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Chlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. A mouse model has been developed in our laboratory to better understand the effect of cold-induced stress on chlamydia genital infection and immune response. However, the stress mechanism affecting the host response to Chlamydia muridarum genital infection remains unclear. Here, we demonstrate a role for the beta2-adrenergic receptor (β2-AR), which binds noradrenaline and modulates the immune response against chlamydia genital infection in a mouse model. A successful β2-AR homozygous knockout (KO) mouse model was used to study the infection and analyze the immune response. Our data show that stressed mice lacking the β2-AR are less susceptible to C. muridarum genital infection than controls. A correlation was obtained between lower organ load and higher interferon-gamma production by CD4+ and CD8+ cells of the KO mice. Furthermore, exposure of CD4+ T cells to noradrenaline alters the production of cytokines in mice during C. muridarum genital infection. This study suggests that the blockade of β2-AR signaling could be used to increase resistance to chlamydia genital infection. We value the β2-AR KO as a viable model that can provide reproducible results in investigating medical research, including chlamydia genital infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Purpose: Recent studies demonstrated a contribution of adrenoceptors (ARs) to osteoarthritis (OA) pathogenesis. Several AR subtypes are expressed in joint tissues and the β2-AR subtype seems to play a major role during OA progression. However, the importance of β2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in β2-AR-deficient ( Adrb2 -/- ) mice after surgical OA induction., Methods: OA was induced by destabilization of the medial meniscus (DMM) in male wildtype (WT) and Adrb2 -/- mice. Cartilage degeneration and synovial inflammation were evaluated by histological scoring. Subchondral bone remodeling was analyzed using micro-CT. Osteoblast (alkaline phosphatase - ALP) and osteoclast (cathepsin K - CatK) activity were analyzed by immunostainings. To evaluate β2-AR deficiency-associated effects, body weight, sympathetic tone (splenic norepinephrine (NE) via HPLC) and serum leptin levels (ELISA) were determined. Expression of the second major AR, the α2-AR, was analyzed in joint tissues by immunostaining., Results: WT and Adrb2 -/- DMM mice developed comparable changes in cartilage degeneration and synovial inflammation. Adrb2 -/- DMM mice displayed elevated calcified cartilage and subchondral bone plate thickness as well as increased epiphyseal BV/TV compared to WTs, while there were no significant differences in Sham animals. In the subchondral bone of Adrb2 -/- mice, osteoblasts activity increased and osteoclast activity deceased. Adrb2 -/- mice had significantly higher body weight and fat mass compared to WT mice. Serum leptin levels increased in Adrb2 -/- DMM compared to WT DMM without any difference between the respective Shams. There was no difference in the development of meniscal ossicles and osteophytes or in the subarticular trabecular microstructure between Adrb2 -/- and WT DMM as well as Adrb2 -/- and WT Sham mice. Number of α2-AR-positive cells was lower in Adrb2 -/- than in WT mice in all analyzed tissues and decreased in both Adrb2 -/- and WT over time., Conclusion: We propose that the increased bone mass in Adrb2 -/- DMM mice was not only due to β2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, β2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rösch, Muschter, Taheri, El Bagdadi, Dorn, Meurer, Zaucke, Schilling, Grässel, Straub and Jenei-Lanzl.)

The autonomic nervous system has been studied for its involvement in the control of macrophages; however, the mechanisms underlying the interaction between the adrenergic receptors and alternatively activated macrophages (M2) remain obscure. Using FVB wild-type and beta 2 adrenergic receptors knockout, we found that β2-AR deficiency alleviates hepatobiliary damage in mice infected with C. sinensis . Moreover, β2-AR-deficient mice decrease the activation and infiltration of M2 macrophages and decrease the production of type 2 cytokines, which are associated with a significant decrease in liver fibrosis in infected mice. Our in vitro results on bone marrow-derived macrophages revealed that macrophages from Adrb2 -/- mice significantly decrease M2 markers and the phosphorylation of ERK/mTORC1 induced by IL-4 compared to that observed in M2 macrophages from Adrb2 +/+ . This study provides a better understanding of the mechanisms by which the β2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Koda, Zhang, Zhou, Xu, Li, Liu, Liu, Lv, Wang, Shi, Gao, Yu, Li, Xu, Chen, Tekengne, Adzika, Tang, Sun, Zheng and Yan.)

Caco-2 cells are increasingly used to study the absorption of drugs and nutrients, including D-glucose, an important nutrient that mainly gets absorbed from the intestine by the sodium/glucose cotransporter 1 (SGLT1). However, disadvantages of Caco-2 cells for such studies have been reported, e.g., D-glucose cannot elicit translocation of the intracellular pool of SGLT1 to the apical membrane, the origin of the cells affects glucose uptake, and Caco-2 cells exhibit heterogeneity. This study aimed to characterize SGLT1-mediated glucose transport across Caco-2 cell monolayers. We found that at lower glucose concentrations (5 mM) SGLT1 contributes more to total glucose transport than at higher (10 mM) glucose concentrations, suggesting contributions by another transporter at higher glucose concentrations. This contrasts with the in vivo situation, where SGLT1 dominant glucose transporter at all glucose concentrations. We also tested whether known regulators like sugars or catecholamines can stimulate glucose transport across Caco-2 cell monolayers. Neither epinephrine nor 2-deoxy-D-glucose could stimulate glucose transport. Moreover, the epinephrine could not induce accumulation of cyclic adenosine monophosphate (cAMP) in Caco-2 cells, indicating the absence of a functional β 2 -adrenoceptor in Caco-2 cells, which could explain the lack of epinephrine effect on glucose transport. Also, Caco-2 cells may lack some kinases required for increased SGLT1 transport. Overall, SGLT1-mediated glucose transport and its regulation in Caco-2 cells differ from that in vivo, and caution is advised when extrapolating glucose transport results obtained with this model to the in vivo situation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands., Competing Interests: Declaration of Interests A provisional patent application has been filed by the President and Fellows of Harvard College based on this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs) 1,2 , but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics 3,4 , cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism.

