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Phosphorylation of myofilament proteins critically regulates beat-to-beat cardiac contraction and is typically altered in heart failure (HF). β-Adrenergic activation induces phosphorylation in numerous substrates at the myofilament. Nevertheless, how cardiac β-adrenoceptors (βARs) signal to the myofilament in healthy and diseased hearts remains poorly understood. The aim of this study was to uncover the spatiotemporal regulation of local βAR signaling at the myofilament and thus identify a potential therapeutic target for HF. Phosphoproteomic analysis of substrate phosphorylation induced by different βAR ligands in mouse hearts was performed. Genetically encoded biosensors were used to characterize cyclic adenosine and guanosine monophosphate signaling and the impacts on excitation-contraction coupling induced by β1AR ligands at both the cardiomyocyte and whole-heart levels. Myofilament signaling circuitry was identified, including protein kinase G1 (PKG1)-dependent phosphorylation of myosin light chain kinase, myosin phosphatase target subunit 1, and myosin light chain at the myofilaments. The increased phosphorylation of myosin light chain enhances cardiac contractility, with a minimal increase in calcium (Ca2+) cycling. This myofilament signaling paradigm is promoted by carvedilol-induced β1AR-nitric oxide synthetase 3 (NOS3)-dependent cyclic guanosine monophosphate signaling, drawing a parallel to the β1AR-cyclic adenosine monophosphate-protein kinase A pathway. In patients with HF and a mouse HF model of myocardial infarction, increasing expression and association of NOS3 with β1AR were observed. Stimulating β1AR-NOS3-PKG1 signaling increased cardiac contraction in the mouse HF model. This research has characterized myofilament β1AR-PKG1-dependent signaling circuitry to increase phosphorylation of myosin light chain and enhance cardiac contractility, with a minimal increase in Ca2+ cycling. The present findings raise the possibility of targeting this myofilament signaling circuitry for treatment of patients with HF.

Cardioselective β-blockade is generally well tolerated in practice and contraindications to this therapy are uncommon. β-blockers are a diverse therapeutic class, and their individual tolerability profiles are influenced strongly by their pharmacodynamic effects across different adrenergic receptors. Bisoprolol, probably the β-blocker with the highest selectivity for blockade of β1- vs. β2-adrenoceptors, does not block β2-adrenoceptors to an appreciable extent at doses in therapeutic use. Side-effects often attributed to β-blockers, such as erectile dysfunction and adverse metabolic effects are uncommon with bisoprolol and other β-blockers used at doses which only block β1-adrenoceptors. Cautious use of a cardioselective β-blocker is not contraindicated in people with chronic obstructive pulmonary disease or asthma and the outcomes benefits of β-blockers in patients with coronary heart disease or heart failure are also apparent in patients with concurrent COPD. Starting with a low dose and titrating upwards carefully is important for optimising the tolerability of a β-blocker. Most people with hypertension will receive combination antihypertensive therapy in practice, and the low-dose combination therapy approach provides a useful strategy for optimising the efficacy and tolerability of a regimen that includes a β-blocker, compared with up-titrating an existing monotherapy. [ABSTRACT FROM AUTHOR]

Cardiac dysfunction is involved in disorders of energy metabolism. High‐titre autoantibodies against the β1‐adrenoceptor (β1‐AAs) have been reported to exist in patients with cardiac dysfunction; however, the mechanism by which β1‐AAs affect cardiac function is unknown. This study aimed to determine whether β1‐AAs disturb myocardium energy metabolism and cause cardiac dysfunction. β1‐AA monoclonal antibodies (β1‐AAmAbs) were successfully pre‐synthesized by hybridoma clones and used in all experiments. β1‐AAmAbs impaired cardiac function and induced a myocardial metabolic disturbance, as evidenced by decreased left ventricular ejection fraction and fractional shortening. In addition, β1‐AAmAbs decreased the adenosine triphosphate level and increased cardiac energy consumption (rate–pressure product). We further showed that the effects of β1‐AAmAbs on heart tissue might involve the mitochondria and metabolic pathways via the β1‐adrenoceptor based on an immunoprecipitation and mass spectrometry. Additionally, we found that β1‐AAmAbs impaired myocardial mitochondrial structure, decreased the membrane potential, and induced insufficient mitophagy. In conclusion, β1‐AAmAb‐induced cardiac dysfunction is partly due to a disturbance in myocardial energy metabolism. [ABSTRACT FROM AUTHOR]

