproterenol activated the downstream signaling of SNS to accelerate OTM from 300 to 540 μm after 7 d of force application. Adrb1/2(-/-) mice showed significantly reduced OTM distance (19.5 μm) compared with the wild-type mice (107.6 μm) after 7 d of force application. Histopathologic analysis showed that the number of Adrb2-positive cells increased in the compressive region of periodontal ligament after orthodontic force was applied on rats. Mechanistically, mechanical compressive force upregulated Adrb2 expression in primary-cultured human periodontal ligament cells (PDLCs) through the elevation of intracellular Ca(2+) concentration. Activation of Adrb2 in PDLCs increased the RANKL/OPG ratio and promoted the peripheral blood mononuclear cell differentiation to osteoclasts in the cocultured system. Upregulation of Adrb2 in PDLCs promoted osteoclastogenesis, which accelerated OTM through Adrb2-enhanced bone resorption. In summary, this study suggests that mechanical force-induced Adrb2 activation in PDLCs contributes to SNS-regulated OTM., (© International & American Associations for Dental Research.)

β-Adrenergic agonists (β-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of β-agonists, a β2-adrenergic receptor (β2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant β2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of β-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized β2-AR proteins by β-agonists. The IC50 and limit of detection values for ractopamine were 30.38μgL(-1) and 5.20μgL(-1), respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of β-agonists in animal feeds., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Experiences with a high degree of emotional salience are better remembered than events that have little emotional context and the amygdala is thought to play an important role in this enhancement of memory. Visual recognition memory relies on synaptic plasticity in the perirhinal cortex but little is known about the mechanisms that may underlie emotional enhancement of this form of memory. There is good evidence that noradrenaline acting via β-adrenoceptors (β-ADRs) can enhance memory consolidation. In the present study we examine the role of β-ADRs in synaptic plasticity at the amygdala-perirhinal pathway (LA-PRh) and compare this to mechanisms of intra-perirhinal (PRh-PRh) synaptic plasticity. We demonstrate that activity-dependent PRh-PRh long-term potentiation (LTP) does not rely on β1- or β2-ADRs and that LA-PRh LTP relies on β1-ADRs but not β2-ADRs. We further demonstrate that application of the β-ADR agonist isoprenaline produces lasting PRh-PRh potentiation but only transient potentiation at the LA-PRh input. However, at the LA-PRh input, combining stimulation that is subthreshold for LTP induction with isoprenaline results in long-lasting potentiation. Isoprenaline-induced and isoprenaline plus subthreshold stimulation-induced potentiation in the PRh-PRh and LA-PRh inputs, respectively were both dependent on activation of NMDARs (N-methyl-D-aspartate receptors), voltage-gated calcium channels and PKA (protein kinase A). Understanding the mechanisms of amygdala-perirhinal cortex plasticity will allow a greater understanding of how emotionally-charged events are remembered., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)