Β-adrenergic Agonist Research Articles

Sympathetic nervous system hyperactivity-induced oxidative stress promoting endothelial dysfunction is dependent on the NADPH oxidases/SGLT2 crosstalk: potential role in cardiogenic shock

Abstract Background Hyperactivity of the sympathetic nervous system (SNS) promotes cardiac dysfunction, that can contribute to heart failure and even cardiogenic shock. Endothelial dysfunction associated with increased formation of reactive oxygen species (ROS) has been suggested to promote cardiac dysfunction and heart failure. Our previous results showed that SGLT2 and ROS derived from endothelial NADPH oxidases contribute to cardiac dysfunction. Purpose Therefore, this study examined whether cardiogenic shock and hyperactivation of the SNS involve NADPH oxidases and SGLT2 in the development of endothelial dysfunction using in vitro and in vivo approaches. Methods Peripheral blood from patients with cardiogenic shock and healthy controls was drawn by vein puncture into tubes containing sodium citrate. Cultured human microvascular endothelial cells (HMEC-1) were stimulated with either 100 nM of the β-adrenergic agonist isoproterenol or with heparinized (10 U/ml) patient plasma. Male wild-type mice and mice lacking the NADPH oxidase subunit p22phox in the endothelium (p22phox ecKO, 11-week-old) were treated with isoproterenol (100 mg/kg) for five consecutive days and sacrificed at day 14. ROS levels were assessed by dihydroethidium fluorescence, and the expression levels of target genes and proteins by RT-qPCR and Western blot, respectively. siRNA approaches were used to down-regulate the NADPH oxidase subunit p22phox or SGLT2. Results Exposure of HMEC-1 to either plasma from patients suffering from cardiogenic shock or isoproterenol stimulated the formation of ROS. ROS levels were reduced by N-acetylcysteine (an antioxidant) and silencing of p22phox, by empagliflozin (a selective SGLT2 inhibitor) and silencing of SGLT2 as well as by metoprolol (a beta1 adrenergic receptor antagonist) and ICI-118,551 (a beta2 adrenergic receptor antagonist). Cardiogenic shock patient plasma and isoproterenol upregulated the NADPH oxidase subunits p22phox and Nox4 as well as SGLT2. These responses were prevented by silencing of either p22phox or SGLT2, and by empagliflozin. In addition to improved cardiac function and blunted SGLT2 expression, p22phox ecKO mice were protected against isoproterenol-induced endothelial dysfunction and senescence, characterized by upregulation of eNOS, nitrotyrosine, VCAM-1 and p16 in the left ventricle. Conclusion SNS hyperactivity and cardiogenic shock-derived patient plasma cause a feed forward loop between NADPH oxidases and SGLT2, promoting pro-oxidant responses and endothelial dysfunction via activation of both beta1 and beta2 adrenergic receptors. The NADPH oxidase/SGLT2 crosstalk also contributes to SNS hyperactivity-induced senescence, cardiac and endothelial dysfunction. Thus, the endothelial NADPH oxidases/SGLT2 crosstalk appears to have a major role in cardiac diseases associated with SNS hyperactivity such as cardiogenic shock. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Deutsches Herzzentrum München

The CaV1.2 G406R mutation decreases synaptic inhibition and alters L-type Ca2+ channel-dependent LTP at hippocampal synapses in a mouse model of Timothy Syndrome

Genetic alterations in autism spectrum disorders (ASD) frequently disrupt balance between synaptic excitation and inhibition and alter plasticity in the hippocampal CA1 region. Individuals with Timothy Syndrome (TS), a genetic disorder caused by CaV1.2 L-type Ca2+ channel (LTCC) gain-of function mutations, such as G406R, exhibit social deficits, repetitive behaviors, and cognitive impairments characteristic of ASD that are phenocopied in TS2-neo mice expressing G406R. Here, we characterized hippocampal CA1 synaptic function in male TS2-neo mice and found basal excitatory transmission was slightly increased and inhibitory transmission strongly decreased. We also found distinct impacts on two LTCC-dependent forms of long-term potentiation (LTP) synaptic plasticity that were not readily consistent with LTCC gain-of-function. LTP induced by high-frequency stimulation (HFS) was strongly impaired in TS2-neo mice, suggesting decreased LTCC function. Yet, CaV1.2 expression, basal phosphorylation, and current density were similar for WT and TS2-neo. However, this HFS-LTP also required GABAA receptor activity, and thus may be impaired in TS2-neo due to decreased inhibitory transmission. In contrast, LTP induced in WT mice by prolonged theta-train (PTT) stimulation in the presence of a β-adrenergic receptor agonist to increase CaV1.2 phosphorylation was partially induced in TS2-neo mice by PTT stimulation alone, consistent with increased LTCC function. Overall, our findings provide insights regarding how altered CaV1.2 channel function disrupts basal transmission and plasticity that could be relevant for neurobehavioral alterations in ASD.This article is part of the Special Issue on ‘L-type calcium channel mechanisms in neuropsychiatric disorders’.

