Structural Insights into the Dynamic Process of ß2-Adrenergic Receptor Signaling.

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Control of lipolysis by a population of oxytocinergic sympathetic neurons.

Oxytocin (OXT), a nine-amino-acid peptide produced in the hypothalamus and released by the posterior pituitary, has well-known actions in parturition, lactation and social behaviour1, and has become an intriguing therapeutic target for conditions such as autism and schizophrenia2. Exogenous OXT has also been shown to have effects on body weight, lipid levels and glucose homeostasis1,3, suggesting that it may also have therapeutic potential for metabolic disease1,4. It is unclear, however, whether endogenous OXT participates in metabolic homeostasis. Here we show that OXT is a critical regulator of adipose tissue lipolysis in both mice and humans. In addition, OXT serves to facilitate the ability of ß-adrenergic agonists to fully promote lipolysis. Most surprisingly, the relevant source of OXT in these metabolic actions is a previously unidentified subpopulation of tyrosine hydroxylase-positive sympathetic neurons. Our data reveal that OXT from the peripheral nervous system is an endogenous regulator of adipose and systemic metabolism.

Modulation of fast sodium current in airway smooth muscle cells by exchange protein directly activated by cAMP.

Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by NaV1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, INa, was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a ß-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca2+] was buffered by inclusion of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of INa was not due to Ca2+-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation V1/2, the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates INa in mouse ASM via Epac, but not PKA.NEW & NOTEWORTHY ß-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.

ß-Adrenergic signaling drives structural and functional maturation of mouse cardiomyocytes.

Cardiac maturation represents the last phase of heart development and is characterized by morphofunctional alterations that optimize the heart for efficient pumping. Its understanding provides important insights into cardiac regeneration therapies. Recent evidence implies that adrenergic signals are involved in the regulation of cardiac maturation, but the mechanistic underpinnings involved in this process are poorly understood. Herein, we explored the role of ß-adrenergic receptor (ß-AR) activation in determining structural and functional components of cardiomyocyte maturation. Temporal characterization of tyrosine hydroxylase and norepinephrine levels in the mouse heart revealed that sympathetic innervation develops during the first 3 wk of life, concurrent with the rise in ß-AR expression. To assess the impact of adrenergic inhibition on maturation, we treated mice with propranolol, isolated cardiomyocytes, and evaluated morphofunctional parameters. Propranolol treatment reduced heart weight, cardiomyocyte size, and cellular shortening, while it increased the pool of mononucleated myocytes, resulting in impaired maturation. No changes in t-tubules were observed in cells from propranolol mice. To establish a causal link between ß-AR signaling and cardiomyocyte maturation, mice were subjected to sympathectomy, followed or not by restoration with isoproterenol treatment. Cardiomyocytes from sympathectomyzed mice recapitulated the salient immaturity features of propranolol-treated mice, with the additional loss of t-tubules. Isoproterenol rescued the maturation deficits induced by sympathectomy, except for the t-tubule alterations. Our study identifies the ß-AR stimuli as a maturation promoting signal and implies that this pathway can be modulated to improve cardiac regeneration therapies.NEW & NOTEWORTHY Maturation involves a series of morphofunctional alterations vital to heart development. Its regulatory mechanisms are only now being unveiled. Evidence implies that adrenergic signaling regulates cardiac maturation, but the mechanisms are poorly understood. To address this point, we blocked ß-ARs or performed sympathectomy followed by rescue experiments with isoproterenol in neonatal mice. Our study identifies the ß-AR stimuli as a maturation signal for cardiomyocytes and highlights the importance of this pathway in cardiac regeneration therapies.

The mechanism of 25-hydroxycholesterol-mediated suppression of atrial ß1-adrenergic responses.