O-GlcNAcylation was first found by Torres and Hart in monocytes. It is a dynamic and reversible post-translational modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation is increased in diabetic cardiomyopathy (DCM) patients and it has been reported that OGT plays an important role in the regulation of cardiac gene transcription, cell cycle and calcium homeostasis. The purpose of this study is to investigate the effects of OGT on signal transduction and function of β1-adrenoceptor (β1AR) in adult rat cardiomyocytes. We found that after overexpressing OGT by adenovirus vector in adult rat cardiomyocytes, cAMP formation and phosphorylation of phospholamban (PLB) at Ser16 (p16-PLB) were decreased under isoprenaline (ISO) stimulation. Over expression of OGT increased the intracellular [Ca2+i and deteriorated the death of cardiomyocytes induced by prolonged stimulation with ISO. β1-adrenoceptor was overexpressed using a plasmid vector and then co-immunoprecipitation (co-IP) followed by Western blot was employed to define the O-GlcNAcylation of β1-adrenoceptor. The results showed that O-GlcNAcylation of β1-adrenoceptor was increased in OGT overexpressed cells, and there was no significant change in the formation of cAMP and phosphorylation of PLB after β1-adrenoceptor was blocked by CGP20712A. Given that OGT affects the signal transduction of β1-adrenoceptor in adult rat cardiomyocytes by increasing the O-GlcNAcylation of β1-adrenoceptor, the mechanism revealed in this study indicates that OGT and β1AR may be therapeutic targets in patients undergoing diabetic cardiomyopathy. • OGT overexpression increased the O-GlcNAcylation of β1-adrenoceptor. • OGT overexpression could downregulate the formation of cAMP and phosphorylation of phospholamban. • Overexpression of OGT increased the intracellular [Ca2+i and deteriorated the death of cardiomyocytes induced by prolonged stimulation with ISO. [ABSTRACT FROM AUTHOR]

New Findings What is the central question of this study? High titres of autoantibodies against the second extracellular loop of the β1-adrenergic receptor (β1-AAs) can be detected in the sera of patients with ventricular arrhythmias, but a causal relationship between β1-AAs and ventricular arrhythmias has not been established., What is the main finding and its importance? Monoclonal β1-AAs (β1-AR mAbs) were used in the experiments. We showed that β1-AR mAbs increased susceptibility to ventricular arrhythmias and induced repolarization abnormalities. Antibody adsorption of β1-AAs will be a potential new therapeutic strategy for ventricular arrhythmias in patients with high titres of β1-AAs., High titres of autoantibodies against the second extracellular loop of the β1-adrenergic receptor (β1-AAs) can be detected in sera from patients with ventricular arrhythmias, but a causal relationship between β1-AAs and ventricular arrhythmias has not been established. In this work, ECGs of guinea-pigs and isolated guinea-pig hearts were recorded. Ventricular tachycardia (VT) and ventricular fibrillation (VF) were evoked by programmed electrical stimulation of the left ventricular epicardium of isolated guinea-pig hearts. The monophasic action potential and effective refractory period of the left ventricle were recorded in paced isolated guinea-pig hearts. Furthermore, to increase the specificity, monoclonal autoantibodies against the second extracellular loop of the β1-adrenergic receptor (β1-AR mAbs) were used in all experiments. The results showed that β1-AR mAbs induced premature ventricular contractions in guinea-pigs and isolated guinea-pig hearts. In addition, β1-AR mAbs decreased the threshold of VT/VF and prolonged the duration of VT/VF. Furthermore, β1-AR mAbs shortened the corrected QT interval and effective refractory period, and prolonged late-phase repolarization of the monophasic action potential (MAPD90−30). These changes in electrophysiological parameters might be attributed, at least in part, to the arrhythmogenicity of β1-AR mAbs. [ABSTRACT FROM AUTHOR]