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on βarr-dependent β2AR signaling. Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background and Aim: β 2 -Adrenoceptors (β 2 -ARs) are G protein-coupled receptors (GPCRs) expressed in the major insulin target tissues. The interplay between β 2 -AR and insulin pathways is involved in the maintenance of glucose homeostasis. The aim of this study was to explore the consequences of β 2 -ARs deletion on insulin sensitivity and insulin signaling cascade in metabolically active tissues., Methods and Results: We evaluated glucose homeostasis in skeletal muscle and liver of β 2 -AR-null mice (β 2 -AR -/- ) by performing in vivo (glucose tolerance test and insulin tolerance test) and ex vivo (glucose uptake and glycogen determination) experiments. β 2 -AR gene deletion is associated with hepatic insulin resistance and preserved skeletal muscle insulin sensitivity. Importantly, we demonstrate that hepatic β 2 -AR regulates insulin-induced AKT activation via Grb2-mediated SRC recruitment through a G i -independent mechanism., Conclusions: β-AR stimulation contributes to the development of early stages of insulin resistance progression in the liver. Our findings indicate that the cross-talk between β 2 -AR and insulin signaling represents a fundamental target towards the development of novel therapeutic approaches to treat type 2 diabetes and metabolic syndrome., (Copyright © 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background: Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity., Methods: High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking β 2 -adrenergic receptor (β 2 AR) or β-arrestin2. Wild-type mice fed with high-fat diet were treated with a β-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function., Results: High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and β-arrestin2-dependent transactivation of a β 2 AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of β 2 AR or GRK2, or genetic deletion of β 2 AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction., Conclusions: These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β 2 AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM., (© 2016 American Heart Association, Inc.)

Background: Immune cell-mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through β-adrenergic receptors (βARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown., Methods: Wild-type (WT) mice were irradiated followed by isoform-specific βAR knockout (βARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression., Results: β2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. β2ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via β-arrestin-dependent β2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to β2AR activity, and spleens from human tissue donors treated with β-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of β2AR in β2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice., Conclusions: Immune cell-expressed β2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration., (© 2016 American Heart Association, Inc.)

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2h, host defenses against bacterial growth are suppressed 2-3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidation (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (β2-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related β2-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related β2-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.

Aims: Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP production, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). The intracellular regional dynamics of cAMP production and hydrolysis modulate downstream signals resulting in different biological responses. The interplay between beta receptors (βARs) signalling and phosphodiesterase 5 (PDE5) activity remains to be addressed., Methods and Results: Using combined strategies with pharmacological inhibitors and genetic deletion of PDEs and βAR isoforms, we revealed a specific pool of cAMP that is under dual regulation by PDE2 and, indirectly, PDE5 activity. Inhibition of PDE5 with sildenafil produces a cGMP-dependent activation of PDE2 that attenuates cAMP generation induced by βAR agonists, with concomitant modulation of stimulated contraction rate and calcium transients. PDE2 haploinsufficiency abolished the effects of sildenafil. The negative chronotropic effect of PDE5 inhibition through PDE2 activation was also observed in sinoatrial node tissue from adult mice. PDE5 inhibition selectively lowered contraction rate stimulated by β2AR, but not β1AR activation, supporting a compartmentalization of the cGMP-modulated pool of cAMP., Conclusion: These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

The counter-regulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well studied, whereas the details of insulin regulation of β adrenergic receptor (βAR) signaling pathway in heart remain unknown. Here, we characterize a novel signaling pathway of insulin receptor (IR) to G protein-coupled receptor kinase 2 (GRK2) in the heart. Insulin stimulates recruitment of GRK2 to β2AR, which induces β2AR phosphorylation at the GRK sites of serine 355/356 and subsequently β2AR internalization. Insulin thereby suppresses βAR-induced cAMP-PKA activities and contractile response in neonatal and adult mouse cardiomyocytes. Deletion of insulin receptor substrate 2 (IRS2) disrupts the complex of IR and GRK2, which attenuates insulin-mediated β2AR phosphorylation at the GRK sites and β2AR internalization, and the counter-regulation effects of insulin on βAR signaling. These data indicate the requirements of IRS2 and GRK2 for insulin to stimulate counter-regulation of βAR via β2AR phosphorylation and internalization in cardiomyocytes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Inhibition of TNFα protects the retina against diabetic-like changes in rodent models. The mechanism by which TNFα induces deleterious retinal changes is not known. Previously, we have shown that TNFα can inhibit normal insulin signal transduction, leading to increased apoptosis in both retinal endothelial cells (REC) and Müller cells. Additionally, β2-adrenergic receptor knockout mice (β2KO) have increased TNFα levels and decreased insulin receptor activity. In this study, we hypothesized that inhibition of TNFα in β2KO mice would increase normal insulin signaling, leading to improved retinal function., Methods: C57BL6 or β2KO mice were left untreated or treated with etanercept (0.3 mg/kg subcutaneously, 3× a week) for 2 months. Electroretinogram analyses were done before treatment was initiated and after two months of treatment with etanercept on all mice. Western blot or ELISA analyses were done on whole retinal lysates from all four groups of mice for TNFα, suppressor of cytokine signaling 3 (SOCS3), insulin receptor, and apoptotic proteins., Results: Etanercept significantly reduced TNFα levels in β2KO mice, leading to increased insulin receptor phosphorylation on tyrosine 1150/1151. SOCS3 levels were increased in β2KO mice, which were reduced after etanercept treatment. Pro-apoptotic proteins were reduced in etanercept-treated β2KO mice. Etanercept improved ERG amplitudes in β2KO mice., Conclusions: Inhibition of TNFα by etanercept protects the retina likely through reduced TNFα-mediated insulin resistance, leading to reduced apoptosis.

