β3-AR Agonist Research Articles

Protective effects of (R)-enantiomers but not (S)-enantiomers of β2-adrenergic receptor agonists against acute colitis: The role of β2AR

The outcomes of ulcerative colitis (UC) treatment remain unsatisfactory. Salbutamol is a β2-adrenergic receptor (β2AR) agonist that is frequently used to treat human airway diseases, and it is a chiral drug with (RS)-isomers. However, the effects of (RS)-enantiomers of this drug on acute ulcerative colitis remain unknown. The present work determined and compared the effects of different chiral β2AR agonists in acute colitis. Acute colitis was established in mice with 3% dextran sulfate sodium and the mice were orally administered different salbutamol isomers. Body weight loss, colon length, disease activity index (DAI), and colon histopathology were assessed. Inflammatory cytokine levels were detected by ELISA. Colonic biopsies were collected from colitis patients. 16S rDNA amplicon sequencing was carried out to assess the composition and relative abundance of the gut microbiome. The expression of M1 and M2 macrophage markers in the colon were assessed by immunofluorescence staining and Western blotting. The results revealed that (R)-salbutamol prevented body weight loss and colonic shortening, decreased the DAI and histopathological scores, and reduced splenomegaly and inflammatory cytokine levels significantly better than (RS)-salbutamol and (S)-salbutamol. (R)-salbutamol downregulated levels of inflammatory protein in LPS-induced human colon tissue specimens. Furthermore, (R)-salbutamol ameliorated gut dysbiosis and macrophage polarization in mice with colitis. The β2AR antagonist ICI-118551 reversed the effect of (R)-salbutamol in ameliorating acute colitis. Taken together, (R)-salbutamol ameliorated the mice with acute colitis, which can serve as a new candidate or lead compound for UC treatment.

Long-Acting β2 Adrenergic Receptor Agonist Ameliorates Imiquimod-Induced Psoriasis-Like Skin Lesion by Regulating Keratinocyte Proliferation and Apoptosis.

Psoriasis is a chronic inflammatory disease that affects approximately 1%–5% of the population worldwide. Considering frequent relapse, adverse drug reactions, and large costs of treatment, it is urgent to identify new medications for psoriasis. Keratinocytes play an essential role during psoriasis development, and they express high levels of β2-Adrenergic receptor (β2-AR), which increases intracellular cAMP levels when activated. Increased level of cAMP is associated with the inhibition of epidermal cell proliferation. In the present study, we observed the effect of salmeterol, a long-acting β2-AR agonist, on the proliferation and apoptosis of keratinocytes as well as imiquimod-induced psoriasis-like skin lesions in mice. As phosphodiesterase 4 (PDE4) inhibitors increases intracellular cAMP concentration by inhibiting its inactivation, we further explored the synergetic effect of a PDE4 inhibitor and salmeterol on psoriasis-like skin lesions in mice. Our results indicated that salmeterol effectively inhibited the proliferation of HaCaT cells induced by TNF-α and serum, and this effect was accompanied by significantly increased apoptosis and CREB phosphorylation, which were reversed by the PKA inhibitor, H89. Salmeterol ameliorated imiquimod-induced psoriasis-like skin lesions in mice, but salmeterol combined with a PDE4 inhibitor had no synergetic effect in improving skin lesions in mice. Of note, the synergistic effects of anti-proliferation and induction of apoptosis in HaCaT cells appeared by inhibiting ERK signaling. In summary, salmeterol, a long-acting β2-AR agonist, alleviates the severity of psoriasis via inhibiting the proliferation and promoting apoptosis of keratinocytes, partially by activating the cAMP/PKA signaling pathway.