To develop a novel β1-adrenoceptor selective radiotracer, 99mTc-labeld N-(2-aminoethyl)-3-(4-(2-hydroxy-3-(isopropylaminopropoxy)phenyl)propanamide ([ 99mTc] 1) was prepared by incorporating [ 99mTcO4 ]− into the precursor 3. Radiotracer [ 99mTc] 1 was optimized in several reaction conditions for kit-type preparation of 99mTc-radiopharmaceuticals. The radiochemical yield was around 95% (non-decay corrected) within 30 s labeling time and radiochemical purity showed over 98%. In image studies, [ 99mTc] 1 provided a good heart accumulation, whereas liver and lung uptakes showed relatively low at initial time. Furthermore, [ 99mTc] 1 images at 25 min post-injection provided a clearly visible cardiac image with high contrast. To confirm the β1-adrenoceptor-binding specificity of [ 99mTc] 1, a blocking experiment was performed. In vivo blocking with excess esmolol (18 mg/kg) or atenolol (2 mg/kg) resulted in significantly reduced radioactivity uptake in the heart around 92 and 76%, respectively. The results suggested that [ 99mTc] 1 might be of worth applying to the assessment of β1-adrenoceptor expression in the heart diseases. [ABSTRACT FROM AUTHOR]

Activation of β 1 -adrenoceptor stimulates myocardial membrane destabilization in isoproterenol induced rats. Male albino Wistar rats were pre and co-treated with 7-hydroxycoumarin (16 mg/kg body weight) daily for 8 days. Myocardial infarction was induced into rats by the subcutaneous administration of isoproterenol (100 mg/kg body weight) at an interval of 24 h daily for a period of two days (7th and 8th day). The levels/activities of serum cardiac troponin-T, lactate dehydrogenase and the concentrations of heart lipid peroxidation products were significantly increased and the antioxidant status was significantly decreased in isoproterenol induced rats. Furthermore, the activity of sodium/potassium-dependent adenosine triphosphatase was significantly decreased and the activities of calcium and magnesium-dependent adenosine triphosphatases were significantly increased in the heart of isoproterenol induced myocardial infarcted rats. Isoproterenol induced rats also revealed increased concentrations of sodium and calcium and decreased concentrations of potassium in the heart. 7-hydroxycoumarin pre- and co-treatment showed considerable impact on all biochemical parameters assessed. Also, 7-HC greatly reduced the infarct size of the myocardium. The in vitro study confirmed its potent free radical scavenging activity. Thus, the present study revealed that 7-HC attenuates myocardial membrane destabilization by reinstating the activities/levels of adenosine triphosphatases and minerals in isoproterenol induced rats by inhibiting oxidative stress. These effects are attributed to the membrane stabilizing and free radical scavenging properties of 7-hydroxycoumarin. [ABSTRACT FROM AUTHOR]

We wanted to assess whether β-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively., Urothelial cells were cultured for 24 h in the presence of the β-adrenoceptor agonists isoprenaline (β1/2/3), dobutamine (β1), salbutamol (β2), BRL 37344 (β3), CGP 12177 (a partial β-agonist) or β-adrenoceptor antagonists (metoprolol; β1, propranolol; β1/2). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay ( ATCC). Western blot and immunocytochemistry were used for detection of the β1-adrenoceptor., Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of β1-adrenoceptors in both urothelial cell lines., The present data show that the urothelium expresses atypical β1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation. [ABSTRACT FROM AUTHOR]

