Determination of Agonistically Acting Autoantibodies to the Adrenergic Beta-1 Receptor by Cellular Bioassay.

Agonistically acting autoantibodies directed against the adrenergic beta-1 receptor (beta1-AABs) are a pathogenic factor in diseases of the heart and circulatory system such as dilated cardiomyopathy. Here we describe the detection of such functionally active beta1-AABs from serum samples using spontaneously beating neonatal rat cardiomyocytes, which express the fully functional adrenergic beta-1 receptor coupled with the signal transduction pathway that induces chronotropy. With serum samples added (containing beta1-AABs), an increased positive chronotropic effect is caused that can be blocked by the subsequent addition of specific beta-blockers (abolishing this chronotropic response). The return to the basal beat rate of the cells by the addition of a beta-blocker proves the adrenergic beta-1 receptor specificity of the serum sample.

Comprehensive analysis of the atenolol - DNA complex by viscometric, molecular docking and spectroscopic techniques.

This paper discusses multi-spectroscopic and molecular docking analysis of the interaction between atenolol (ATN) and deoxyribose nucleic acid (DNA) using alizarin (ALZ) as a spectroscopic probe. ATN is a ß1 -receptor antagonist belonging to the ß-blocker class of molecules. Experimental findings that were based on different spectroscopic analysis, melting studies, viscometric analysis, 1 H nuclear magnetic resonance and circular dichroism studies revealed the presence of a grove-binding mode. The effect of ionic strength was also studied, and observations suggested that electrostatic interaction also played a minor role during interaction. Molecular docking analysis suggested that the dominant force for the grove-binding phenomenon was hydrogen bonding between the 24-H residue of ATN and O of the 10-G residue, and the 40-H residue of ATN and N of the 17-A base residue. Competitive binding study of the ALZ-DNA complex with ATN showed that, despite an increase in the amount of ATN in the ALZ-DNA complex, the overall absorbance remained unchanged. The decrease in fluorescence in the ALZ-DNA system may be due to new non-fluorescent ATN-DNA-ALZ complex formation.

AMPKα2 deficiency enhanced susceptibility to ventricular arrhythmias in mice by the role of ß-adrenoceptor signaling.

AMP-activated protein kinase-α2 is the main catalytic subunit of the heart, which is mainly located in cardiac myocytes. The effect of AMP-activated protein kinase-α2 on the cardiac electrophysiology is barely studied. From the previous study, it is possible that AMP-activated protein kinase-α2 may have some effect on the electrophysiology of the heart. To prove the hypothesis, we used the AMP-activated protein kinase-α2 knockout (AMPKα2-/-) mice to estimate the electrophysiological characteristics of AMPKα2-/- mice and try to find the mechanism between them. We used AMP-activated protein kinase-α2 gene knockout (AMPKα2-/-) mice and control wild-type mice as the experimental animals. In the experiment, we measured the monophasic action potential duration and test the inducibility to ventricular arrhythmia in isolated mice heart with and without ß-adrenoceptor antagonist metoprolol. Meanwhile, plasma concentration of catecholamine was collected. We found that AMPKα2-/- significantly shortened 90% repolarization of monophasic action potential (MAP) (MAPD90) than wild-type (47.4 ± 2.6 ms vs. 55.5 ± 2.4 ms, n = 10, P < 0.05) and were more vulnerable to be induced to ventricular arrhythmias (70% (7/10) vs. 10% (1/10), P < 0.05), accompanied by the higher concentration of catecholamine (epinephrine: 1.75 ± 0.18 nmol/L vs. 0.68 ± 0.10 nmol/L n = 10, P < 0.05; norepinephrine: 9.56 ± 0.71 nmol/L vs. 2.52 ± 0.31 nmol/L n = 10, P < 0.05). The shortening of MAPD90 and increased inducibility to ventricular arrhythmias of AMPKα2-/- could almost be abolished when perfusion with ß-adrenoceptor antagonist metoprolol. It indicated that the ß-adrenoceptor activation resulting from catecholamine release was mainly responsible for the relating changes of electrophysiology of AMPKα2-/-. It had great clinical significance, as in patients who had problem with AMP-activated protein kinase-α2 gene, we might use ß-adrenoceptor antagonists as the prevention of arrhythmias in future. Impact statement As far as we know, this is the first time the role of AMP-activated protein kinase-α2 (AMPKα2) on the cardiac electrophysiology is explored, and we found that the ß-adrenoceptor activation resulting from catecholamine release was mainly responsible for the changes of electrophysiology related to the absence of AMPKα2. This has great clinical significance, as in patients who have problems with AMPKα2 gene, we may use ß-adrenoceptor antagonists for the prevention of arrhythmias in future.