David O. Warner, M.D., Recipient of the 2022 Excellence in Research Award

David O. Warner, M.D., Recipient of the 2022 Excellence in Research Award

Gut Microbiome and Metabolome Modulation by Maternal High-Fat Diet and Thermogenic Challenge.

The gut microbiota plays a critical role in energy homeostasis and its dysbiosis is associated with obesity. Maternal high-fat diet (HFD) and β-adrenergic stimuli alter the gut microbiota independently; however, their collective regulation is not clear. To investigate the combined effect of these factors on offspring microbiota, 20-week-old offspring from control diet (17% fat)- or HFD (45% fat)-fed dams received an injection of either vehicle or β3-adrenergic agonist CL316,243 (CL) for 7 days and then cecal contents were collected for bacterial community profiling. In a follow-up study, a separate group of mice were exposed to either 8 °C or 30 °C temperature for 7 days and blood serum and cecal contents were used for metabolome profiling. Both maternal diet and CL modulated the gut bacterial community structure and predicted functional profiles. Particularly, maternal HFD and CL increased the Firmicutes/Bacteroidetes ratio. In mice exposed to different temperatures, the metabolome profiles clustered by treatment in both the cecum and serum. Identified metabolites were enriched in sphingolipid and amino acid metabolism in the cecum and in lipid and energy metabolism in the serum. In summary, maternal HFD altered offspring’s response to CL and altered microbial composition and function. An independent experiment supported the effect of thermogenic challenge on the bacterial function through metabolome change.

Study of the biological relevance of Wnt/β-catenin signaling pathway and β-adrenergic regulation in osteoblastic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can invade the bone site, proliferate and differentiate into cartilage and bone. Therefore, they serve as an important target for developing modalities to enhance local and systemic bone formation. It has been demonstrated that the Wnt/β-catenin signaling pathway plays a key role in the movement of MSCs toward osteoblast lineage. β-Catenin, an indispensable mediator of canonical Wnt signaling, is a transcriptional factor regulating the mesenchymal precursor's differentiation to mature osteoblasts. On the other hand, the β-adrenergic nervous system negatively impacts the osteogenic differentiation of MSCs. β-Adrenergic receptors (β-AR) are the dominant synergistic receptors on MSCs. It is established that catecholamines activate β-adrenergic receptors and inhibit osteoblastogenesis through an unknown molecular mechanism. This study evaluated the β-adrenergic system mediates inhibition of osteoblastogenesis through the inhibition of the Wnt/β-catenin signaling pathway. Hence, we have treated MSCs with a β-adrenergic agonist (Isoproterenol) and a β-adrenergic antagonist (propranolol). Next, on days 7 and 14 post-treatment, we have examined mineralization, osteogenic markers and the expression of genes related to Wnt signaling. Our findings suggested that isoproterenol (β-adrenergic agonist) inhibits the differentiation of mesenchymal stem cells to osteoblasts via stimulation of β2-adrenergic receptor and the subsequent inhibition of Wnt β-catenin signaling by lowering the expression of β-catenin. Nevertheless, the SOST expression did not exhibit a meaningful pattern to support that Isoproterenol inhibits the Wnt signaling pathway by increasing sclerostin expression.