As expressed predominantly in cardiac tissue, beta1-adrenoceptor (β 1 -AR) is broadly accepted as one of the main targets for drugs against cardiovascular ailments. However, the discovery of β 1 -AR ligand is gravely challenged due to the lack of efficient screening method. This work developed a general strategy for pursuing β 1 -AR ligands from the herbal extract by immobilizing haloalkane dehalogenase (Halo)-tagged β 1 -AR onto microspheres coated with 6-chlorohexanoic acid, and applying the immobilized β 1 -AR in the analysis of ligand-receptor interaction. The morphology was characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). The chromatographic specificity of the immobilized receptor column was evaluated by determining the association constants of atenolol, esmolol and metoprolol using stepwise frontal analysis plus injection amount-dependent method. The potential ligands binding to β 1 -AR was screened by collecting the peak with retention time longer than the void time, and identified the collection by reverse phase liquid chromatography coupled with tandem mass spectrometry. The association constants of the three drugs to β 1 -AR were (3.33 ± 0.29)× 106 M−1, (2.33 ± 0.23)× 106 M−1 and (2.06 ± 0.03)× 106 M−1, indicating a desired specificity of the immobilized receptor for recognizing its ligands. Molecular docking showed that van der Waals, hydrogen bonds, and hydrophobic interactions were the principal interaction forces for the receptor-drug complexes. Benzoylmesaconine was screened as the potential ligand of β 1 -AR in Radix Aconiti Lateralis Praeparata extract. The association constant of the ligand was (1.06 ± 0.02)× 105 M−1, hinting structural modification may be required before clinical application. The immobilized β 1 -AR is possible to provide a rapid method for screening potential ligands in herbal extract. • Preparation of β 1 -AR stationary phase by the bioorthogonal click reaction. • Revealing the binding mechanism between drugs and β 1 -AR by chromatographic methods. • Screening the bioactive compounds targeting β 1 -AR from herbal medicine. [ABSTRACT FROM AUTHOR]

Abstract: The prefrontal cortex (PFC) plays a critical role in cognitive functions, including working memory, attention regulation and behavioral inhibition. Microinjection of β1-adrenoceptor (β1-AR) agonist into the PFC has been shown to impair PFC cognitive function. However, the underlying cellular and molecular mechanisms have not been determined yet. In the present study, we tested the hypothesis that β1-AR mediated modulation of excitatory synaptic transmission contributes to PFC dysfunction. We found that 1) the β1-AR agonist Dobutamine (Dobu) suppressed the amplitude evoked excitatory postsynaptic currents (eEPSCs). 2) Dobu induced a significant suppression of the frequency and amplitude of miniature EPSCs (mEPSCs). 3) Dobu-suppressed glutamate release was mediated via decreasing release probability and the number of releasable vesicles. 4) Dobu suppressed inward currents evoked by puff application of glutamate or NMDA via postsynaptic PKA-dependent pathway. The present study indicates that β1-AR activation suppresses excitatory synaptic transmission in medial PFC (mPFC) via both pre- and post-synaptic PKA-dependent mechanisms. Our results may provide a cellular and molecular mechanism that helps explain β1-AR-induced PFC dysfunction. [Copyright &y& Elsevier]

• Preparaion of a new stationary phase by immobilizing the beta1-adrenoceptor (β 1 -AR) onto ibrutinib-coated microspheres. • Exploration of the binding mechanism between a ligand and the β 1 -AR by the thermodynamic analysis. • Development of a chromatographic method for pursuing β 1 -AR ligand from complex matrices. The discovery of beta1-adrenoceptor (β 1 -AR) ligands is viewed as an enormous demand for fighting ailments mediated by the receptor including cardiovascular diseases. Such pursuit is gravely challenged due to the lack of lead screening methods with high efficiency. This work developed a chromatographic method for pursuing β 1 -AR ligand from the herbal extract by fusing epidermal growth factor receptor (EGFR) as a tag at its C-terminus to stably express the fusion receptor in E. coli , immobilizing the expressed EGFR-tagged β 1 -AR onto ibrutinib-derivatized amino microspheres, and applying the immobilized receptor in the analysis of ligand-receptor interaction and herbal extract. Comprehensive characterizations like X-ray photoelectron spectroscopy and retention behaviors of canonical drugs demonstrated high specificity and good stability of the immobilized β 1 -AR prepared through the covalent reaction between the EGFR and ibrutinib decorated on the microsphere surface. Frontal analysis of atenolol, metoprolol, and esmolol confirmed their bindings to β 1 -AR with association constants of 1.07 × 104, 6.54 × 103, and 1.45 × 104 M−1. The thermodynamic analysis provided proof of electrostatic interaction, hydrogen bonds, and van der Waals force driving those interactions. Pulegone was recognized as a bioactive compound that specifically binding to β 1 -AR from the extract of Ziziphora clinopodioides Lam by analyzing the retention peak through reverse-phase high performance liquid chromatography coupled with tandem mass spectrometry. These results, taken together, indicated that the current method is possible to provide an alternative for discovering β 1 -AR ligands with high efficiency from complex matrices like herbal extract. [ABSTRACT FROM AUTHOR]