Metoprolol decreases the plasma exposure of metformin via the induction of liver, kidney and muscle uptake in rats.

Drug interactions are one of the commonest causes of side effects, particularly in long-term therapy. The aim of the current study was to investigate the possible effects of metoprolol on the pharmacokinetics of metformin in rats and to clarify the mechanism of drug interaction. In this study, rats were treated with metformin alone or in combination with metoprolol. Plasma, urine and tissue concentrations of metformin were determined by HPLC. Western blotting and real-time qPCR were used to evaluate the expression of rOCTs and rMATE1. The results showed that, after single or 7-day repeated administration, the plasma concentrations of metformin in the co-administration group were significantly decreased compared with that in the metformin group. However, the parameter V/F of metformin in the co-administration group was markedly increased compared with that in the metformin group. The hepatic, renal and muscular Kp of metformin were markedly elevated after co-administration with metoprolol. Consistently, metformin uptake in rat kidney slices was significantly induced by metoprolol. In addition, multiple administrations of metoprolol significantly reduced the expression of rMATE1 in rat kidney as well as the urinary excretion of metformin. Importantly, after long-term administration, lactic acid and uric acid levels in the co-administration group were increased by 25% and 26%, respectively, compared with that in the metformin group. These results indicate that metoprolol can decrease the plasma concentration of metformin via the induction of hepatic, renal and muscular uptake, and long-term co-administration of metformin and metoprolol can cause elevated lactic acid and uric acid levels. Copyright © 2016 John Wiley & Sons, Ltd.

Screening ß1AR inhibitors by cell membrane chromatography and offline UPLC/MS method for protecting myocardial ischemia.

A high expression ß1AR/cell membrane chromatography (ß1AR-CMC) and offline UPLC/MS method has been developed for screening active ingredients from Coptis chinensis. In this study, the fractions retained by CMC column were separated and identified by UPLC/MS system. Using metoprolol as a positive control drug, coptisine from C. chinensis was identified as the active component which could inhibit ß1AR. Compared with the control group: coptisine could attenuate the infarct size and release malondialdehyde (MDA) while increasing superoxide dismutase (SOD) activity, suggesting a role in reducing myocardial injury. In vitro, coptisine could decrease apoptosis, showing their protective effects upon cardiomyocytes. This ß1AR-CMC-offline-UPLC/MS method can be applied for screening active components acting on ß1AR from traditional Chinese medicines.

Structure-Based Prediction of G-Protein-Coupled Receptor Ligand Function: A ß-Adrenoceptor Case Study.