Accumulation of the microtubule-binding protein tau is a key event in several neurodegenerative disorders referred to as tauopathies, which include Alzheimer's disease, frontotemporal lobar degeneration, Pick's disease, progressive supranuclear palsy and corticobasal degeneration. Thus, understanding the molecular pathways leading to tau accumulation will have a major impact across multiple neurodegenerative disorders. To elucidate the pathways involved in tau pathology, we removed the gene encoding the beta-2 adrenergic receptors (β2ARs) from a mouse model overexpressing mutant human tau. Notably, the number of β2ARs is increased in brains of AD patients and epidemiological studies show that the use of beta-blockers decreases the incidence of AD. The mechanisms underlying these observations, however, are not clear. We show that the tau transgenic mice lacking the β2AR gene had a reduced mortality rate compared with the parental tau transgenic mice. Removing the gene encoding the β2ARs from the tau transgenic mice also significantly improved motor deficits. Neuropathologically, the improvement in lifespan and motor function was associated with a reduction in brain tau immunoreactivity and phosphorylation. Mechanistically, we provide compelling evidence that the β2AR-mediated changes in tau were linked to a reduction in the activity of GSK3β and CDK5, two of the major tau kinases. These studies provide a mechanistic link between β2ARs and tau and suggest the molecular basis linking the use of beta-blockers to a reduced incidence of AD. Furthermore, these data suggest that a detailed pharmacological modulation of β2ARs could be exploited to develop better therapeutic strategies for AD and other tauopathies., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Muscles can be injured in different ways and the trauma and subsequent loss of function and physical capacity can impact significantly on the lives of patients through physical impairments and compromised quality of life. The relative success of muscle repair after injury will largely determine the extent of functional recovery. Unfortunately, regenerative processes are often slow and incomplete, and so developing novel strategies to enhance muscle regeneration is important. While the capacity to enhance muscle repair by stimulating β2-adrenoceptors (β-ARs) using β2-AR agonists (β2-agonists) has been demonstrated previously, the exact role β-ARs play in regulating the regenerative process remains unclear. To investigate β-AR-mediated signaling in muscle regeneration after myotoxic damage, we examined the regenerative capacity of tibialis anterior and extensor digitorum longus muscles from mice lacking either β1-AR (β1-KO) and/or β2-ARs (β2-KO), testing the hypothesis that muscles from mice lacking the β2-AR would exhibit impaired functional regeneration after damage compared with muscles from β1-KO or β1/β2-AR null (β1/β2-KO) KO mice. At 7 days post-injury, regenerating muscles from β1/β2-KO mice produced less force than those of controls but muscles from β1-KO or β2-KO mice did not exhibit any delay in functional restoration. Compared with controls, β1/β2-KO mice exhibited an enhanced inflammatory response to injury, which delayed early muscle regeneration, but an enhanced myoblast proliferation later during regeneration ensured a similar functional recovery (to controls) by 14 days post-injury. This apparent redundancy in the β-AR signaling pathway was unexpected and may have important implications for manipulating β-AR signaling to improve the rate, extent and efficacy of muscle regeneration to enhance functional recovery after injury.

Three types of beta adrenergic receptors (ARβ1-3) mediate the sympathetic activation of brown adipose tissue (BAT), the key thermogenic site for mice which is also present in adult humans. In this study, we evaluated adaptive thermogenesis and metabolic profile of a mouse with Arβ2 knockout (ARβ2KO). At room temperature, ARβ2KO mice have normal core temperature and, upon acute cold exposure (4 °C for 4 h), ARβ2KO mice accelerate energy expenditure normally and attempt to maintain body temperature. ARβ2KO mice also exhibited normal interscapular BAT thermal profiles during a 30-min infusion of norepinephrine or dobutamine, possibly due to marked elevation of interscapular BAT (iBAT) and of Arβ1, and Arβ3 mRNA levels. In addition, ARβ2KO mice exhibit similar body weight, adiposity, fasting plasma glucose, cholesterol, and triglycerides when compared with WT controls, but exhibit marked fasting hyperinsulinemia and elevation in hepatic Pepck (Pck1) mRNA levels. The animals were fed a high-fat diet (40% fat) for 6 weeks, ARβ2KO mice doubled their caloric intake, accelerated energy expenditure, and induced Ucp1 expression in a manner similar to WT controls, exhibiting a similar body weight gain and increase in the size of white adipocytes to the WT controls. However, ARβ2KO mice maintain fasting hyperglycemia as compared with WT controls despite very elevated insulin levels, but similar degrees of liver steatosis and hyperlipidemia. In conclusion, inactivation of the ARβ2KO pathway preserves cold- and diet-induced adaptive thermogenesis but disrupts glucose homeostasis possibly by accelerating hepatic glucose production and insulin secretion. Feeding on a high-fat diet worsens the metabolic imbalance, with significant fasting hyperglycemia but similar liver structure and lipid profile to the WT controls., (© 2014 Society for Endocrinology.)

Objective: Sustained β-adrenergic stimulation is a hallmark of sympathetic hyperactivity in cardiovascular diseases. It is associated with oxidative stress and altered vasoconstrictor tone. This study investigated the β-adrenoceptor subtype and the signaling pathways implicated in the vascular effects of β-adrenoceptor overactivation., Methods and Results: Mice lacking the β1- or β2-adrenoceptor subtype (β1KO, β2KO) and wild-type (WT) were treated with isoproterenol (ISO, 15 μg.g(-1) x day(-1), 7 days). ISO significantly enhanced the maximal vasoconstrictor response (Emax) of the aorta to phenylephrine in WT (+34%) and β1KO mice (+35%) but not in β2KO mice. The nitric oxide synthase (NOS) inhibitor L-NAME abolished the differences in phenylephrine response between the groups, suggesting that ISO impaired basal NO availability in the aorta of WT and β1KO mice. Superoxide dismutase (SOD), pertussis toxin (PTx) or PD 98,059 (p-ERK 1/2 inhibitor) incubation reversed the hypercontractility of aortic rings from ISO-treated WT mice; aortic contraction of ISO-treated β2KO mice was not altered. Immunoblotting revealed increased aortic expression of Giα-3 protein (+50%) and phosphorylated ERK1/2 (+90%) and decreased eNOS dimer/monomer ratio in ISO-treated WT mice. ISO enhanced the fluorescence response to dihydroethidium (+100%) in aortas from WT mice, indicating oxidative stress that was normalized by SOD, PTx and L-NAME. The ISO effects were abolished in β2KO mice., Conclusions: The β2-adrenoceptor/Giα signaling pathway is implicated in the enhanced vasoconstrictor response and eNOS uncoupling-mediated oxidative stress due to ISO treatment. Thus, long-term β2-AR activation might results in endothelial dysfunction.

There is about 30% higher risk of the myocardial infarction in patients diagnosed with multiple sclerosis (MS) than in people without MS. Increased risk of cardiovascular disease development positively correlates with levels of serum markers of an endothelial dysfunction, and may give rise to a global cerebral hypoperfusion. It appears that these complications precede progressive loss of axons, which mechanisms are complex and should be linked to a loss of β2 adrenergic receptors on astrocytes of demyelinating lesions. Consequence of this deficiency, the cause of which is not known yet, is a decline in energy metabolism of axons. Moreover, the loss of these receptors is linked to a reduced redistribution of potassium ions by astrocytes, glutamate excitotoxicity and increase of calcium ion concentration in the axon with subsequent activation of necrotic processes. In addition to immunological aspects we should take into account also parameters of the functional state of endothelium when appropriate targeted therapy for patient is considered.