Collagen VI ablation in zebrafish causes neuromuscular defects during developmental and adult stages

Collagen VI (COL6) is an extracellular matrix protein exerting multiple functions in different tissues. In humans, mutations of COL6 genes cause rare inherited congenital disorders, primarily affecting skeletal muscles and collectively known as COL6-related myopathies, for which no cure is available yet. In order to get insights into the pathogenic mechanisms underlying COL6-related diseases, diverse animal models were produced. However, the roles exerted by COL6 during embryogenesis remain largely unknown. Here, we generated the first zebrafish COL6 knockout line through CRISPR/Cas9 site-specific mutagenesis of the col6a1 gene. Phenotypic characterization during embryonic and larval development revealed that lack of COL6 leads to neuromuscular defects and motor dysfunctions, together with distinctive alterations in the three-dimensional architecture of craniofacial cartilages. These phenotypic features were maintained in adult col6a1 null fish, which displayed defective muscle organization and impaired swimming capabilities. Moreover, col6a1 null fish showed autophagy defects and organelle abnormalities at both embryonic and adult stages, thus recapitulating the main features of patients affected by COL6-related myopathies. Mechanistically, lack of COL6 led to increased BMP signaling, and direct inhibition of BMP activity ameliorated the locomotor activity of col6a1 null embryos. Finally, treatment with salbutamol, a β2-adrenergic receptor agonist, elicited a significant amelioration of the neuromuscular and motility defects of col6a1 null fish embryos. Altogether, these findings indicate that this newly generated zebrafish col6a1 null line is a valuable in vivo tool to model COL6-related myopathies and suitable for drug screenings aimed at addressing the quest for effective therapeutic strategies for these disorders.

Mechanisms of β-adrenergic receptors agonists in mediating pro and anti-apoptotic pathways in hyperglycemic Müller cells.

The current study aimed to investigate the stimulatory effect of beta-adrenergic receptors (β-ARs) on brain derived neurotropic factor (BDNF) and cAMP response element binding protein (CREB). Human Müller cells were cultured in low and high glucose conditions. Cells were treated with xamoterol (selective agonist for β1-AR), salmeterol (selective agonist for β2-AR), isoproterenol (β-ARs agonist) and propranolol (β-ARs antagonist), at 20µM concentration for 24h. Western Blotting assay was performed for the gene expression analysis. DNA damage was evaluated by TUNEL assay. DCFH-DA assay was used tocheck the level of reactive oxygen species (ROS). Cytochrome C release was measured by ELISA. Xamoterol, salmeterol and isoproterenol showed no effect on Caspase-8 but it reduced the apoptosis and increased the expression of BDNF in Müller cells. A significant change in the expression of caspase-3 was observed in cells treated with xamoterol and salmeterol as compared to isoproterenol. Xamoterol, salmeterol and isoproterenol significantly decreased the reactive oxygen species (ROS) when treated for 24 hours. Glucose-induced cytochrome c release was disrupted in Müller cells. β-ARs, stimulated by agonist play a protective role in hyperglycemic Müller cells, with the suppression of glucose-induced caspase-3 and cytochrome c release. B-Ars may directly mediate the gene expression of BDNF.

Simultaneous determination of eight β-adrenergic agonists in human urine by an isotope dilution-online clean-up system coupled with liquid chromatography-tandem mass spectrometry.

β-Adrenergic agonist compounds are medicines that open up the lung's medium and large airways. β-Adrenergic agonist compounds have been illegally or legally used to increase lean muscle mass in meat animals, bodybuilding, weight-loss programs, and athletes. Developing a rapid analytical approach for determining β-adrenergic agonist compounds in biological samples is crucial for individual exposure assessment. This study established an analytical method for simultaneously measuring eight β-adrenergic agonist compounds in human urine, including clenbuterol, terbutaline, salbutamol, ractopamine, zilpaterol, cimaterol, tulobuterol, and fenoterol. Two hundred microliters of a urine sample were added to eight deuterium-labeled internal standard mixtures and glucuronidase/arylsulfatase for enzymatic hydrolysis, and were then analyzed using an online clean-up system coupled with a liquid chromatography-tandem mass spectrometry system (LC-MS/MS). The limit of quantification ranged from 0.03 to 0.12ng/mL urine for the eight β-adrenergic agonist compounds. The relative standard deviations (RSD) of the within-run and between-run precisions were less than 10%, and the relative accuracy errors were less than 17% in the three-level spiked artificial urine samples. Two hundred eighty human urine samples collected from the general population in Taiwan were assessed to demonstrate the capability and feasibility of this method. The detection frequencies were 33% for clenbuterol, 5% for ractopamine, and less than 5% for the others. We concluded that the isotope dilution-online clean-up system coupled with LC-MS/MS method is a valuable analytical method for investigating urinary β-adrenergic agonist compounds in humans and is valuable for human biomonitoring studies.