Abstract 2191: Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction

Abstract Bone metastasis is a major cause of morbidity and mortality for breast cancer patients. Bone is a unique metastatic microenvironment because of complex interactions among numerous distinct cell types such as osteo-blasts, -clasts, -cytes and marrow immune cells in physiological and pathological conditions. Imbalanced bone homeostasis by diverse factors such as the sympathetic nerve system (SNS) activation, potentially contribute to the progression of bone metastasis, yet precise mechanisms remain unclear. We investigated the effects of beta 2 adrenergic receptor (β2AR) activation in osteoblasts induced by prolonged SNS stimulation on anti-tumoral immunity in bone metastasis. We treated female Balb/C with chronic immobilization stress (CIS; 2 h. daily) and found that CIS increased syngeneic intra-tibial 4T1 bone metastasis growth as well as the number of CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) counterbalancing anti-tumoral cytotoxic T cells in bone microenvironment. A β2AR-specific inhibitor (ICI-118551) or MDSC depletion using anti-Gr1 antibodies reversed CIS-induced bone metastatic tumor growth. More importantly, 2-week CIS pre-treatment increased EDU+ MDSC proliferation in the bone marrow, contributing to subsequent bone metastatic tumor growth and micro-metastatic seeding in intra-tibial and intra-cardiac tumor injection models, respectively. When CIS was administered after the establishment of bone metastatic tumors, the pro-tumorigenic effects were marginal. In osteoblast-specific β2AR knockout mice (Adrb2flox/flox; murine 2.3kb Col1-CreC57BL6 mice), CIS did not increase MDSC proliferation compared with littermate floxed controls, suggesting that β2AR in osteoblasts upregulates MDSC in bone microenvironment. RNA-sequencing transcriptome analysis of saline- or clenbuterol (a selective β2AR agonist)-treated murine osteoblasts showed that interleukin-6 (IL6) was the most significantly increased cytokine. Indeed, CIS or clenbuterol treatment increased IL-6 expression in vivo (immunohistochemistry) and in vitro (cultured osteoblasts), respectively. MDSC isolated from the bone marrow of CIS-treated mice had significantly increased STAT3 phosphorylation by flow cytometry and the expression of immuno-suppressive functional molecules such as arginase 1 (Arg1) and indolamine 2,3-dioxygenase 1 (Ido1) compared to those from control mice. Our data collectively demonstrated that SNS-dependent β2AR in osteoblasts expands and activates MDSC in the pre-metastatic bone microenvironment via the IL6-STAT3-Arg1/IDO1 pathway, creating a fertile soil for bone metastasis progression. Our data provided the first direct evidence for anti-tumoral immunity and pro-tumorigenic effects of autonomous nerve system dysfunction specific to the metastatic bone microenvironment. Citation Format: Eun Jung Lee, Da Hyeon Yun, Seungpil Jung, Serk In Park. Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2191.

201-LB: Overexpression of GOT1 in Brown Adipose Tissue Enhances Thermogenesis and Glucose Metabolism by Activating the Malate-Aspartate Shuttle

Brown adipose tissue (BAT) is characterized by thermogenic and glucose-consuming properties. BAT takes up large amounts of circulating glucose during cold exposure or pharmacological stimulation of β3-adrenergic receptors (β3AR) , but glucose oxidation in mitochondria only modestly contributes as a fuel source for thermogenesis. How glucose metabolism supports thermogenesis is incompletely understood. Here, we identify a novel role of the malate-aspartate shuttle (MAS) in enhancing thermogenesis, glucose uptake and glycolysis in brown adipose tissue. We found that glutamic-oxaloacetic transaminase 1 (GOT1) , a key enzyme of the MAS, exists at very low levels in BAT but is markedly induced by cold or β3AR agonists, while other MAS enzymes (GOT2 and MDH1/2) remain unchanged. Enforced expression of Got1 in BAT increased energy expenditure in mice and further enhanced thermogenesis upon cold exposure or β3-adrenergic stimulation. Moreover, mice overexpressing Got1 in BAT exhibited enhanced glucose tolerance. Mechanistically, Got1 overexpression stimulated mitochondrial uncoupled respiration. MAS-driven thermogenesis in turn led to AMPK activation, which likely explains enhanced glucose uptake and glycolysis. Collectively, our data demonstrate a novel GOT1-MAS-AMPK axis that regulates thermogenesis and glucose metabolism in cold-activated BAT. Disclosure C. Park: None. H. Cheng: None. S. R. Lee: None. K. Eilertsen: None. J. Chang: None. Funding NIH (DK104748, 1P30GM118430, P30DK072476)

Targeting the Beta-2-Adrenergic Receptor and the Risk of Developing Alzheimer's Disease: A Retrospective Inception Cohort Study.