The spectacular advances in G-protein-coupled receptor (GPCR) structure determination have opened up new possibilities for structure-based GPCR ligand discovery. The structure-based prediction of whether a ligand stimulates (full/partial agonist), blocks (antagonist), or reduces (inverse agonist) GPCR signaling activity is, however, still challenging. A total of 31 ß1 (ß1R) and ß2 (ß2R) adrenoceptor crystal structures, including antagonist, inverse agonist, and partial/full agonist-bound structures, allowed us to explore the possibilities and limitations of structure-based prediction of GPCR ligand function. We used all unique protein-ligand interaction fingerprints (IFPs) derived from all ligand-bound ß-adrenergic crystal structure monomers to post-process the docking poses of known ß1R/ß2R partial/full agonists, antagonists/inverse agonists, and physicochemically similar decoys in each of the ß1R/ß2R structures. The systematic analysis of these 1920 unique IFP-structure combinations offered new insights into the relative impact of protein conformation and IFP scoring on selective virtual screening (VS) for ligands with a specific functional effect. Our studies show that ligands with the same function can be efficiently classified on the basis of their protein-ligand interaction profile. Small differences between the receptor conformation (used for docking) and reference IFP (used for scoring of the docking poses) determine, however, the enrichment of specific ligand types in VS hit lists. Interestingly, the selective enrichment of partial/full agonists can be achieved by using agonist IFPs to post-process docking poses in agonist-bound as well as antagonist-bound structures. We have identified optimal structure-IFP combinations for the identification and discrimination of antagonists/inverse agonist and partial/full agonists, and defined a predicted IFP for the small full agonist norepinephrine that gave the highest retrieval rate of agonists over antagonists for all structures (with an enrichment factor of 46 for agonists and 8 for antagonists on average at a 1% false-positive rate). This ß-adrenoceptor case study provides new insights into the opportunities for selective structure-based discovery of GPCR ligands with a desired function and emphasizes the importance of IFPs in scoring docking poses.

Esmolol added in repeated, cold, oxygenated blood cardioplegia improves myocardial function after cardiopulmonary bypass.

OBJECTIVE: This study investigated if the ß-receptor blocking agent esmolol, added to standard oxygenated blood cardioplegia, improved myocardial function after weaning from bypass. DESIGN: A block-randomized, blinded study. SETTING: A university laboratory. PARTICIPANTS: Twenty anesthetized pigs, Norwegian Landrace. INTERVENTIONS: After cardiopulmonary bypass, cardiac arrest was induced with cold (12°C), oxygenated blood cardioplegia, enriched with either esmolol or vehicle, repeated every 20 minutes. After 100 minutes the heart was reperfused and weaned. MEASUREMENTS AND MAIN RESULTS: Left ventricular function was evaluated with pressure-volume loops, local myocardial function with multilayer strain and strain rate by epicardial short-axis tissue Doppler imaging. One hour after declamping, preload recruitable stroke work did not differ between groups, but increased to 72±3 mmHg in esmolol-treated animals v 57±4 mmHg (p<0.001) in controls after 3 hours. Radial peak ejection strain rate also was increased by esmolol; 6.0±1.0 s(-1)v 2.9±0.3 s(-1) (p<0.001) in subendocardium and 3.9±0.5 s(-1)v 2.3±0.2 s(-1) (p<0.005) in the midmyocardium. Cardiac index was increased, 4.0±0.2 L/min/m(2) by esmolol v 3.3±0.1 L/min/m(2) for controls (p<0.05). Isovolumetric relaxation time constant was reduced by esmolol, 23±1 ms v 26±1 ms (p<0.025). Troponin-T did not differ and was 339±48 ng/L for the esmolol group and 357±55 ng/L for the control group (p = 0.81). CONCLUSIONS: Esmolol added to blood cardioplegia preserved systolic cardiac function during the first 3 hours after reperfusion in a porcine model with 100 minutes of cardioplegic arrest.

Metoprolol oxidation polymorphism in Brazilian elderly cardiac patients.

OBJECTIVES: The purpose of this study was to phenotype the CYP2D6 in elderly with heart disease classified as extensive metabolizer or poor metabolizers (PM) of metoprolol, develop and validate the method of analysis of metoprolol tartrate and its metabolite in urine using HPLC, and identify potential correlations between anthropometric factors with metabolic ratios of metoprolol/α-OH metoprolol in urine. METHODS: The sample was composed of 130 elderly individuals with a previously identified type of heart condition, with normal renal and hepatic functions. The urine of all the patients were collected 0-8 h after the administration of a pill of 100 mg of metoprolol to determine concentrations of metoprolol and α-hydroxymetoprolol. Those patients presenting a metabolic ratio greater than 12.6 were phenotyped as PM. KEY FINDINGS: The median age of patients was 71.0 years, with a minimum of 60 and maximum of 93 years old. Three patients (2.3%) were phenotyped as PM of metoprolol different from the rate (7-10%) of PM existing in the Caucasian population. CONCLUSIONS: Most of the studied individuals were women, and the proportion of elderly with heart disease classified as PM was smaller than what is usually found among Caucasian populations.