Biochar-supported Cu nanocluster as an electrochemical ultrasensitive interface for ractopamine sensing.

Electrochemical sensors actually involve an electrocatalytic process involving an efficient and selective energy conversion that is related to the morphology and size of the interface of the modified materials. Ultrasmall nanoclusters or single atoms generate a greater catalytic ability than normal nanomaterials. In this study biochar-supported Cu nanoclusters (CuNCs@CNFs) were fabricated via a carbon confinement synthesis method toward ultrasensitive electrochemical sensing of ractopamine (RAC). RAC is a β-adrenergic receptor agonist that is illegally used as a feed additive to significantly improve muscle accretion, resulting in RAC accumulation in meat-based food products. The unique structure of CuNCs@CNFs and the interconnectivity between the CuNCs and the CNFs enable the nanocomposite to significantly enhance conductivity and electrocatalytic activity. Using the CuNCs@CNFs-based sensor, RAC was determined with a high sensitivity of 1641 μA μM-1 cm-2. The feasibility of detecting RAC in spiked meat samples was also carried out with satisfactory recoveries ranging from 91.39% to 94.58%.

Budget Impact Analysis of Vibegron for the Treatment of Overactive Bladder in the USA.

BackgroundOveractive bladder (OAB) is associated with considerable clinical and economic burden. Treatment of patients with OAB using anticholinergics is limited by tolerability issues and increased anticholinergic burden, which is associated with increased risk of dementia and falls/fractures. This analysis assessed the budget impact of introducing the β3-adrenergic agonist vibegron for the treatment of patients with OAB from US commercial payor and Medicare perspectives.MethodsA budget impact model (BIM) with a 5-year time horizon was developed using a top-down, prevalence-based approach and projected market shares for 1-million-member US commercial and Medicare plans. The BIM included vibegron, mirabegron, and anticholinergics, incorporating changes in clinical outcomes (efficacy, drug–drug interactions, anticholinergic burden (ACB), OAB-related comorbidities, and adverse events (AEs)). Costs per member per month (PMPM) and per treated member per month (PTMPM) were determined. One-way sensitivity analyses quantified the impact of changes in key variables.ResultsThe introduction of vibegron was associated with a modest increase in PMPM cost over 5 years of $0.12 (range for years 1‒5, $0.01‒$0.26) for commercial payors and $0.24 ($0.01‒$0.52) for Medicare (PTMPM cost: $2.70 ($0.17‒$4.85) and $3.15 ($0.19‒$5.82), respectively). Costs were partially offset by savings related to decreased third-line treatment use, yearly decreases in AE and comorbidity incidence, reduced drug–drug interactions, and reduced ACB associated with vibegron introduction. PMPM costs were most sensitive to vibegron market share assumptions, OAB prevalence, and vibegron persistence at 1 month for private payors and Medicare and additionally vibegron persistence at 12 months for Medicare.ConclusionsVibegron may address unmet needs in treating OAB and is a useful addition to health plans while minimizing risks of anticholinergic AEs, ACB, and drug–drug interactions, which may partially offset increased pharmacy costs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40273-022-01163-5.

Supplemental treatment to atropine improves the efficacy to reverse nerve agent induced bronchoconstriction

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction.PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the β2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect.In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.

Impact of R-Carvedilol on β2-Adrenergic Receptor-Mediated Spontaneous Calcium Release in Human Atrial Myocytes