Highlights: [•] β1- and β2-adrenoceptors (ARs) are expressed in different interneurons in the mPFC. [•] These interneurons include PV-, CR-, CB-, SST- and Reelin-immunoreactive cells. [•] CR-ir cells are less likely to express β1/2-ARs than the other types of cells. [•] SST-ir cells are more likely to express β2-AR than the other types of interneurons. [Copyright &y& Elsevier]

Abstract: The effects of intra-accumbal infusion of selective agonists for the β-adrenoceptor subtypes on the noradrenaline and dopamine efflux in the nucleus accumbens of freely moving rats were investigated, using in vivo microdialysis. Neither β1-(dobutamine: 0.06 and 0.12pmol) nor β2-adrenoceptor agonist (salbutamol: 0.36 and 3.6pmol) altered the basal noradrenaline and dopamine efflux in the nucleus accumbens. Co-administration of 0.06pmol of dobutamine with salbutamol (3.6pmol) did not affect the noradrenaline levels, but it increased the dopamine efflux to approximately 120%. Co-administration of 0.12pmol of dobutamine with salbutamol (0.36 or 3.6pmol) also increased DA efflux to approximately 120% without affecting noradrenaline levels. The non-selective β-adrenoceptor antagonist l-propranolol (1200pmol) that did not alter the basal noradrenaline and dopamine levels, suppressed the dopamine efflux, induced by co-administration of dobutamine (0.12pmol) and salbutamol (3.6pmol). The doses mentioned are the total amount of drug over the 60-min infusion period. The present results support our previously reported conclusion that stimulation of accumbal β-adrenoceptors which are suggested to be postsynaptically located on accumbal dopaminergic terminals, can enhance the dopamine efflux in the nucleus accumbens. The present study also provides in vivo neurochemical evidence that concomitant, but not separate, activation of accumbal β1- and β2-adrenoceptors synergistically increases the accumbal dopamine efflux. [Copyright &y& Elsevier]

Aquilaria agallocha Roxb. (Thymelaeceae) heartwood is used in cardiac failure; however, its cardiotonic properties are poorly understood at a pharmacological level. This study investigated methanol extract of A. agallocha for presence of glycosides and its effects on force of contraction, heart rate and coronary flow on rabbit isolated heart. Furthermore, contribution of β1- adrenoceptors and/or L-type Ca2+ channels in A. agallocha-induced cardiac effects was explored. A. agallocha extract, digoxin, verapamil and metoprolol were administered in a retrograde manner. Force of contraction and heart rate were recorded with a force transducer attached to the heart. Coronary flow was measured from collected effluent. The extract induced a significant increase in force of contraction (p<0.001), decrease in heart rate (p<0.05) and coronary flow (p<0.001). Metoprolol (p<0.01) and verapamil (p<0.01) significantly inhibited the plant extract-induced effects. Thus, A. agallocha exhibited cardiotonic effects, most likely via cardiac glycosides, involving β1- adrenoceptors and L-type Ca2+ channels. [ABSTRACT FROM AUTHOR]