Investigation of nebivolol as a novel therapeutic agent for the treatment of Alzheimer's disease.

Nebivolol is a selective ß1 adrenergic receptor antagonist with nitric oxide-mediated vasodilatory properties utilized in the treatment of hypertension. Previously, nebivolol was shown to modulate amyloid-ß protein precursor processing in vitro. In this study, we investigated the in vivo effects of nebivolol on the modulation of amyloid neuropathology in the Tg2576 mouse model of Alzheimer's disease (AD). We found that nebivolol is brain bioavailable and can be readily detected in the brain following three weeks of treatment at a dose of 1 mg/kg/day. Moreover, this treatment regime resulted in a significant reduction of amyloid-ß neuropathology in the brain, and this reduction was inversely correlated with plasma levels of amyloid-ß. Chronic nebivolol treatment of Tg2576 mice with established amyloid neuropathology and cognitive impairments significantly reduced brain amyloid content but failed to improve cognitive function. Our study demonstrates that nebivolol is highly tolerable and safe and can significantly reduce amyloid neuropathology in the brain, which could be one of the most important parameters for primary prevention of AD. Our studies support the continued investigation of nebivolol for the treatment of AD at very early stages of the disease.

The common Arg389gly ADRB1 polymorphism affects heart rate response to the ultra-short-acting ß(1) adrenergic receptor antagonist esmolol in healthy individuals.

The ß1-adrenergic receptor (ß1AR) Arg389Gly polymorphism affects responses to orally administered ß1AR antagonists (ß-blockers) in vivo. However, the effect of this polymorphism on the early heart rate response to ß-blockers has not been evaluated. The aim of this study was to determine the effect of the Arg389Gly polymorphism on the inhibition of exercise-induced tachycardia by esmolol, an ultra-short-acting intravenously administered ß1AR antagonist. Healthy nonsmoking White individuals were enrolled on the basis of their ADRB1 genotype, including carriers of 0, 1 or 2 Arg389 alleles (n=9 in each group, total 27, 18 men). Placebo and esmolol were infused consecutively for 10 min each, separated by 30 min. At the end of each infusion, participants performed dynamic handgrip exercise. Heart rate and blood pressure were compared among three ADRB1 genotypes. Carriers of 0, 1, or 2 Arg389 alleles varied significantly in both exercise-induced tachycardia during esmolol (P(ANOVA)=0.030) and esmolol inhibition of exercise-induced tachycardia [0.78±7.70, 5.11±4.05, 10.22±9.78 bpm, respectively (P=0.014)]. The early effect of esmolol on exercise-induced tachycardia was significantly greater among Arg389 than in Gly389 homozygote healthy individuals (NCT01388036).

Simultaneous determination of metoprolol and α-hydroxymetoprolol in human plasma using excitation-emission matrix fluorescence coupled with second-order calibration methods.

BACKGROUND: Metoprolol (MET) is a ß1-adrenoceptor antagonist, which is widely used in the treatment of cardiovascular diseases, and α-hydroxymetoprolol (α-OHM) is its hydroxylated metabolite. Owing to their similar structures, optimization of the condition for the chromatography approach, which is in common use for determination, is both time consuming and laborious. RESULTS: A new and effective strategy that combines the excitation-emission matrix fluorescence with second-order calibration methods was developed for simultaneous determination of MET and α-OHM in human plasma. CONCLUSION: Although the fluorescence spectra of MET and α-OHM overlapped and a large number of unknown and uncalibrated fluorescent components coexisted, the developed method enables accurate concentrations together with reasonable resolution of excitation and emission profiles for the analytes of interest. An additional advantage of the proposed method is that there is no need for separation and sample pretreatment, in addition to lower cost than traditional methods.