A hallmark of atrial fibrillation is an excess of spontaneous calcium release events, which can be mimicked by β1- or β2-adrenergic stimulation. Because β1-adrenergic receptor blockers (β1-blockers) are primarily used in clinical practice, we here examined the impact of β2-adrenergic stimulation on spontaneous calcium release and assessed whether the R- and S-enantiomers of the non-selective β- blocker carvedilol could reverse these effects. For this purpose, human atrial myocytes were isolated from patients undergoing cardiovascular surgery and subjected to confocal calcium imaging or immunofluorescent labeling of the ryanodine receptor (RyR2). Interestingly, the β2-adrenergic agonist fenoterol increased the incidence of calcium sparks and waves to levels observed with the non-specific β-adrenergic agonist isoproterenol. Moreover, fenoterol increased both the amplitude and duration of the sparks, facilitating their fusion into calcium waves. Subsequent application of the non β-blocking R-Carvedilol enantiomer reversed these effects of fenoterol in a dose-dependent manner. R-Carvedilol also reversed the fenoterol-induced phosphorylation of the RyR2 at Ser-2808 dose-dependently, and 1 µM of either R- or S-Carvedilol fully reversed the effect of fenoterol. Together, these findings demonstrate that β2-adrenergic stimulation alone stimulates RyR2 phosphorylation at Ser-2808 and spontaneous calcium release maximally, and points to carvedilol as a tool to attenuate the pathological activation of β2-receptors.

Paper-Based Fluidic Sensing Platforms for β-Adrenergic Agonist Residue Point-of-Care Testing

The illegal use of β-adrenergic agonists during livestock growth poses a threat to public health; the long-term intake of this medication can cause serious physiological side effects and even death. Therefore, rapid detection methods for β-adrenergic agonist residues on-site are required. Traditional detection methods such as liquid chromatography have limitations in terms of expensive instruments and complex operations. In contrast, paper methods are low cost, ubiquitous, and portable, which has led to them becoming the preferred detection method in recent years. Various paper-based fluidic devices have been developed to detect β-adrenergic agonist residues, including lateral flow immunoassays (LFAs) and microfluidic paper-based analytical devices (μPADs). In this review, the application of LFAs for the detection of β-agonists is summarized comprehensively, focusing on the latest advances in novel labeling and detection strategies. The use of μPADs as an analytical platform has attracted interest over the past decade due to their unique advantages and application for detecting β-adrenergic agonists, which are introduced here. Vertical flow immunoassays are also discussed for their shorter assay time and stronger multiplexing capabilities compared with LFAs. Furthermore, the development direction and prospects for the commercialization of paper-based devices are considered, shedding light on the development of point-of-care testing devices for β-adrenergic agonist residue detection.

Agonists in the Extended Conformation Stabilize the Active State of β-Adrenoceptors

In this study, we conducted a comparative analysis of the structure of agonists and antagonists of transmembrane (TM) β-adrenoceptors (β-ARs) and their interactions with the β-ARs and proposed the mechanism of receptor activation. A characteristic feature of agonist and antagonist molecules is the presence of a hydrophobic head (most often, one or two aromatic rings) and a tail with a positively charged amino group. All β-adrenergic agonists have two carbon atoms between the aromatic ring of the head and the nitrogen atom of the amino group. In antagonist molecules, this fragment can be either reduced or increased to four atoms due to the additional carbon and oxygen atoms. The agonist head, as a rule, has two H-bond donors or acceptors in the para- and meta-positions of the aromatic rings, while in the antagonist heads, these donors/acceptors are absent or located in other positions. Analysis of known three-dimensional structures of β-AR complexes with agonists showed that the agonist head forms two H-bonds with the TM5 helix, and the tail forms an ionic bond with the D3.32 residue of the TM3 helix and one or two H-bonds with the TM7 helix. The tail of the antagonist can form similar bonds, but the interaction between the head and the TM5 helix is much weaker. As a result of these interactions, the agonist molecule acquires an extended “strained string” conformation, in contrast to the antagonist molecule, which has a longer, bended, and flexible tail. The “strained string” of the agonist interacts with the TM6 helix (primarily with the W6.48 residue) and turns it, which leads to the opening of the G protein-binding site on the intracellular side of the receptor, while flexible and larger antagonist molecules do not have the same effect on the receptor.