Background:Animal studies suggested that β2-Adrenergic receptors (β2AR) may be a potential target for the treatment of Alzheimer’s disease (AD).Objective:This retrospective inception cohort study aimed to assess the association between antagonists and agonists of the β2AR and the risk of starting treatment for AD in older adults.Methods:A retrospective inception cohort study was conducted among older adults who initiated either non-selective βAR antagonists or selective β2AR agonists using the University Groningen IADB.nl prescription database (study period 1994–2019). For each exposed cohort, two reference cohorts (A and B) were matched on age at index date. The main outcome was defined as at least two prescriptions for cholinesterase inhibitors (rivastigmine, galantamine, and donepezil) and/or memantine. Cox proportional hazard regression models were used to estimate hazard ratios (HR).Results:The risk of developing AD was elevated among patients exposed to non-selective βAR antagonists (A: aHR 3.303, 95% CI 1.230–8.869, B: aHR 1.569, 95% CI 0.560–4.394) and reduced among patients exposed to selective β2AR agonists (A: aHR 0.049, 95% CI 0.003–0.795, B: aHR 0.834, 95% CI 0.075–9.273) compared to reference patients.Conclusion:These findings suggest that exposure to non-selective βAR antagonists is associated with an increased risk for developing AD whereas there may be a decreased risk for developing AD after exposure to selective β2AR agonists.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein

β2-adrenoreceptor (β2AR) agonists have been associated with a decreased risk of developing Parkinson’s disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of β2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

Characterization of immune cells of stressed beta2-adrenergic receptor homozygous knockout mouse model during Chlamydia muridarum genital infection

Abstract The relationship of stress in chlamydia genital disease pathogenesis and its influence on the host immune response remains unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone norepinephrine (NE), is known to impair the function of immune cells. This study was undertaken to determine whether cold-induced stress (CIS) results in differential viability percent of immune cells β2-AR KO and WT C57BL/6J treated with NE, β2-AR agonist(Fenoterol), or antagonist(ICI 118,551), and patterns of cytokine production. CIS was induced by immersing mice in cold water for five minutes daily for 21 days. Total CD4+, CD8+, differentiated bone marrow-derived monocytes were purified by negative selection and treated with chemicals for one hour before proliferation for 48 h proliferation. The vi-cell viability analyzer showed that NE results in a 30 and 40 % decrease in viability of WT C57BL/6J CD4+ T and CD8+ cells, respectively, compared to KO (p<0.05). Similarly, NE and Fenoterol resulted in a 20 and 27% decrease in viability of bone marrow-derived monocytes compared to the β2-AR KO mice. Cells treated with ICI 118, 551 showed the slightest reduction in viability (12%) compared to those treated with NE. ELISA results show decreased IL-4 and increased IFN-g secretion by CD4+ T cells of β2-AR KO compared to WT. The secretion of TNF-a by CD8+ cells and bone marrow cells treated with NE and β2-AR agonist or antagonist was high in all treatment groups. This study provides insights for the first time to better understand the NE-β2-AR pathway during chlamydia genital infection. Supported by WV-INBRE of NIH Grant P20GM103434 and NIH grant # 1R15AI124156-01 given to BSC.