25-Hydroxycholesterol (25HC) is a biologically active oxysterol, whose production greatly increases during inflammation by macrophages and dendritic cells. The inflammatory reactions are frequently accompanied by changes in heart regulation, such as blunting of the cardiac ß-adrenergic receptor (AR) signaling. Here, the mechanism of 25HC-dependent modulation of responses to ß-AR activation was studied in the atria of mice. 25HC at the submicromolar levels decreased the ß-AR-mediated positive inotropic effect and enhancement of the Ca2+ transient amplitude, without changing NO production. Positive inotropic responses to ß1-AR (but not ß2-AR) activation were markedly attenuated by 25HC. The depressant action of 25HC on the ß1-AR-mediated responses was prevented by selective ß3-AR antagonists as well as inhibitors of Gi protein, Gßγ, G protein-coupled receptor kinase 2/3, or ß-arrestin. Simultaneously, blockers of protein kinase D and C as well as a phosphodiesterase inhibitor did not preclude the negative action of 25HC on the inotropic response to ß-AR activation. Thus, 25HC can suppress the ß1-AR-dependent effects via engaging ß3-AR, Gi protein, Gßγ, G protein-coupled receptor kinase, and ß-arrestin. This 25HC-dependent mechanism can contribute to the inflammatory-related alterations in the atrial ß-adrenergic signaling.

Milrinone as Compared with Dobutamine in the Treatment of Cardiogenic Shock.

BACKGROUND: Cardiogenic shock is associated with substantial morbidity and mortality. Although inotropic support is a mainstay of medical therapy for cardiogenic shock, little evidence exists to guide the selection of inotropic agents in clinical practice. METHODS: We randomly assigned patients with cardiogenic shock to receive milrinone or dobutamine in a double-blind fashion. The primary outcome was a composite of in-hospital death from any cause, resuscitated cardiac arrest, receipt of a cardiac transplant or mechanical circulatory support, nonfatal myocardial infarction, transient ischemic attack or stroke diagnosed by a neurologist, or initiation of renal replacement therapy. Secondary outcomes included the individual components of the primary composite outcome. RESULTS: A total of 192 participants (96 in each group) were enrolled. The treatment groups did not differ significantly with respect to the primary outcome; a primary outcome event occurred in 47 participants (49%) in the milrinone group and in 52 participants (54%) in the dobutamine group (relative risk, 0.90; 95% confidence interval [CI], 0.69 to 1.19; P = 0.47). There were also no significant differences between the groups with respect to secondary outcomes, including in-hospital death (37% and 43% of the participants, respectively; relative risk, 0.85; 95% CI, 0.60 to 1.21), resuscitated cardiac arrest (7% and 9%; hazard ratio, 0.78; 95% CI, 0.29 to 2.07), receipt of mechanical circulatory support (12% and 15%; hazard ratio, 0.78; 95% CI, 0.36 to 1.71), or initiation of renal replacement therapy (22% and 17%; hazard ratio, 1.39; 95% CI, 0.73 to 2.67). CONCLUSIONS: In patients with cardiogenic shock, no significant difference between milrinone and dobutamine was found with respect to the primary composite outcome or important secondary outcomes. (Funded by the Innovation Fund of the Alternative Funding Plan for the Academic Health Sciences Centres of Ontario; ClinicalTrials.gov number, NCT03207165.).

Olodaterol promotes thermogenesis in brown adipocytes via regulation of the ß2-AR/cAMP/PKA signaling pathway.