The role of the β2AR (β2 adrenergic receptor) after stroke is unclear as pharmacological manipulations of the β2AR have produced contradictory results. We previously showed that mice deficient in the β2AR (β2KO) had smaller infarcts compared with WT (wild-type) mice (FVB) after MCAO (middle cerebral artery occlusion), a model of stroke. To elucidate mechanisms of this neuroprotection, we evaluated changes in gene expression using microarrays comparing differences before and after MCAO, and differences between genotypes. Genes associated with inflammation and cell deaths were enriched after MCAO in both genotypes, and we identified several genes not previously shown to increase following ischaemia (Ccl9, Gem and Prg4). In addition to networks that were similar between genotypes, one network with a central core of GPCR (G-protein-coupled receptor) and including biological functions such as carbohydrate metabolism, small molecule biochemistry and inflammation was identified in FVB mice but not in β2KO mice. Analysis of differences between genotypes revealed 11 genes differentially expressed by genotype both before and after ischaemia. We demonstrate greater Glo1 protein levels and lower Pmaip/Noxa mRNA levels in β2KO mice in both sham and MCAO conditions. As both genes are implicated in NF-κB (nuclear factor κB) signalling, we measured p65 activity and TNFα (tumour necrosis factor α) levels 24 h after MCAO. MCAO-induced p65 activation and post-ischaemic TNFα production were both greater in FVB compared with β2KO mice. These results suggest that loss of β2AR signalling results in a neuroprotective phenotype in part due to decreased NF-κB signalling, decreased inflammation and decreased apoptotic signalling in the brain.

As they age, mice deficient for the β2-adrenergic receptor (Adrb2(-/-) ) maintain greater trabecular bone microarchitecture, as a result of lower bone resorption and increased bone formation. The role of β1-adrenergic receptor signaling and its interaction with β2-adrenergic receptor on bone mass regulation, however, remains poorly understood. We first investigated the skeletal response to mechanical stimulation in mice deficient for β1-adrenergic receptors and/or β2-adrenergic receptors. Upon axial compression loading of the tibia, bone density, cancellous and cortical microarchitecture, as well as histomorphometric bone forming indices, were increased in both Adrb2(-/-) and wild-type (WT) mice, but not in Adrb1(-/-) nor in Adrb1b2(-/-) mice. Moreover, in the unstimulated femur and vertebra, bone mass and microarchitecture were increased in Adrb2(-/-) mice, whereas in Adrb1(-/-) and Adrb1b2(-/-) double knockout mice, femur bone mineral density (BMD), cancellous bone volume/total volume (BV/TV), cortical size, and cortical thickness were lower compared to WT. Bone histomorphometry and biochemical markers showed markedly decreased bone formation in Adrb1b2(-/-) mice during growth, which paralleled a significant decline in circulating insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGF-BP3). Finally, administration of the β-adrenergic agonist isoproterenol increased bone resorption and receptor activator of NF-κB ligand (RANKL) and decreased bone mass and microarchitecture in WT but not in Adrb1b2(-/-) mice. Altogether, these results demonstrate that β1- and β2-adrenergic signaling exert opposite effects on bone, with β1 exerting a predominant anabolic stimulus in response to mechanical stimulation and during growth, whereas β2-adrenergic receptor signaling mainly regulates bone resorption during aging., (Copyright © 2012 American Society for Bone and Mineral Research.)

Background and Purpose: Inhalation of a β-adrenoceptor agonist (β-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of β-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this β(2) -adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by β(2) -adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional β-agonist-induced β(2) -adrenoceptor desensitization in the context of allergic inflammatory airway disease., Experimental Approach: We combined chronic allergen exposure with repeated β-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of β(2) -adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of β(2) -adrenoceptors (KO). Associated inflammatory changes - cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and β(2) -adrenoceptor density (radioligand binding) - were also measured. KEY RESULTS β(2) -Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype., Conclusions and Implications: This animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic β- agonist treatment on β-adrenoceptor function in asthma. Such information may guide the clinical use of β-agonists and provide insight into development of novel β-adrenoceptor ligands for the treatment of asthma., (Published 2011. This article is a U. S. Government work and is in the public domain in the USA.)

The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.

Background/aims: β(2)-adrenoceptor (β(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal β(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional β(2)-AR (β(2)KO), focusing on the role of NO and superoxide anion., Methods and Results: Isolated thoracic aortas from β(2)KO and wild-type mice (WT) were studied. β(2)KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between β(2)KO and WT mice. Basal NO availability was reduced in aortas from β(2)KO mice. Incubation of β(2)KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. β(2)KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in β(2)KO aortas., Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of β(2)KO mice. This study extends the knowledge of the relevance of the endogenous activity of β(2)-AR to the maintenance of the vascular physiology., (Copyright © 2012 S. Karger AG, Basel.)

The nervous system is classically organized into sympathetic and parasympathetic systems acting in opposition to maintain physiological homeostasis. Here, we report that both systems converge in the activation of β2-adrenoceptors of splenic regulatory lymphocytes to control systemic inflammation. Vagus nerve stimulation fails to control serum TNF levels in either β2-knockout or lymphocyte-deficient nude mice. Unlike typical suppressor CD25(+) cells, the transfer of CD4(+)CD25(-) regulatory lymphocytes reestablishes the anti-inflammatory potential of the vagus nerve and β2-agonists to control inflammation in both β2-knockout and nude mice. β2-Agonists inhibit cytokine production in splenocytes (IC(50)≈ 1 μM) and prevent systemic inflammation in wild-type but not in β2-knockout mice. β2-Agonists rescue wild-type mice from established polymicrobial peritonitis in a clinically relevant time frame. Regulatory lymphocytes reestablish the anti-inflammatory potential of β2-agonists to control systemic inflammation, organ damage, and lethal endotoxic shock in β2-knockout mice. These results indicate that β2-adrenoceptors in regulatory lymphocytes are critical for the anti-inflammatory potential of the parasympathetic vagus nerve, and they represent a potential pharmacological target for sepsis.