Diesel exhaust particles alter cAMP dynamics in human bronchial epithelial cells

Diesel exhaust particles (DEP) are one of the main causes of air pollution and are associated with lung disorders. DEP exposure may promote several pathological processes, including epithelial‐to‐mesenchymal transition (EMT), inflammation and oxidative stress, which contribute to a decline in lung function. Cyclic AMP (cAMP) has been reported to alleviate lung dysfunction. The impact of DEP on cAMP signaling is largely unknown. Human bronchial epithelial (BEAS‐2B) cells were exposed to different concentrations (≤ 300 µg/ml) for up to 72 h. Markers of oxidative stress, inflammation, EMT, components of mitochondrial bioenergetics and cAMP signaling were evaluated. DEP increased gene expression of Nrf1, thioredoxin, HO‐1 and SOD‐2 (oxidative stress markers), and interleukin (IL)‐6 and IL‐8 (inflammatory markers), as well as the EMT marker transforming growth factor‐β1 (both mRNA and protein). Epithelial cell markers (E‐cadherin, ZO‐1) were reduced by DEP, whereas mesenchymal markers (β‐catenin, collagen‐1) were increased. DEP reduced both basal and ATP‐linked respiration as well as the glycolytic capacity of mitochondria. Concomitantly, DEP profoundly altered mitochondrial morphology and increased the gene expression of A‐kinase anchoring protein (AKAP) 1 (a known mitochondrial scaffold protein). Furthermore, DEP significantly affected Epac1 mRNA levels and appeared to increase Epac1 protein, but did not alter the expression of phospho‐PKA substrates. Interestingly, DEP increased gene expression levels of several AC family members (AC3, AC6 and AC7), the β2‐adrenoceptor (β2‐AR) and prostanoid E receptor subtype 4 (EP4). Importantly, real‐time measurements of cAMP demonstrated that DEP strongly reduced cAMP induced by forskolin (AC activator), fenoterol (β2‐AR agonist) and a stable EP4 agonist, implicating that DEP might limit the therapeutic effects of β2‐AR agonists used to treat lung disorders. Our findings suggest that DEP promotes oxidative stress, inflammation, and EMT. These profound alterations in cellular behavior were accompanied by changes in mitochondrial dysfunction and real‐time cAMP dynamics, which may contribute to lung dysfunction induced by air pollution exposure.

Nicotine Alters cAMP Signaling in Arterial Myocytes

Arterial myocytes receive many inputs from sympathetic nerves that regulate vascular reactivity by activating GS protein‐coupled receptors (GSPCRs). The ubiquitous second messenger 3′,5′‐ cyclic adenosine monophosphate (cAMP) is synthesized by adenylyl cyclases (ACs) in response to the activation of various GSPCRs and plays a critical role in integrating the myriad of signals elicited. Importantly, arterial myocyte cAMP signaling has been traditionally linked with relaxation. We found that arterial myocytes from mice exposed to secondhand smoke (SHS) had an attenuated cAMP synthesis in response to the β‐adrenergic receptor (βAR) agonist isoproterenol (ISO) (unpublished data), which could alter vascular reactivity. Tobacco smoke contains over 7,000 chemicals, including nicotine. Nicotine, the primary addictive component in tobacco cigarettes is a strong sympathomimetic with significant vasoactive effects and have been shown to alter cellular cAMP production. However, how nicotine impacts cAMP signaling in the vasculature, particularly in arterial myocytes, and the implications of these changes in vascular reactivity are unclear. We are tackling this knowledge gap by expressing a FRET‐based cAMP biosensor in cultured arterial myocytes to determine cAMP signaling in response to nicotine/cotinine. We also developed a mouse expressing a new cAMP sensor specifically in SM (CamperSM) to investigate cAMP dynamics in native VSM/arteries. We hypothesize that nicotine impairs receptor‐mediated cAMP signaling in arterial myocytes leading to altered vascular reactivity. Our data using unpassaged arterial myocytes expressing a cytosolic FRET‐based cAMP sensor exposed to cotinine (the primary metabolite of nicotine) show a reduction in ISOinduced cAMP production. Similar results were observed in arterial myocytes from CAMPERSM mice infused with nicotine. These results suggest similar effects of cotinine in vitro or nicotine in vivo on βAR‐mediated cAMP synthesis in arterial myocytes. Intriguingly, we found that inhibition of phosphodiesterases rescue cAMP synthesis in CAMPERSM arterial myocytes exposed to nicotine. We correlated the nicotine‐induced reduction in arterial myocytes cAMP synthesis with impaired vasodilation in response to ISO. Our data suggest that nicotine impairs receptormediated cAMP signaling in arterial myocytes leading to altered vascular reactivity. Nicotineinduced cAMP dysregulation leading to altered arterial myocytes excitability could be an underlying factor contributing to cardiovascular disease in individuals exposed to SHS and users of nicotine containing products.