The escalating incidence of metabolic pathologies such as obesity and diabetes mellitus underscores the imperative for innovative therapeutics targeting lipid metabolism modulation. Within this context, augmenting thermogenic processes in adipose cells emerges as a viable therapeutic approach. Given the limitations of previous ß3-adrenergic receptor (ß3-AR) agonist treatments in human diseases, there is an increasing focus on therapies targeting the ß2-adrenergic receptor (ß2-AR). Olodaterol (OLO) is a potent ß2-AR agonist that is a potential novel pharmacological candidate in this area. Our study explores the role and underlying mechanisms of OLO in enhancing brown adipose thermogenesis, providing robust evidence from in vitro and in vivo studies. OLO demonstrated a dose-dependent enhancement of lipolysis, notably increasing the expression of Uncoupling Protein 1 (UCP1) and raising the rate of oxygen consumption in primary brown adipocytes. This suggests a significant increase in thermogenic potential and energy expenditure. The administration of OLO to murine models noticeably enhanced cold-induced nonshivering thermogenesis. OLO elevated UCP1 expression in the brown adipose tissue of mice. Furthermore, it promoted brown adipocyte thermogenesis by activating the ß2-AR/cAMP/PKA signaling cascades according to RNA sequencing, western blotting, and molecular docking analysis. This investigation underscores the therapeutic potential of OLO for metabolic ailments and sheds light on the intricate molecular dynamics of adipocyte thermogenesis, laying the groundwork for future targeted therapeutic interventions in human metabolic disorders.

The ß-Blocker Carvedilol and Related Aryloxypropanolamines Promote ERK1/2 Phosphorylation in HEK293 Cells with K A Values Distinct From Their Equilibrium Dissociation Constants as ß 2-Adrenoceptor Antagonists: Evidence for Functional Affinity.

The determination of affinity by using functional assays is important in drug discovery because it provides a more relevant estimate of the strength of interaction of a ligand to its cognate receptor than radioligand binding. However, empirical evidence for so-called, "functional affinity" is limited. Herein, we determined whether the affinity of carvedilol, a ß-adrenoceptor antagonist used to treat heart failure that also promotes extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, differed between these two pharmacological activities. Four structurally related ß-adrenoceptor antagonists (alprenolol, carazolol, pindolol, propranolol) that also activated ERK1/2 were included as comparators to enhance our understanding of how these drugs work in the clinical setting. In HEK293 cells stably expressing the human ß 2-adrenoceptor carvedilol and related aryloxypropanolamines were partial agonists of ERK1/2 phosphorylation with potencies ([A]50s) that were lower than their equilibrium dissociation constants (K Bs) as ß 2-adrenoceptor antagonists. As the [A]50 of a partial agonist is a good approximation of its K B, then these data indicated that the affinities of carvedilol and related ligands for these two activities were distinct. Moreover, there was a significant negative rank order correlation between the [A]50 of each ligand to activate ERK1/2 and their intrinsic activities (i.e., as intrinsic activity for ERK1/2 phosphorylation increased, so did affinity). Genome editing revealed that the transducer that coupled the ß 2-adrenoceptor to ERK1/2 phosphorylation in response to carvedilol and other ß 2-adrenoceptor antagonists was Gαs. Collectively, these data support the concept of "functional affinity" and indicate that the ability of the ß 2-adrenoceptor to recruit Gαs may influence the affinity of the activating ligand. SIGNIFICANCE STATEMENT: In HEK293 cells overexpressing the human ß2-adrenoceptor carvedilol and four related aryloxypropanolamines behaved as ß2-adrenoceptor antagonists and partial agonists of ERK1/2 phosphorylation with rank orders of affinity that were distinct. These data imply that carvedilol and other ß-blockers can stabilize the ß2-adrenoceptor in different affinity conformations that are revealed when functionally distinct responses are measured. This is the basis for the pharmacological concept of "functional affinity."

The renal vasodilatation from ß-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels.

The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.

Development of novel ß2-adrenergic receptor agonists for the stimulation of glucose uptake - The importance of chirality and ring size of cyclic amines.

ß2-Adrenergic receptor (ß2AR) agonists have been reported to stimulate glucose uptake (GU) by skeletal muscle cells and are therefore highly interesting as a possible treatment for type 2 diabetes (T2D). The chirality of compounds often has a great impact on the activity of ß2AR agonists, although this has thus far not been investigated for GU. Here we report the GU for a selection of synthesized acyclic and cyclic ß-hydroxy-3-fluorophenethylamines. For the N-butyl and the N-(2-pentyl) compounds, the (R) and (R,R) (3d and 7e) stereoisomers induced the highest GU. When the compounds contained a saturated nitrogen containing 4- to 7-membered heterocycle, the (R,R,R) enantiomer of the azetidine (8a) and the pyrrolidine (9a) had the highest activity. Altogether, these results provide pivotal information for designing novel ß2AR agonist for the treatment of T2D.