ß1 -adrenergic receptor autoantibodies from heart failure patients enhanced TNF-α secretion in RAW264.7 macrophages in a largely PKA-dependent fashion.

Autoantibodies against the second extracellular loop of ß(1) -adrenergic receptor (ß(1) -AA) not only contribute to increased susceptibility to heart failure, but also play a causative role in myocardial remodeling through their catecholamine-like effects via binding with the ß(1) -adrenergic receptor. The current study was designed to determine whether ß(1) -AA isolated from the sera of heart failure patients could cause TNF-α secretion from the murine macrophage-like cell line RAW264.7. Blood samples were collected from 40 patients who had suffered heart failure, as well as from 40 healthy subjects. The titer of ß(1) -AA and the level of TNF-α were detected using ELISA. The effect of ß(1) -AA on murine macrophage-like cell line RAW264.7 proliferation was detected by CCK-8 kits and CFSE assay. Western blot assay was used to analyze the expression of phospho-VASP. ß(1) -AA appeared more frequently in patients with heart failure than in healthy subjects. The ß(1) -AA isolated from heart failure patients promoted an increase of TNF-α levels, which could be completely blocked by the selective ß(1) -adrenergic receptor antagonist metoprolol and the second extracellular loop of ß(1) -adrenergic receptor (ß(1) -AR-EC(II) ), but only partially inhibited by PKA inhibitor H89. Furthermore, the ß(1) -AA could enhance the proliferation of RAW264.7 cells in vitro. Meanwhile, the expression of phospho-VASP was markedly increased in the presence of ß(1) -AA. These results demonstrate for the first time that the ß(1) -AA isolated from heart failure patients could bind with ß(1) -AR on the surface of RAW264.7 cells, causing the release of TNF-α largely in a PKA-dependent fashion.

Randomized study on the efficacy and safety of landiolol, an ultra-short-acting ß1-adrenergic blocker, in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention.

BACKGROUND: It is still controversial whether intravenous administration of ß-blocker in the very acute phase of acute myocardial infarction (AMI) is beneficial. Landiolol is an ultra-short-acting ß-blocker that has less effect on blood pressure, but little is known about its efficacy and safety for patients with AMI undergoing primary percutaneous coronary intervention (PCI). METHODS AND RESULTS: A consecutive 96 patients with AMI not manifesting cardiogenic shock were prospectively randomized to landiolol (n = 47) or a control group (n = 49). Continuous administration of landiolol (3 µg·kg(-1)·min(-1) for 24 h) was done just after PCI in the landiolol group, but not in the control group. Heart rate decreased by 9.4 ± 1.7 beats/min after initiation of landiolol (P<0.01), but was unchanged in the control group. Left ventricular ejection fraction assessed 6 months later was greater than that at 2 weeks in the landiolol group (52.0 ± 1.5 vs. 49.1 ± 1.5%, P = 0.01), but remained unchanged in the control group. Left ventricular end-diastolic volume index assessed 6 months later was increased compared with that at 2 weeks in the control group (78.0 ± 2.7 vs. 72.5 ± 2.8 ml/m(2), P = 0.02), whereas it was unchanged in the landiolol group. CONCLUSIONS: Early intravenous administration of landiolol in patients with AMI undergoing PCI is safe and has the potential to improve cardiac function and inhibit cardiac remodeling in the chronic phase.

Nebivolol for the treatment of heart failure.