Kidney targeting of formoterol containing polymeric nanoparticles improves recovery from ischemia reperfusion-induced acute kidney injury in mice

The β2 adrenergic receptor agonist, formoterol, is an inducer of mitochondrial biogenesis and restorer of mitochondrial and kidney function in acute and chronic models of kidney injury. Unfortunately, systemic administration of formoterol has the potential for adverse cardiovascular effects, increased heart rate, and decreased blood pressure. To minimize these effects, we developed biodegradable and biocompatible polymeric nanoparticles containing formoterol that target the kidney, thereby decreasing the effective dose, and lessen cardiovascular effects while restoring kidney function after injury. Male C57Bl/6 mice, treated with these nanoparticles daily, had reduced ischemia-reperfusion-induced serum creatinine and kidney cortex kidney injury molecule-1 levels by 78% and 73% respectively, compared to control mice six days after injury. With nanoparticle therapy, kidney cortical mitochondrial number and proteins reduced by ischemic injury, recovered to levels of sham-operated mice. Tubular necrosis was reduced 69% with nanoparticles treatment. Nanoparticles improved kidney recovery even when the dosing frequency was reduced from daily to two days per week. Finally, compared to treatment with formoterol-free drug alone, these nanoparticles did not increase heart rate nor decrease blood pressure. Thus, targeted kidney delivery of formoterol-containing nanoparticles is an improvement in standard formoterol therapy for ischemia-reperfusion-induced acute kidney injuries by decreasing the dose, dosing frequency, and cardiac side effects.

P-800 Plerixafor for endogenous mobilisation of mesenchymal stem cells to induce endometrial regeneration

Abstract Study question Will the treatment with plerixafor alone or associated with β3 agonist cause endometrial regeneration and consequently improve the reproductive outcome of mice with thin endometrium? Summary answer The combined treatment with plerixafor and β3 agonist improved the reproductive outcomes, indicating a potential therapeutic option for infertility related to thin endometrium. What is known already Thin endometrium represents a huge challenge in assisted reproduction, since it is related to decreased endometrial receptivity and consequent implantation failure. The treatments with mesenchymal stem cells (MSC) have regenerative properties that improve endometrial thickness and expression of receptivity biomarkers. Plerixafor is a CXCR4 antagonist receptor that disrupts the CXCL12-CXCR4 axis, which is directly related to stem cell migration, and has been used in tissue regeneration studies to increase the influx of stem cells to the site of injury. In addition, its combined use with β3 adrenergic agonist receptors leads to selective recruitment of MSC with better regenerative results. Study design, size, duration Experiments were carried out on female C57Bl/6 mice (age 9 weeks; 20g). Forty-eight individuals were assigned for either thin endometrium induction or sham procedure; then, mice in each group were randomly divided into three treatment subgroups (n = 8): (1) Placebo - phosphate-buffered saline, (2) plerixafor, (3) plerixafor and BRL37344 (rodent-selective β3 adrenergic receptor agonist). Two estrous cycles after modelling and treatment, all groups were mated with proven C57Bl/6 male mice for four weeks. Participants/materials, setting, methods Thin endometrium was induced by direct injection (50 μl) of 95% ethanol in the surgically exposed uterus, while Phosphate-buffered saline (PBS) was used in the sham procedure. Regarding the treatments, the groups received: (1) PBS (5 mg/kg sc) single-dose after surgery, (2) plerixafor (5 mg/kg sc) single-dose after surgery, (3) BRL37344 (rodent-selective β3 adrenergic receptor agonist) (10 mg/kg sc) for four consecutive days previous to surgery plus plerixafor (5 mg/kg sc) single-dose. Main results and the role of chance The pregnancy rate and litter size were analysed in order to establish the impact on the reproductive outcomes. In addition, a macroscopic anatomic analysis was performed on the offspring to evaluate possible teratogenic effects. Concerning pregnancy rates, 23 out of 24 sham mice got pregnant (despite the treatment group). The thin endometrium model reproduced the literature results, with under 30% of failure in inducing endometrial damage, represented by a pregnancy rate of 25% (2/8) in the PBS group in comparison to all sham groups (p < 0,05). The plerixafor plus β3 agonist treatment completely restored the endometrial function, promoting pregnancy rates similar to all sham groups (p > 0,05). There was no difference between the plerixafor alone group and all the other groups (p > 0.05) in terms of pregnancy, although a clinically relevant difference between placebo and plerixafor alone group was found (25% versus 62,5%, respectively). The litter size was similar between the treatment groups, except for the plerixafor group with a smaller number of newborns. Regarding the teratogenic potential of the tested drugs (plerixafor and β3 agonist), we did not observe any relevant macroscopic malformations (internal and external) in the treated groups. Limitations, reasons for caution There is a need to establish the biological mechanisms responsible for endometrial recovery. Moreover, this study provided treatment for an acute injury; therefore, future work should evaluate the application on long term endometrial damage. The long term effect of the single-dose treatment should also be assessed. Wider implications of the findings Our findings indicate a potential therapy for thin endometrium. Plerixafor and β3 agonist treatment resulted in an expressive improvement in reproductive outcomes, without a detrimental impact on offspring. Moreover, this treatment utilises two clinical approved classes of drugs, which could reduce the timeline and cost of translational applications in future. Trial registration number not applicable

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.