The short-acting beta-2 adrenoceptor agonists (β(2)-agonists), such as salbutamol, are effective bronchodilators used to treat asthma. They lack significant anti-inflammatory effect in vivo as well as on isolated alveolar macrophages even though they exhibit this effect on freshly isolated monocytes. The purpose of this study was to determine if this observation is related to a change in the expression and/or function of surface β(2)-receptors during the differentiation of these cells to macrophages. Purified monocytes, cultured for 1-48 h were pre-treated with the β(2)-agonists (salbutamol or procaterol) or PGE(2) before being stimulated with bacterial lipopolysaccharide (LPS). Subsequently, the amount of TNF-α (a typical inflammatory mediator) released over 24 h, as well as agonist-stimulated cAMP, were determined by enzyme immunoassays. Western blotting techniques were used to study the expression of the membrane β(2)-receptor protein. Results showed that in freshly isolated human monocytes, both the β(2)-agonists and PGE(2) significantly inhibited LPS-induced TNF-α release as well as increased intracellular cAMP. After culturing adherent monocytes for 24-48 h, the ability of the β(2)-agonists to produce both effects was completely lost, whereas that of PGE(2) was essentially intact. Western blotting data showed a near complete loss of surface expression of β(2)-receptors in cells cultured for ≥24 h. These results show that as human monocytes adhere to surfaces to begin differentiation into macrophages, they selectively lose their surface β(2)-receptors and hence become insensitive to the anti-inflammatory effect of β(2)-agonists. This may explain why β(2)-agonists lack significant anti-inflammatory effect on alveolar macrophages or in clinical asthma., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Xamoterol, a partial β(1)-adrenergic receptor agonist, has been reported to impair the retrieval of hippocampus-dependent spatial reference memory in rats. In contrast, xamoterol restores memory retrieval in gene-targeted mice lacking norepinephrine (NE) and in a transgenic mouse model of Down syndrome in which NE levels are reduced. Restoration of retrieval by xamoterol in these two models complements the observation that NE and β(1) signaling are required for hippocampus-dependent retrieval of contextual and spatial reference memory in wild-type mice and rats. Additional evidence indicates that cAMP-mediated PKA and Epac signaling are required for the retrieval of hippocampus-dependent memory. As a result, we hypothesized that xamoterol has effects in addition to the stimulation of β(1) receptors that, at higher doses, act to counter the effects of β(1) signaling. Here we report that xamoterol-induced disruption of memory retrieval depends on β(2)-adrenergic receptor signaling. Interestingly, the impairment of memory retrieval by xamoterol is blocked by pretreatment with pertussis toxin, an uncoupling agent for G(i/o) signaling, suggesting that β(2) signaling opposes β(1) signaling during memory retrieval at the level of G protein and cAMP signaling. Finally, similar to the time-dependent roles for NE, β(1), and cAMP signaling in hippocampus-dependent memory retrieval, xamoterol only impairs retrieval for several days after training, indicating that its effects are also limited by the age of the memory. We conclude that the disruption of memory retrieval by xamoterol is mediated by G(i/o)-coupled β(2) signaling, which opposes the G(s)-coupled β(1) signaling that is transiently required for hippocampus-dependent emotional memory retrieval.

Background and Purpose: There is much evidence supporting the role of β₂-adrenoceptors (β₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β₂AR knock-out mice (β₂AR-/-) in vivo and explored possible molecular mechanisms in vitro., Experimental Approach: Femoral artery resection (FAR) was performed in wild-type and β₂AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human β₂AR (ADβ₂AR) was used to reinstate β₂ARs in β₂AR-/- mice. Molecular mechanisms were investigated in mouse-derived aortic endothelial cells (EC) in vitro, focusing on NFκB activation and transcriptional activity., Results: Angiogenesis was severely impaired in β₂AR-/- mice subjected to FAR, but was restored by gene therapy with ADβ₂AR. The proangiogenic responses to a variety of stimuli were impaired in β₂AR-/- EC in vitro. Moreover, removal of β₂ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates βARs) nor TNFα induced NFκB activation in β₂AR(-/-) EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in β₂AR(-/-) ECs. ADβ₂AR administration restored β₂AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα., Conclusions and Implications: Our results suggest that β₂ARs control angiogenesis through the tight regulation of nuclear transcriptional activity., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β(1)/β(2)AR double knockout mice. We further showed the specificity of β(2)AR in this crosstalk as treatment with formoterol, a β(2)AR-selective agonist, but not dobutamine, a predominantly β(1)AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR (the mammalian target of rapamycin), P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β(2)AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β(2)AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions.

Although adrenergic receptors (AR) and hyperhomocysteinemia (HHcy) are implicated in heart failure, their role in diabetic cardiomyopathy is not completely understood. We tested the hypothesis that glucose mediated depletion of beta2-AR and HHcy impair contractile function of cardiomyocytes leading to diabetic cardiomyopathy. To prove the hypothesis, cardiac function was assessed in 12week male diabetic Ins2+/- Akita and C57BL/6J mice by echocardiography, pressure-volume loop, and contractile function of cardiomyocytes. The results revealed cardiac dysfunction in Akita. To investigate the mechanism, the levels of beta2-AR, GLUT4, sarcoplasmic reticulum calcium ATP-ase-isoform 2 (SERCA-2) and homocysteine (Hcy) metabolic enzymes-cystathionine beta synthase (CBS), cystathionine gamma lyase (CTH), and methyl tetrahydrofolate reductase (MTHFR) were determined in the heart. It revealed down-regulation of beta2-AR, GLUT4, SERCA-2, CBS, CTH, and MTHFR in Akita. Attenuation of beta2-AR in hyperglycemic condition was also confirmed in cardiomyocytes at in vitro level. Interestingly, the ex vivo treatment of cardiomyocytes with beta2-AR antagonist deteriorated whereas beta-AR agonist ameliorated contractile function. It points to the involvement of beta2-AR in diabetic cardiomyopathy. We conclude that degradation of beta2-AR and impairment of Hcy metabolism is implicated in diabetic cardiomyopathy., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Expression of beta(2)-adrenoceptors (beta(2)-ARs) within the nociceptive system suggested their potential implication in nociception and pain. Recently, we demonstrated that these receptors are essential for neuropathic pain treatment by antidepressant drugs. The aim of the present study was to investigate whether the stimulation of beta(2)-ARs could in fact be adequate to alleviate neuropathic allodynia. Neuropathy was induced in mice by sciatic nerve cuffing. We demonstrate that chronic but not acute stimulation of beta(2)-ARs with agonists such as clenbuterol, formoterol, metaproterenol and procaterol suppressed neuropathic allodynia. By using a pharmacological approach with the beta(2)-AR antagonist ICI 118,551 or a transgenic approach with mice deficient for beta(2)-ARs, we confirmed that the antiallodynic effect of these agonists was specifically related to their action on beta(2)-ARs. We also showed that chronic treatment with the beta(1)-AR agonist xamoterol or with the beta(3)-AR agonist BRL 37344 had no effect on neuropathic allodynia. Chronic stimulation of beta(2)-ARs, but not beta(1)- or beta(3)-ARs, by specific agonists is thus able to alleviate neuropathic allodynia. This action of beta(2)-AR agonists might implicate the endogenous opioid system; indeed chronic clenbuterol effect can be acutely blocked by the delta-opioid receptor antagonist naltrindole. Present results show that beta(2)-ARs are not only essential for the antiallodynic action of antidepressant drugs on sustained neuropathic pain, but also that the stimulation of these receptors is sufficient to relieve neuropathic allodynia in a murine model. Our data suggest that beta(2)-AR agonists may potentially offer an alternative therapy to antidepressant drugs for the chronic treatment of neuropathic pain.