Circadian rhythm affects the magnitude of contact hypersensitivity response in mice.

The circadian rhythm controls multiple biological processes, including immune responses; however, its impact on cutaneous adaptive immune response remains unclear. We used a well-established cutaneous type IV allergy model, contact hypersensitivity (CHS). We induced CHS using dinitrofluorobenzene (DNFB). Mice were sensitized and elicited with DNFB in the daytime or at night. In mice, a nocturnally active animal, we found that ear swelling increased when mice were sensitized at night compared with in the daytime. In addition, cell proliferation and cytokine production in the draining lymph nodes (LNs) were promoted when sensitized at night. We hypothesized that these differences were due to the oscillation of leukocyte distribution in the body through the circadian production of adrenergic hormones. Administration of a β2-adrenergic receptor (β2AR) agonist salbutamol in the daytime decreased the number of immune cells in blood and increased the number of immune cells in LNs. In contrast, a β2AR antagonist ICI18551 administration at night increased the number of immune cells in blood and decreased the number of immune cells in LNs. Accordingly, the severity of CHS response was exacerbated by salbutamol administration in the daytime and attenuated by ICI18551 administration at night. Our study demonstrated that the magnitude of adaptive CHS response depends on the circadian rhythm and this knowledge may improve the management of allergic contact dermatitis (ACD) in humans.

Production of CD4+ T Cell Subsets in a Beta2-Adrenergic Receptor Knockout Stress Mouse Model during Chlamydia muridarum Genital Infection

Kaitlyn Cook*, Tesfaye Belay, Ph.D*., *Department of Applied Science and Mathematics, Bluefield State College, Bluefield WV 24701 Production of CD4+ T Cell Subsets in a Beta2-Adrenergic Receptor Knockout Stress Mouse Model during Chlamydia muridarum Genital Infection
 Chlamydia is the most widely spread sexually transmitted disease, with more than one hundred million new cases occurring every year worldwide. Studies show the effect of stress promoting infection, but little is known about Chlamydia and the impact of stress. Cold-induced stress was used to simulate stress in mice to understand the relationship between the stress hormone norepinephrine (NE) and the immune system. This study aims to determine the role of beta2-adrenergic receptor (β2-AR) under stressful conditions during infection. We hypothesize the β2-AR suppresses T helper cell 1 (Th1) and promotes the production of Th2 cytokines. Following stressing, infection, sacrificing, and dissection of mice, the spleen, lymph node, and genital tract cells were proliferated for 72 hours. ELISA data for IL-13 concentration shows stressed knockout (KO) mice had 273.6pg/mL compared to stressed wildtype (WT) mice with 120.6pg/mL. Various treatment of CD4+ T cells resulted in production of TNF- α, ranging from 595pg/mL to 398.7pg/mL. Viability of CD4+ T cells treated with β2-AR agonist Fenoterol, and antagonist ICI 118,551 were analyzed. The viability of CD4+ T cells treated with NE was 70.7% in KO, compared to 69.1% and 70.2% for the agonist and antagonist, respectively. NE, Fenoterol, and ICI 118,551 treatment resulted in 72.2%, 83.6% and 84.4% respectively for WT. No significant difference is found between the agonist and antagonist. Chlamydia muridarum isolation from the genital tract of treatment groups is underway. The ELISA data implies that β2-AR mice restored increased production of protective cytokines more than their WT counterparts.