Pharmacological treatment of asthma in Sweden from 2005 to 2015.

OBJECTIVE: Despite access to effective therapies many asthma patients still do not have well-controlled disease. This is possibly related to underuse of inhaled corticosteroids (ICS) and overuse of short-acting ß2-agonists (SABA). Our aim was to investigate longitudinal trends and associated factors in asthma treatment. METHODS: Two separate cohorts of adults with physician-diagnosed asthma were randomly selected from 14 hospitals and 56 primary health centers in Sweden in 2005 (n = 1182) and 2015 (n = 1225). Information about symptoms, maintenance treatment, and use of rescue medication was collected by questionnaires. Associations between treatment and sex, age, smoking, education, body mass index (BMI), physical activity, allergic asthma, and symptom control were analyzed using Pearson's chi2-test. Odds ratios (ORs) were calculated using logistic regression. RESULTS: Maintenance treatment with ICS together with long-acting ß2-agonists (LABA) and/or montelukast increased from 39.2% to 44.2% (p = 0.012). The use of ICS + LABA as-needed increased (11.1-18.9%, p < 0.001), while SABA use decreased (46.4- 41.8%, p = 0.023). Regular treatment with ICS did not change notably (54.2-57.2%, p = 0.14). Older age, former smoking, and poor symptom control were related to treatment with ICS + LABA/montelukast. In 2015, 22.7% reported daily use of SABA. A higher step of maintenance treatment, older age, obesity, shorter education, current smoking, allergic asthma, low or very high physical activity, and a history of exacerbations were associated with daily SABA use. CONCLUSIONS: The use of ICS + LABA both for maintenance treatment and symptom relief has increased over time. Despite this, the problem of low use of ICS and high use of SABA remains.

Inhaled ß2 Adrenergic Agonists and Other cAMP-Elevating Agents: Therapeutics for Alveolar Injury and Acute Respiratory Disease Syndrome?

Inhaled long-acting ß-adrenergic agonists (LABAs) and short-acting ß-adrenergic agonists are approved for the treatment of obstructive lung disease via actions mediated by ß2 adrenergic receptors (ß2-ARs) that increase cellular cAMP synthesis. This review discusses the potential of ß2-AR agonists, in particular LABAs, for the treatment of acute respiratory distress syndrome (ARDS). We emphasize ARDS induced by pneumonia and focus on the pathobiology of ARDS and actions of LABAs and cAMP on pulmonary and immune cell types. ß2-AR agonists/cAMP have beneficial actions that include protection of epithelial and endothelial cells from injury, restoration of alveolar fluid clearance, and reduction of fibrotic remodeling. ß2-AR agonists/cAMP also exert anti-inflammatory effects on the immune system by actions on several types of immune cells. Early administration is likely critical for optimizing efficacy of LABAs or other cAMP-elevating agents, such as agonists of other Gs-coupled G protein-coupled receptors or cyclic nucleotide phosphodiesterase inhibitors. Clinical studies that target lung injury early, prior to development of ARDS, are thus needed to further assess the use of inhaled LABAs, perhaps combined with inhaled corticosteroids and/or long-acting muscarinic cholinergic antagonists. Such agents may provide a multipronged, repurposing, and efficacious therapeutic approach while minimizing systemic toxicity. SIGNIFICANCE STATEMENT: Acute respiratory distress syndrome (ARDS) after pulmonary alveolar injury (e.g., certain viral infections) is associated with ∼40% mortality and in need of new therapeutic approaches. This review summarizes the pathobiology of ARDS, focusing on contributions of pulmonary and immune cell types and potentially beneficial actions of ß2 adrenergic receptors and cAMP. Early administration of inhaled ß2 adrenergic agonists and perhaps other cAMP-elevating agents after alveolar injury may be a prophylactic approach to prevent development of ARDS.