The present study investigated cardiac function in hearts of mice with total deficiency of the beta1-, beta2- and beta3-adrenoceptors (TKO) in comparison to wildtype mice (WT). We investigated cardiac morphology and echocardiographic function, measured protein expression of Ca2+-regulatory proteins, SERCA 2a activity, myofibrillar function, and performed running wheel tests. Heart weight and heart-to-body weight ratio were significantly smaller in TKO as compared to WT. This was accompanied by a decrease in the size of the cardiomyocytes in TKO. Heart rate and ejection fraction were significantly diminished in TKO as compared to WT. Protein expressions of SERCA 2a, ryanodine receptor and Na+/Ca2)-exchanger were similar in TKO and WT mice, but phospholamban protein expression was increased. PKA-dependent phosphorylation of phospholamban at serine 16 was absent and CaMKII-dependent phosphorylation at threonine 17 was decreased in TKO. All alterations were paralleled by a decrease in SERCA 2a-activity. A similar maximal calcium-dependent tension but an increased myofibrillar calcium-sensitivity was measured in TKO as compared to WT. We did not observe relevant functional impairments of TKO in running wheel tests. In the absence of beta-agonistic stimulation, SERCA 2a activity is mainly regulated by alterations of phospholamban expression and phosphorylation. The decreased SERCA 2a activity following beta-adrenoceptor deficiency may be partly compensated by an increased myofibrillar calcium-sensitivity.

Objective: Tricyclic antidepressants (TCAs) are one of the first-line pharmacological treatments against neuropathic pain. TCAs increase the extracellular concentrations of noradrenaline and serotonin by blocking the reuptake transporters of these amines. However, the precise downstream mechanism leading to the therapeutic action remains identified. In this work, we evaluated the role of adrenergic receptors (ARs) in the action of TCAs., Methods: We used pharmacological and genetic approaches in mice to study the role of ARs in the antiallodynic action of the TCA nortriptyline. Peripheral neuropathy was induced by the insertion of a polyethylene cuff around the main branch of the sciatic nerve. The specific role of beta(2)-AR was evaluated by studying beta(2)-AR(-/-) mice. We used von Frey filaments to assess mechanical allodynia., Results: The antiallodynic action of nortriptyline was not affected by cotreatment with the alpha(2)-AR antagonist yohimbine, the beta(1)-AR antagonists atenolol or metoprolol, or the beta(3)-AR antagonist SR 59230A. On the contrary, the beta-AR antagonists propranolol or sotalol, the beta(1)/beta(2)-AR antagonists alprenolol or pindolol, or the specific beta(2)-AR antagonist ICI 118,551 blocked the action of nortriptyline. The effect of nortriptyline was also totally absent in beta(2)-AR-deficient mice., Interpretation: Stimulation of beta(2)-AR is necessary for nortriptyline to exert its antiallodynic action against neuropathic pain. These findings provide new insight into the mechanism by which antidepressants alleviate neuropathic pain. Our results also raise the question of a potential incompatibility between beta-blockers that affect beta(2)-AR and antidepressant drugs in patients treated for neuropathic pain.

Background: Several beta-adrenergic receptor (betaAR) antagonists have been shown to have neuroprotective effects against cerebral ischemia. However, clenbuterol, a beta(2)AR agonist, was shown to have neuroprotective activity by increasing nerve growth factor expression. We used beta(2)AR knockout mice and a beta(2) selective antagonist to test the effect of loss of beta(2)ARs on outcome from transient focal cerebral ischemia., Methods: Ischemia was induced by the intraluminal suture method, for 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Neurological score was determined at 24 h reperfusion and infarct size was determined by cresyl violet or 2,3,5-triphenyltetrazolium chloride staining. beta(2)AR knockout mice and wild-type congenic FVB/N controls were studied, as well as 2 groups of wild type mice given either ICI 118,551 (0.2 mg/kg) or 0.9% saline intraperitoneally 30 min before MCAO (n = 10 per group). Changes in expression of heat shock protein (Hsp)72 after ischemia were examined by immunohistochemistry and western blots., Results: Compared with wild type littermates, infarct volume was decreased by 22.3% in beta(2)AR knockout mice (39.7 +/- 10.7 mm(3) vs 51.0 +/- 11.4 mm(3), n = 10/group, P = 0.034) after 60 min of MCAO followed by 24 h reperfusion. Pretreatment with a beta(2)AR selective antagonist, ICI 118,551, also decreased infarct size significantly, by 25.1%, compared with the saline control (32.8 +/- 11.9 mm(3) vs 43.8 +/- 10.3 mm(3), n = 10/group, P = 0.041). Neurological scores were also significantly improved in mice lacking the beta(2)AR or pretreated with ICI 118,551. After cerebral ischemia, total levels of Hsp72 and the number of Hsp72 immunopositive cells were greater in mice lacking beta(2) AR., Conclusion: Brain injury is reduced and neurological outcome improved after MCAO in mice lacking the beta(2)AR, or in wild type mice pretreated with a selective beta(2)AR antagonist. This is consistent with a shift away from prosurvival signaling to prodeath signaling in the presence of beta(2)AR activation in cerebral ischemia. Protection is associated with higher levels of Hsp72, a known antideath protein. The effect of beta(2)AR signaling in the setting of cerebral ischemia is complex and warrants further study.