Antagonism of α1-adrenoceptors by β3-adrenergic agonists: Structure–function relations of different agonists in prostate smooth muscle contraction

Effects of β3-adrenergic agonists on prostate smooth muscle contraction are poorly characterized, although mirabegron is used for treatment of lower urinary tract symptoms. Off-target effects of several β3-adrenergic agonists include antagonism of α1-adrenoceptors. Proposed, but unconfirmed explanations include phenylethanolamine backbones, found in some β3-adrenergic agonists and imparting interaction with catecholamine binding pockets of adrenoceptors. Here, we examined effects of β3-adrenergic agonists on contractions of human prostate tissues, including ZD7114 (without phenylethanolamine moiety), ZD2079 (phenylethanolamine backbone), BRL37344 and CL316243 (chloride-substituted phenylethanolamine deriatives). Prostate tissues were obtained from radical prostatectomy. Contractions by α1-adrenergic agonists and electric field stimulation (EFS) were studied in an organ bath. ZD7114 (10 µM) right-shifted concentration responses curves for α1-adrenergic agonists, resulting in increased EC50 values for phenylephrine, methoxamine and noradrenaline up to one magnitude, without affecting Emax values. ZD7114 (10 µM) inhibited EFS-induced contractions, resulting in reduced Emax values. All effects of ZD7114 were resistant to the β3-adrenergic antagonist L-748337, including increases in EC50 values for α1-adrenergic agonists, up to more than two magnitudes. Using 10 µM, neither ZD2079, BRL37344 or CL316243 affected α1-adrenergic or EFS-induced contractions. At escalated concentrations, BRL37344 (200 µM) right-shifted concentration response curves for phenylephrine, increased EC50 values for phenylephrine, and inhibited EFS-induced contractions, while CL316243 (300 µM) did not affect phenylephrine- or EFS-induced contractions. In conclusion, phenylethanolamine backbones are not decisive to impart α1-adrenoceptor antagonism to β3-agonists. Effects of β3-adrenergic agonists on prostate smooth muscle contraction are limited to off-target effects, including α1-adrenoceptor antagonism by ZD7114 and BRL37344.

Cytokine-Mediated STAT3 Transcription Supports ATGL/CGI-58-Dependent Adipocyte Lipolysis in Cancer Cachexia

Adipose tissue inflammation is observed in multiple metabolically-altered states including cancer-associated cachexia and obesity. Although cachexia is a syndrome of adipose loss and obesity is a disease of adipose excess, both pathologies demonstrate increases in circulating levels of IL-6 family cytokines, β-adrenergic signaling, and adipocyte lipolysis. While β-adrenergic-stimulated adipocyte lipolysis is well described, there is limited mechanistic insight into how cancer cachexia-associated inflammatory cytokines contribute to adipocyte lipolysis under pathologic conditions. Here, we set out to compare adipocyte lipolysis signaling by cancer cachexia-associated IL-6 family cytokines (IL-6 and LIF) to that of the β-adrenergic agonist isoproterenol. Unlike isoproterenol, the IL-6 family of cytokines required JAK/STAT3-dependent transcriptional changes to induce adipocyte lipolysis. Furthermore, cachexia-associated cytokines that used STAT3 to induce lipolysis were primarily dependent on the lipase ATGL and its cofactor CGI-58 rather than lipases HSL and MAGL. Finally, administration of JAK but not β-adrenergic inhibitors suppressed adipose STAT3 phosphorylation and associated adipose wasting in a murine model of cancer cachexia characterized by increased systemic IL-6 family cytokine levels. Combined, our results demonstrate how the IL-6 family of cytokines diverge from β-adrenergic signals by employing JAK/STAT3-driven transcriptional changes to promote adipocyte ATGL/CGI-58-dependent lipolysis contributing to adipose wasting in cancer cachexia.