Cardiac Protein Kinase D1 ablation alters the myocytes ß-adrenergic response.

ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.

The Isoleucine at Position 118 in Transmembrane 2 Is Responsible for the Selectivity of Xamoterol, Nebivolol, and ICI89406 for the Human ß1-Adrenoceptor.

Known off-target interactions frequently cause predictable drug side-effects (e.g., ß1-antagonists used for heart disease, risk ß2-mediated bronchospasm). Computer-aided drug design would improve if the structural basis of existing drug selectivity was understood. A mutagenesis approach determined the ligand-amino acid interactions required for ß1-selective affinity of xamoterol and nebivolol, followed by computer-based modeling to provide possible structural explanations. 3H-CGP12177 whole cell binding was conducted in Chinese hamster ovary cells stably expressing human ß1, ß2, and chimeric ß1/ß2-adrenoceptors (ARs). Single point mutations were investigated in transiently transfected cells. Modeling studies involved docking ligands into three-dimensional receptor structures and performing molecular dynamics simulations, comparing interaction frequencies between apo and holo structures of ß1 and ß2-ARs. From these observations, an ICI89406 derivative was investigated that gave further insights into selectivity. Stable cell line studies determined that transmembrane 2 was crucial for the ß1-selective affinity of xamoterol and nebivolol. Single point mutations determined that the ß1-AR isoleucine (I118) rather than the ß2 histidine (H93) explained selectivity. Studies of other ß1-ligands found I118 was important for ICI89406 selective affinity but not that for betaxolol, bisoprolol, or esmolol. Modeling studies suggested that the interaction energies and solvation of ß1-I118 and ß2-H93 are factors determining selectivity of xamoterol and ICI89406. ICI89406 without its phenyl group loses its high ß1-AR affinity, resulting in the same affinity as for the ß2-AR. The human ß1-AR residue I118 is crucial for the ß1-selective affinity of xamoterol, nebivolol, and ICI89406 but not all ß1-selective compounds. SIGNIFICANCE STATEMENT: Some ligands have selective binding affinity for the human ß1 versus the ß2-adrenoceptor; however, the molecular/structural reason for this is not known. The transmembrane 2 residue isoleucine I118 is responsible for the selective ß1-binding of xamoterol, nebivolol, and ICI89406 but does not explain the selective ß1-binding of betaxolol, bisoprolol, or esmolol. Understanding the structural basis of selectivity is important to improve computer-aided ligand design, and targeting I118 in ß1-adrenoceptors is likely to increase ß1-selectivity of drugs.

Development and Characterization of a Highly Selective Turn-On Fluorescent Ligand for ß3-Adrenergic Receptor.

For the precise visualization of GPCR, subtype selectivity of turn-on fluorescent ligands is of major relevance. Although there are many thriving ß-adrenergic receptors (ß-ARs) probes, none of them are selective to the ß3-subtype, which severely limits the development of ß3-AR investigations. Using a polyethylene glycol (PEG) chain to conjugate the Py-5 fluorophore with mirabegron, we present here a highly selective fluorescent ligand, H2, for ß3-AR. It was established by the radioligand and NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) binding experiments that molecule H2 has a substantially higher affinity for ß3-AR than the other two subtypes (1/3, 45-fold; 2/3, 16-fold). More crucially, when molecule H2 was incubated with ß3-AR, the turn-on fluorescent signals could be quickly released. The subsequent investigations, which included cell imaging, tissue imaging, and flow-cytometry analysis, proved that molecule H2 may make it possible to quickly and accurately fluorescently identify ß3-AR at different levels. We offer a prospective fluorescent turn-on ligand with exceptional selectivity for ß3-AR as a result of our combined efforts.