Abstract: The cyclic AMP/protein kinase A signaling pathway is thought to be involved in neural differentiation of mesenchymal stem cells. In the present study, we examined the involvement of β-adrenoceptor signaling on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells. Mouse iPS cells were cultured on ultra-low-attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 1μM) and/or the β-adrenoceptor agonist l-isoproterenol (0.3 or 1μM) were added to the EB cultures for 4 days, then EBs were plated on gelatin-coated plates and cultured for 7 or 14 days. Subtype-specific antibody staining revealed that mouse iPS cells express β1-adrenoceptors predominantly. Although treatment with l-isoproterenol alone did not affect the expression of Nestin (a specific marker for neural progenitor cells), l-isoproterenol significantly enhanced ATRA-induced Nestin expression. Pretreatment of EBs with either atenolol (a selective β1-adrenoceptor antagonist) or H89 (a protein kinase A inhibitor) significantly inhibited the l-isoproterenol-enhancement of ATRA-induced Nestin expression. In addition, the l-isoproterenol treatment significantly enhanced ATRA-induced expression of NeuN (a neuron-specific nuclear protein). These findings suggest that β1-adrenoceptor stimulation enhances ATRA-induced neural differentiation of mouse iPS cells. [Copyright &y& Elsevier]

Abstract: Purpose: To evaluate the effect of active immunization with a peptide corresponding to the second extracellular loop of the human beta1-adrenoceptors (β1-AR) on the reactivity of Wistar rat isolated aorta. Methods: Nine-week-old Wistar rats were actively immunized for 3months with a peptide corresponding to the second extracellular loop of the human β1-AR. Specific immunoglobulins G (IgG) were characterized by Elisa and the bicinchonic acid protein assay and their functionality were tested in isolated ventricular cardiomyocytes (IVC) from control rats. Aortic rings isolated from control or immunized rats were mounted in organ baths. Then, contractile curves to phenylephrine (1nM to 300μM) and relaxant curves to acetylcholine (1nM to 100μM) and isoprenaline (1nM to 30μM) were established. Results: Cell shortening increased dose-dependently in rat IVC superfused with IgG containing β1-AR antibodies (10 or 25μg/mL). Isoprenaline-induced positive inotropy was strongly reduced in IgG containing β1-AR antibodies preincubated (3h) IVC. Phenylephrine–and acetylcholine–induced aortic responses were greatly inhibited in immunized rats compared to control ones. However, active immunization did not influence the isoprenaline-mediated relaxation. Conclusions: The present work confirms that β1-AR antibodies directed against the second extracellular loop of β1-AR induce a positive inotropic effect in adult rat IVC. Moreover, we demonstrated, for the first time, that 3-month immunization with β1-AR peptide was associated with altered aortic endothelial function without change in the β-AR-mediated vasorelaxation. [Copyright &y& Elsevier]

Circulating autoantibodies directed against the 2nd extracellular loop (EL-2) of β1-adrenoceptors (β1-AABs) have been detected in the serum of patients with various cardiovascular pathologies. β1-AABs induce agonistic, positive inotropic effects via β1-adrenoceptors (β1ARs). In the mammalian heart, β1-AR can exist in 2 distinct activated configurations (the so-called high- and low-affinity states). The aim of the present study was to investigate whether the action of β1-AAB is dependent on the affinity state of β1AR in isolated ventricular cardiomyocytes of adult Wistar rats. Immunoglobulin G (IgG) containing β1-AAB obtained from animals immunized with a peptide corresponding to the EL-2 of human β1-AR, caused a dose-dependent increase in cell shortening. Isoproterenol-induced inotropy was significantly reduced in cardiomyocytes that had been preincubated with IgG containing β1-AAB and in cardiomyocytes isolated from immunized rats. The negative effects of preincubation with IgG containing β1-AAB on the response to isoproterenol was inhibited in the presence of bisoprolol. CGP 12177A and pindolol-induced inotropy was not affected by IgG preincubation or immunization. No detectable inotropic effect of cell shortening was obtained with IgG containing β1-AAB in the presence of propranolol and 3-isobutyl-1-methylxanthine. The present study demonstrates that β1-AABs have no agonist/antagonist-like effects upon low-affinity state β1-ARs. This result indicates that β1-AABs recognize and stabilize the high-affinity state, but are unable to stabilize and (or) induce the low-affinity state receptor. [ABSTRACT FROM AUTHOR]