Cryo-EM structures of the β3 adrenergic receptor bound to solabegron and isoproterenol

The β3-adrenergic receptor (β3AR) is the most essential drug target for overactive bladder and has therapeutic potentials for the treatments of type 2 diabetes and obesity. Here, we report the cryo-electron microscopy structures of the β3AR-Gs signaling complexes with the selective agonist, solabegron and the nonselective agonist, isoproterenol. Comparison of the isoproterenol-, mirabegron-, and solabegron-bound β3AR structures revealed that the extracellular loop 2 changes its conformation depending on the bound agonist and plays an essential role in solabegron binding. Moreover, β3AR has an intrinsically narrow exosite, regardless of the agonist type. This structural feature clearly explains why β3AR prefers mirabegron and solabegron, as the narrow exosite is suitable for binding with agonists with elongated shapes. Our study deepens the understanding of the binding characteristics of β3AR agonists and may pave the way for developing β3AR-selective drugs.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as β2-adrenergic receptor (β2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Biased β-Agonists Favoring Gs over β-Arrestin for Individualized Treatment of Obstructive Lung Disease.

Signals from G-protein-coupled receptors (GPCRs) are the most frequently targeted pathways of currently prescribed therapeutics. Rather than being a simple switch, it is now evident that a given receptor can directly initiate multiple signals, and biasing to achieve signal selectivity based on agonist structure is possible. Biased agonists could direct therapeutically favorable pathways while avoiding counterproductive or adverse reaction pathways. For obstructive lung diseases, β2-adrenergic receptor (β2AR) agonists act at these receptors on airway smooth muscle (ASM) cells to open the airways by relaxing ASM, improving airflow and morbidity. However, these receptors signal to the G protein Gs (increasing cAMP and promoting relaxation), but also to β-arrestin (promoting desensitization and a loss of effectiveness). Indeed, β-agonist use is associated with adverse events in asthma pathogenesis and clinical outcomes which are related to desensitization. β-agonists favoring Gs coupling over β-arrestin binding would provide a means of tailoring bronchodilator therapy. In this review, we show how combinatorial methods with a 40 million compound agnostic library led to a new class of biased β-agonists that do not desensitize, providing an opportunity to personalize therapy in patients who experience poor efficacy or adverse effects from traditional balanced agonists.

The glucose lowering effects of CL 316,243 dissipate with repeated use and are rescued bycilostamide.

Repeated activation of the beta 3 adrenergic receptor (β3AR) with the agonist CL 316,243 (CL) results in remodeling of white adipose tissue (WAT) characterized by increased mitochondrial enzymes and expression of uncoupling protein 1 (UCP1). β3AR activation also has profound acute metabolic effects including rapidly decreasing blood glucose, secondary to fatty acid‐induced increases in insulin, and increasing energy expenditure. The acute (single dose) effects of β3AR activation have largely been examined in treatment naive animals and under room temperature housing conditions. The current study examined if repeated CL treatment would lead to an attenuation of acute metabolic effects of CL treatment under thermoneutral housing conditions and if this could be rescued with cilostamide, a phosphodiesterase inhibitor. We provide evidence demonstrating that the acute effects of CL to increase serum fatty acids and insulin and reduce blood glucose, but not increases in energy expenditure, are attenuated in mice following repeated treatment with CL. This occurs in parallel with reductions in indices of protein kinase A signaling in WAT including the phosphorylation of hormone sensitive lipase. The findings of attenuated serum fatty acid, insulin, and blood glucose responses were confirmed in both high‐fat fed and UCP1−/− mice repeatedly treated with CL. Desensitization to CL in mice was rescued by cilostamide. Herein, we provide evidence that the glucose lowering, but not thermogenesis inducing, effects of CL are attenuated with repeated treatment and can be rescued by cilostamide. The findings of this study point toward novel adjunct treatment approaches that could be used to maximize therapeutic, glucose lowering effects of β3AR agonists.

Pepducin ICL1-9-Mediated β2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner.

β-Adrenergic receptors (βAR) are essential targets for the treatment of heart failure (HF); however, chronic use of βAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of β2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a β-arrestin (βarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of βarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of β2AR to promote Gi protein/βarr-dependent activation of RhoA/ROCK/PKD signaling.

PlexinA1 activation induced by β2-AR promotes epithelial-mesenchymal transition through JAK-STAT3 signaling in human gastric cancer cells.