The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands.

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

RATIONALE: Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. OBJECTIVE: The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during ß-adrenergic stimulation, and in heart failure. METHODS AND RESULTS: We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and ß-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVß to α1C I-II loop and eliminated ß-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to ß-adrenergic agonists when reconstituted heterologously with ß2B and Rad or transgenically expressed in cardiomyocytes. CONCLUSIONS: Ca2+ channel activity is dynamically modulated under basal conditions, during ß-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVß binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the ß-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by ß-adrenergic agonists/PKA also requires this rigid linker and ß-binding to α1C.

Intracellular ß1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant ßAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant ßAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. CONCLUSIONS: Functional ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.

Adverse events of clenbuterol among athletes: a systematic review of case reports and case series.

Clenbuterol is a potent beta-2 agonist widely misused by professional athletes and bodybuilders. Information on clenbuterol associated adverse events is present in case reports and case series, though it may not be readily available. This systematic review aimed to critically evaluate the evidence of adverse events associated with clenbuterol among athletes. The search strategy was in accordance with PRISMA guidelines. Databases such as PubMed, Science Direct, Scopus, and Google Scholar were searched from 1990 to October 2021 to find out the relevant case reports and case series. There were 23 included studies. Using a suitable scale, the included studies' methodological quality analysis was evaluated. In total, 24 athletes experienced adverse events. Oral ingestion of clenbuterol was the most preferred route among them. The daily administered dose of clenbuterol was ranging from 20 µg to 30 mg. Major adverse events experienced by athletes were supraventricular tachycardia, atrial fibrillation, hypotension, chest pain, myocardial injury, myocarditis, myocardial ischemia, myocardial infarction, cardiomyopathy, hepatomegaly, hyperglycemia, and death. The cardiac-related complications were the most commonly occurring adverse events. Clenbuterol is notorious to produce life-threatening adverse events including death. Lack of evidence regarding the performance-enhancing effects of clenbuterol combined with its serious toxicities questions the usefulness of this drug in athletes.

Protocatechuic acid reverses myocardial infarction mediated by ß-adrenergic agonist via regulation of Nrf2/HO-1 pathway, inflammatory, apoptotic, and fibrotic events.

Myocardial infarction (MI) is an instant ischemic death of cardiomyocytes that remains a major global cause of mortalities. MI is accompanied by oxidative, inflammatory, apoptotic, and fibrotic insults. Protocatechuic acid (PCA) is a polyphenolic compound with various potent biological activities. In this study, we explored the possible cardioprotective role of PCA against isoproterenol (ISO)-mediated MI. Rats were either injected with ISO (85 mg/kg, subcutaneously) or pretreated with PCA (100 or 200 mg/kg, orally). PCA supplementation markedly normalized ISO-induced disturbed cardiac function markers (creatine kinase-MB, lactate dehydrogenase, and troponin T). Notably, PCA administration exerted remarkable increases in glutathione and its derived enzymes, superoxide dismutase, and catalase, as well as decreases in malondialdehyde and nitric oxide levels in the injured cardiac tissue. The molecular findings validated the augmented cellular antioxidative capacity by PCA via increasing the gene expressions of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1. The cardioprotective efficacy of PCA extended to suppress cardiac inflammation as demonstrated by the decreased levels of tumor necrosis factor-alpha, interleukin-1 beta, and nuclear factor kappa B. Additionally, PCA prevented cardiomyocyte loss and fibrosis by decreasing Bax, caspase-3, transforming growth factor-ß1 and matrix metalloproteinase-9, and enhancing B-cell lymphoma 2 and tissue inhibitors of metalloproteinase-3. The cardiac histological screening further confirmed the PCA's protective action. The obtained data recommend PCA as an alternative therapeutic agent to attenuate the molecular, biochemical, and histological alterations associated with MI development.