The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry. Both WT and beta1/beta2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 +/- 1.81 vs. 92.11 +/- 2.62 mmHg) in beta1/beta2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and beta1/beta2ADR-/- mice adapted to changed salt intake without changed MAP. However, the beta1/beta2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the beta-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.

Background and Purpose: The role of beta-adrenoceptors in heart disease remains controversial. Although beta-blockers ameliorate the progression of heart disease, the mechanism remains undefined. We investigated the effect of beta-adrenoceptors on cardiac hypertrophic growth using beta(1)- and beta(2)-adrenoreceptor knockout and wild-type (WT) mice., Experimental Approach: Mice were subjected to aortic banding or sham surgery, and their cardiac function was determined by echocardiography and micromanometry., Key Results: At 4 and 12 weeks after aortic banding, the left ventricle:body mass ratio was increased by 80-87% in wild-type mice, but only by 15% in knockouts, relative to sham-operated groups. Despite the blunted hypertrophic growth, ventricular function in knockouts was maintained. WT mice responded to pressure overload with up-regulation of gene expression of inflammatory cytokines and fibrogenic growth factors, and with severe cardiac fibrosis. All these effects were absent in the knockout animals., Conclusion and Implications: Our findings of a markedly attenuated cardiac hypertrophy and fibrosis following pressure overload in this knockout model emphasize that beta-adrenoceptor signalling plays a central role in cardiac hypertrophy and maladaptation following pressure overload.

Adaptations to the chronic administration of opioids reduce the utility of these drugs in treating pain and support addiction. Recent genetics-based approaches have implicated the beta2 adrenergic receptor (beta2-AR) in controlling some of these responses. We do not know, however, whether this receptor can modulate tolerance, dependence or changes in gene expression caused by chronic opioid administration. For our studies we used C57BL/6 mice and beta2-AR knockout mice in the FVB background. Morphine dose-response relationships were established both prior to and after chronic morphine treatment. In some cases, the selective beta2-AR antagonist butoxamine was administered along with or after morphine. Physical dependence was assessed using naloxone-precipitated withdrawal. The expression of calcitonin gene related peptide (CGRP) and substance P (SP) were measured in spinal cord and dorsal root ganglion (DRG) tissues using both real-time PCR and enzyme-linked immunoassay (ELISA). Both the co-administration of butoxamine with morphine and the administration of butoxamine after chronic morphine reversed morphine tolerance. Morphine failed to cause tolerance in beta2-AR knockout mice. Physical dependence was reduced under the same circumstances. The chronic administration of butoxamine with morphine reduced or eliminated the normally observed up-regulation of CGRP and SP in spinal cord and DRG tissues. Our results suggest that the beta2-AR modulates both opioid tolerance and physical dependence. Activation of beta2-ARs appears to be required for some of the key neurochemical changes which characterize chronic opioid administration. Therefore, beta2-AR antagonists show some promise as agents to enhance chronic opioid analgesic therapy.

The present experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR(-/-)) to assess the role of beta-adrenergic receptors in basal and regulated renin expression and release. On a control diet, plasma renin concentration (in ng angiotensin I per mL per hour), determined in tail vein blood, was significantly lower in beta1/beta2ADR(-/-) than in wild-type (WT) mice (222+/-65 versus 1456+/-335; P<0.01). Renin content and mRNA were 77% and 65+/-5% of WT. Plasma aldosterone (in picograms per mL) was also significantly reduced (420+/-36 in beta1/beta2ADR(-/-) versus 692+/-59 in WT). A low-salt diet (0.03%) for 1 week increased plasma renin concentration significantly in both beta1/beta2ADR(-/-) and WT mice (to 733+/-54 and 2789+/-555), whereas a high-salt diet (8%) suppressed it in both genotypes (to 85+/-24 in beta1/beta2ADR(-/-) and to 676+/-213 in WT). The absolute magnitude of salt-induced changes of plasma renin concentration was markedly greater in WT mice. Acute stimulation of renin release by furosemide, quinaprilat, captopril, or candesartan caused significant increases of plasma renin concentration in both beta1/beta2ADR(-/-) and WT mice, but again the absolute changes were greater in WT mice. We conclude that maintenance of normal levels of renin synthesis and release requires tonic beta-adrenergic receptor activation. In the chronic absence of beta-adrenergic receptor input, the size of the releasable renin pool decreases with a concomitant reduction in the magnitude of the plasma renin concentration changes caused by variations of salt intake or acute stimulation with furosemide, angiotensin-converting enzyme, or angiotensin type 1 receptor inhibition, but regulatory responsiveness is nonetheless maintained.

In wild-type FVB mice, leukocyte recruitment to lipopolysaccharide was sexually dimorphic, with a greater number of leukocytes recruited in females. In male beta(2)-adrenergic receptor knock out mice (bred on a congenic FVB background) the number of leukocytes recruited was increased approximately 4-fold, while in females there was no change, eliminating sexual dimorphism in leukocyte migration. While there were significantly fewer recruited CD62L(+) and CD11a(+) leukocytes in wild-type males, only in male beta-adrenergic receptor knock out mice was there an increase in the number of recruited CD11a(+) leukocytes, again eliminating sexual dimorphism. Thus, leukocyte migration and CD11a(+) adhesion molecule expression in male, but not in female, leukocytes is beta-adrenergic receptor-dependent. Our findings provide support for a role of beta(2)-adrenergic receptor mechanisms in the inflammatory response, and suggest that beta(2)-adrenergic receptor on male leukocytes contributes to sexual dimorphism in the effect of stress on inflammatory diseases.

In the present study, we investigated the IGF system in neonatal astrocytes derived from mice with a targeted disruption of the beta-2 adrenergic receptor (beta(2)AR). beta(2)AR knockout astrocytes demonstrated higher proliferation rates and increased expression of the astrogliotic marker GFAP, as compared with wild-type cells. beta(2)AR deletion also regulated molecules of the IGF system. Although IGF-1 levels remained unaltered, IGF-2 and type 1 IGF receptor expression was increased in beta(2)AR knockout cells. Furthermore, conditioned medium from knockout astrocytes contained lower levels of IGF binding protein-2 and -4. Our data suggest a deficit of beta(2)AR on astrocytes, as previously reported in multiple sclerosis, may have implications on proliferative status of astrocytes, a feature that might be attributed to regulation of IGF mitogenic actions.