Abstract: Background: β1-Adrenoceptors (β1-ARs) are the predominant receptors in regulating heart functions. β1-ARs contain 7-transmembrane domains (7TM), 3 extracellular loops and 3 intracellular loops. Among these loops, the second extracellular loop of β1-AR (β1-AR-ECII) plays an important role in the pathogenesis of heart failure. Methods: (1) Select the sera-negative rats for antibodies against β1-AR-ECII. (2) Detect the level of antibodies against β1-AR-ECII in the process of active immunization with artificial synthetic peptides according to the sequence of human β1-AR-ECII. (3) Observe its long-term role on cardiac structure and function in vivo by immunizing rats. (4) Detect the changes of T lymphocytes subsets in the process of active immunization. Results: The peptides induced the production of specific autoantibody against β1-AR-ECII. Furthermore, immunization with β1-AR-ECII peptide induced both morphological and functional alterations in the hearts of rats, which potentially results in heart failure. In addition, induction of the autoantibodies against β1-AR-ECII increased the CD4 +/CD8 + ratio in the peripheral blood of rats. Discussion: In this study, we determined the effect of anti-β1-AR-ECII autoantibody on morphological and functional cardiomyopathic alterations by immunization of rats with a synthetic peptide corresponding to the β1-AR-ECII for 18months. These results provide further evidence that autoantibody against β1-AR-ECII is involved in the pathogenesis of heart failure. But the underlying mechanisms need further study. [Copyright &y& Elsevier]

Stimulation of β-adrenoceptors activates the canonical adenylate cyclase pathway (via Gs protein) but can also evoke phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1/2) via Gs/Gi switching or β-arrestin-mediated recruitment of Src. In primary cultures of mouse astrocytes, activation of the former of these pathways required micromolar concentrations of the β1/β2-adrenergic agonist isoproterenol, that acted on β1-adrenoceptors, whereas the latter was activated already by nanomolar concentrations, acting on β2 receptors. Protein kinase A activity was required for Gs/Gi switching, which was followed by Ca2+ release from intracellular stores and Giα- and metalloproteinase-dependent transactivation of the epidermal growth factor receptor (EGFR; at its Y1173 phophorylation site), via its receptor-tyrosine kinase, β-arrestin 1/2 recruitment, and MAPK/ERK kinase-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation by Src activation depended on β-arrestin 2, but not β-arrestin 1, was accompanied by Src/EGFR co-precipitation and phosphorylation of the EGFR at the Src-phosphorylated Y845 site and the Y1045 autophosphorylation site; it was independent of transactivation but dependent on MAPK/ERK kinase activity, suggesting EGFR phosphorylation independently of the receptor-tyrosine kinase or activation of Ras or Raf directly from Src. Most astrocytic consequences of activating either pathway (or both) are unknown, but morphological differentiation and increase in glial fibrillary acidic protein in response to dibutyryl cAMP-mediated increase in cAMP depend on Gs/Gi switching and transactivation. [ABSTRACT FROM AUTHOR]