With the medical model shifting from a single biomedical model to a biopsychological-social model, the impact of psychosocial factors on cancer patients has attracted attention. Studies have shown that chronic stress caused by long-term psychological stress, such as anxiety and depression, can promote the malignant progression of tumors by acting on β2-adrenergic receptor (β2-AR). β2-AR can promote tumor migration by activating epithelial-mesenchymal transition (EMT). However, the underlying mechanisms in the regulation of EMT by β2-AR are still unclear. In this study, we established a chronic stress model by treating MGC-803 and SGC-7901 human gastric cancer cells with isoproterenol (ISO), a β2-AR agonist. EMT in the two gastric cancer cell lines was enhanced after ISO treatment. Thereafter, we found that the interaction between β2-AR and PlexinA1 was involved in the process by which chronic stress affects EMT in both MGC-803 and SGC-7901 cells. Moreover, the activation of β2-AR by ISO increased the expression of PlexinA1, activated JAK-STAT3 signaling and further promoted EMT in human gastric cancer cells. Importantly, the knockdown of PlexinA1 by small hairpin RNAs inhibited JAK-STAT3 signaling and abolished the EMT induced by β2-AR. In conclusion, PlexinA1 was an important downstream target of β2-AR, through which β2-AR promoted EMT in human gastric cancer cells by activating JAK-STAT3 signaling.

Regulation of bFGF-induced effects on rat aortic smooth muscle cells by β3-adrenergic receptors

BackgroundBasic fibroblast growth factor (bFGF)-mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury-induced neointima formation and subsequent vascular restenosis, a major event that hinders the long-term success of angioplasty. The function of β3-adrenergic receptors (β3-ARs) in vascular injury-induced neointima formation has not yet been defined. ObjectivesOur current study explored the possible role of β3-ARs in vascular injury-induced neointima formation by testing its effects on bFGF-induced VSMC migration and proliferation. Methodsβ3-AR expression in rat carotid arteries was examined at 14 days following a balloon catheter-induced injury. The effects of β3-AR activation on bFGF-induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular-signal-regulated kinase/mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested. ResultsWe found that vascular injury induced upregulation of β3-ARs in neointima. Pretreatment of VSMCs with a selective β3-AR agonist, CL316,243 significantly potentiated bFGF-induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF-induced cell migration and that inhibiting AKT phosphorylation reduced bFGF-mediated cell proliferation. ConclusionOur results suggest that activation of β3-ARs potentiates bFGF-mediated effects on VSMCs by enhancing bFGF-mediated ERK and AKT phosphorylation and that β3-ARs may play a role in vascular injury-induced neointima formation.

(RS)-bambuterol and its enantiomers: Potential improvement of (R)-bambuterol in mice with colitis

Bambuterol (BMB) has been used clinically to treat asthma due to its bronchodilation activity. However, the effect of BMB on ulcerative colitis (UC) has not been examined. The present work focused on the effects of enantiomeric BMB on UC. Acute UC was induced in mice by 3% dextran sulfate sodium (DSS), and (R)-, (S) and (RS)-BMB were orally administered. Body weight loss and the disease activity index (DAI) were measured once a day. Inflammatory factors were detected by ELISA and qRT-PCR. Histological evaluations of colon samples were performed. IL-6, STAT3, and RORγt pathway-related proteins were analyzed by western blotting. The results verified that colitis severity was dramatically ameliorated by (R)-BMB, which was significantlybetter than the effect of (RS)-BMB or (S)-BMB, as evidenced by body weight loss, DAI, colon length, spleen/body weight ratio and histopathological manifestations. Furthermore, (R)-BMB treatment significantly diminished the levels of inflammatory cytokines and macrophages infiltration in mice with colitis. Besides, treated with (R)-BMB obviously elevated the level of β2AR. In addition, (R)-BMB decreased the expression of IL-6, IL-17, retinoic acid receptor-related orphan receptor-gamma t (RORt), and phosphorylated STAT3 (p-STAT3) in a dose-dependent manner in the colon tissues. The efficacy of (R)-BMB was more notable than aminosalicylic acid (5-ASA). (R)-BMB is either butyrilcholinesterase inhibitor or β2AR agonist which offers new treatment of colitis.

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.