INTRODUCTION: Heart failure (HF) is a common and disabling disease with a high prevalence in the elderly. Beta-blockers are among the mainstay therapies of HF because they antagonize the deleterious effects of the chronic activation of sympathetic nervous system. In large randomized clinical trials, bisoprolol, carvedilol and metoprolol reduced mortality and cardiovascular hospital admission and, hence, are included in current guidelines for HF treatment. AREAS COVERED: Nebivolol is a third-generation ß-blocker with high selectivity for ß1-adrenoceptors selectivity and vasodilating effects. It also shows antioxidant, antiproliferative and antithrombotic properties. Nebivolol is generally well tolerated. Typical ß-blocker-related adverse events are same as that with placebo, except for bradycardia. In addition, it shows no negative effects on chronic obstructive pulmonary disease, erectile function, and glucose and lipid metabolism. The benefits of nebivolol for HF treatment have been evaluated in the SENIORS trial, where it reduced the composite endpoint of mortality and cardiovascular hospital admission. EXPERT OPINION: Nebivolol is a ß-blocker with distinctive characteristics. Initiated at 1.25 mg and titrated up to 10 mg/day, it has shown safety and efficacy in one large outcome trial, when added to standard medical therapy, in elderly patients (≥ 70 years) affected by HF.

Two distinct conformations of helix 6 observed in antagonist-bound structures of a beta1-adrenergic receptor.

The ß(1)-adrenergic receptor (ß(1)AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (ß(1)AR-M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R(3.50) - D/E(6.30) "ionic lock"). Here we compare eight new structures of ß(1)AR-M23, determined from crystallographically independent molecules in four different crystals with three different antagonists bound. These structures are all in the inactive R state and show clear electron density for cytoplasmic loop 3 linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions, there are only two distinct conformations of the cytoplasmic end of helix 6, bent and straight. In the bent conformation, the Arg139-Glu285 salt bridge is present, as in the crystal structure of dark-state rhodopsin. The straight conformation, observed in previously solved structures of ß-receptors, results in the ends of helices 3 and 6 being too far apart for the ionic lock to form. In the bent conformation, the R(3.50)-E(6.30) distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could explain the high basal activity in ß(1)AR compared to rhodopsin. Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an important role in receptor activation.

Pancreatoduodenectomy as a source of human small intestine for Ussing chamber investigations and comparative studies with rat tissue.

A clear understanding of oral drug absorption is an important aspect of the drug development process. The permeability of drug compounds across intact sections of small intestine from numerous species, including man, has often been investigated using modified Ussing chambers. The maintenance of viable, intact tissue is critical to the success of this technique. This study therefore aimed to assess the viability and integrity of tissue from patients undergoing pancreatoduodenectomy, for use in cross-species Ussing chamber studies. Electrical parameters (potential difference, mV; short-circuit current, µA.cm(-2) ; resistance, Ω.cm(2) ) were monitored over the duration of each experiment, as was the permeability of the paracellular marker atenolol. The permeability values (Papp; cm/s × 10(-6) ) for a training-set of compounds, displaying a broad range of physicochemical properties and known human fraction absorbed values, were determined in both rat and human jejunum, as well as Caco-2 cell monolayers. The results indicate that human jejunum sourced from pancreatoduodenectomy remained viable and intact for the duration of experiments. Permeability values generated in rat and human jejunum correlate well (R(2) = 0.86), however the relationship between permeability in human tissue and Caco-2 cells was comparatively weak (R(2) = 0.58). Relating permeability to known human fraction absorbed (hFabs) values results in a remarkably similar relationship to both rat and human jejunum Papp values. It can be concluded that human jejunum sourced from pancreatoduodenectomy is a suitable source of tissue for Ussing chamber permeability investigations. The relationship between permeability and hFabs is comparable to results reported using alternative test compounds.

The structural basis for agonist and partial agonist action on a ß(1)-adrenergic receptor.

ß-adrenergic receptors (ßARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit ßARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) ß(1)-adrenergic receptor (ß(1)AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser(5.42) and Ser(5.46)), but partial agonists only interact with Ser(5.42) (superscripts refer to Ballesteros-Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.