Beta-adrenergic receptors (beta-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (beta1, beta2, and beta3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of beta-ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, beta1-KO, beta2-KO, and beta1/beta2 double KO (DL-KO), to examine the role that beta-AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in beta1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] mice compared with wild-type [WT; 638 (SD 30)] or beta2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking beta1-ARs (beta1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF powers compared with mice with intact beta1-ARs (WT and beta2-KO). These results demonstrate the differential role of beta-AR subtypes in regulating autonomic signaling.

Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the beta2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying beta2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that beta2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.

This study examines the role of the beta2-adrenergic receptor (beta2-AR) in cardioprotection. The beta2-AR couples to Gs and Gi proteins. Gs activates PKA, which phosphorylates the receptor and switches beta2-AR coupling from Gs to Gi. Prior to 20 min of global ischemia, mouse hearts were either perfused for 30 min without treatment (control), treated with 10 nmol/L of isoproterenol (ISO) for 5 min followed by 5 min washout, or preconditioned with 4 cycles of 5 min ischemia and 5 min reflow (PC). Recovery of left ventricular developed pressure (LVDP) and infarct size were measured. Intermittent ISO treatment improved post-ischemic recovery of LVDP (58.5+/-4.8% vs. 22.0+/-6.3% in control) and reduced infarct size (31.0+/-2.4% vs. 53.0+/-4.6% in control). The Gi inhibitor pertussis toxin blocked the ISO-induced improvement in postischemic LVDP and infarct size. To test the role of beta2-AR in PC, we studied mice lacking beta2-AR (beta2-AR-/-) and found that PC had no effect on postischemic LVDP or infarct size in beta2-AR-/-. To test whether PKA is required for the PC and ISO-induced protection, hearts were treated with the PKA inhibitors PKI and H-89. We found that PKI and H-89 blocked the PC- and ISO-induced improvement in postischemic LVDP and infarct size. These data show an important role for beta2-AR in cardioprotection and support the novel hypothesis that preconditioning involves switching of beta2-AR coupling from Gs to Gi.

Bone remodelling, the mechanism by which vertebrates regulate bone mass, comprises two phases, namely resorption by osteoclasts and formation by osteoblasts; osteoblasts are multifunctional cells also controlling osteoclast differentiation. Sympathetic signalling via beta2-adrenergic receptors (Adrb2) present on osteoblasts controls bone formation downstream of leptin. Here we show, by analysing Adrb2-deficient mice, that the sympathetic nervous system favours bone resorption by increasing expression in osteoblast progenitor cells of the osteoclast differentiation factor Rankl. This sympathetic function requires phosphorylation (by protein kinase A) of ATF4, a cell-specific CREB-related transcription factor essential for osteoblast differentiation and function. That bone resorption cannot increase in gonadectomized Adrb2-deficient mice highlights the biological importance of this regulation, but also contrasts sharply with the increase in bone resorption characterizing another hypogonadic mouse with low sympathetic tone, the ob/ob mouse. This discrepancy is explained, in part, by the fact that CART ('cocaine amphetamine regulated transcript'), a neuropeptide whose expression is controlled by leptin and nearly abolished in ob/ob mice, inhibits bone resorption by modulating Rankl expression. Our study establishes that leptin-regulated neural pathways control both aspects of bone remodelling, and demonstrates that integrity of sympathetic signalling is necessary for the increase in bone resorption caused by gonadal failure.

Alveolar epithelial beta-adrenergic receptor (betaAR) activation accelerates active Na+ transport in lung epithelial cells in vitro and speeds alveolar edema resolution in human lung tissue and normal and injured animal lungs. Whether these receptors are essential for alveolar fluid clearance (AFC) or if other mechanisms are sufficient to regulate active transport is unknown. In this study, we report that mice with no beta1- or beta2-adrenergic receptors (beta1AR-/-/beta2AR-/-) have reduced distal lung Na,K-ATPase function and diminished basal and amiloride-sensitive AFC. Total lung water content in these animals was not different from wild-type controls, suggesting that betaAR signaling may not be required for alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with beta1- but not beta2-adrenergic receptors to beta1AR-/-/beta2AR-/- mice indicates that the beta2AR mediates the bulk of beta-adrenergic-sensitive alveolar active Na+ transport. To test the necessity of betaAR signaling in acute lung injury, beta1AR-/-/beta2AR-/-, beta1AR+/+/beta2AR-/-, and beta1AR+/+/beta2AR+/+ mice were exposed to 100% oxygen for up to 204 hours. beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice had more lung water and worse survival from this form of acute lung injury than wild-type controls. Adenoviral-mediated rescue of beta2-adrenergic receptor (beta2AR) function into the alveolar epithelium of beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice normalized distal lung beta2AR function, alveolar epithelial active Na+ transport, and survival from hyperoxia. These findings indicate that betaAR signaling may not be necessary for basal AFC, and that beta2AR is essential for the adaptive physiological response needed to clear excess fluid from the alveolar airspace of normal and injured lungs.

Objective: Clinical studies have demonstrated that hyperglycaemia represents a major risk factor in the development of the endothelial impairment in diabetes, which is the first step in vascular dysfunction. Using non-obese diabetic mice, we have evaluated the role of the adrenergic system and eNOS on progression of the disease, Methods and Results: When glycosuria is high (20 to 500 mg/dL), there is a selective reduction in the response to alpha1 and beta2 agonists but not to dopamine or serotonin. When glycosuria is severe (500 to 1000 mg/dL), there is a complete ablation of the contracture response to the alpha1 receptor agonist stimulation and a marked reduced response to beta2 agonist stimulation. This effect is coupled with a reduced expression of alpha1 and beta2 receptors, which is caused by an inhibition at transcriptional level as demonstrated by RT-PCR. In the severe glycosuria (500 to 1000 mg/dL), although eNOS expression is unchanged, caveolin-1 expression is significantly enhanced, indicating that high glucose plasma levels cause an upregulation of the eNOS endogenous inhibitory tone. These latter results correlate with functional data showing that in severe glycosuria, there is a significant reduction in acetylcholine-induced vasodilatation., Conclusions: Our results show that in diabetes development, there is a progressive selective downregulation of the alpha1 and beta2 receptors. At the same time, there is an increased expression of caveolin-1, the endogenous eNOS inhibitory protein. Thus, caveolin-1 could represent a new possible therapeutic target in vascular impairment associated with diabetes.