Abstract: The structures of porcine and human β2-adrenoceptors differ but the repercussions for porcine cardiac function are unknown. We investigated the function of porcine β2-adrenoceptors in 3 cardiac regions, sinoatrial node, left atrium and right ventricle of newborn piglets. Both (−)-noradrenaline and (−)-adrenaline caused sinoatrial tachycardia: 60±10% and 62±7% of the maximum response (E max) to (−)-noradrenaline (−logEC50 =9.0) and (−)-adrenaline (−logEC50 =7.5) respectively, were resistant to antagonism by the β1-selective CGP20712A (2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide) (300nM) but antagonized by β2-selective ICI118551 (erythro(±)-[1-(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol) (50nM), consistent with mediation through β2-adrenoceptors. The phosphodiesterase3-selective inhibitor cilostamide and phosphodiesterase4-selective inhibitor rolipram did not affect catecholamine chronotropic potencies. Only small CGP20712A-resistant positive inotropic effects of (−)-adrenaline were detected in the left atria (13±2% of E max) and ventricular trabeculae (14±5% of E max). The atrial inotropic responses to (−)-noradrenaline and (−)-adrenaline faded; fades were prevented by rolipram but not cilostamide or concurrent cilostamide+rolipram respectively. (−)-Noradrenaline (ICI118551 present) increased left atrial cAMP levels through β1-adrenoceptors that were markedly enhanced by rolipram but unaffected by cilostamide. Concurrent cilostamide+rolipram uncovered inotropic and cAMP responses to (−)-adrenaline (CGP20712A present). We conclude that sinoatrial β2-adrenoceptors are more important than β1-adrenoceptors in the mediation of tachycardia caused by both (−)-noradrenaline and (−)-adrenaline in the newborn piglet. β2-adrenoceptors have only a minor role in the mediation of left atrial and ventricular inotropic effects of (−)-adrenaline. Catecholamine-evoked tachycardia is not controlled by PDE3 or PDE4. PDE4, but not PDE3, controls the atrial inotropic and cAMP β1-adrenoceptor-mediated responses to (−)-noradrenaline. Both PDE3 and PDE4 blunt left atrial inotropic and cAMP responses to (−)-adrenaline through β2-adrenoceptors. [Copyright &y& Elsevier]

Abstract: Aim of study: Although Zanthoxylum schinifolium has long been used in the traditional oriental medicine, cardiac effects have not well been documented. The aim of the present study was to investigate the effects of aqueous extract of leaves and stems from Zanthoxylum schinifolium (AZS) on inotropic effect and atrial natriuretic peptide (ANP) secretion. Materials and methods: The AZS-induced changes in atrial dynamics, cAMP efflux and atrial ANP secretion were determined in isolated perfused beating rabbit atria. Results: AZS increased atrial pulse pressure, stroke volume, and cAMP efflux concomitantly with inhibition of ANP secretion in a concentration-dependent manner. The AZS-induced increases in atrial dynamics and cAMP efflux, and decrease in ANP secretion were attenuated by pretreatment with propranolol and CGP 20712 but not ICI 118,551. Also, the AZS-induced changes in atrial dynamics and ANP secretion were attenuated by diltiazem and KT 5720. Diltiazem and KT 5720 had not significant effect on the AZS-induced increase in cAMP efflux. Conclusion: These results suggest that AZS elicits a positive inotropic effect and decrease in ANP secretion via β1-adrenoceptor-cAMP-Ca2+ signaling in beating rabbit atria. [Copyright &y& Elsevier]

Background and Objective: Autoimmune mechanisms may contribute to the pathogenesis of periodontal disease. Autoantibodies with the potential to bind and activate β1-adrenoceptors (β1-AR) of human gingival fibroblasts were studied to provide evidence of altered humoral immune response in chronic periodontal disease. Material and Methods: Flow cytometry and enzyme-linked immunosorbent assay using cell culture-adherent gingival fibroblasts and/or their purified membranes and/or a synthetic peptide corresponding to the second extracellular loop of human β1-AR were used to detect serum antibodies. The effects of antibodies from chronic periodontal disease patients on PGE2 generation and CD40 expression were also tested. Results: Circulating immunoglobulin G (IgG) from chronic periodontal disease patients (but not from normal individuals) interacted with the fibroblast surface, activating β1-AR. Atenolol or CGP 20712 (beta 1-AR antagonists) and β1 synthetic peptide inhibited the interaction of IgG with β1-AR. Immunoglobulin G from chronic periodontal disease patients also displayed agonist-like activity associated with specific β1-AR activation, increasing PGE2 generation and CD40 overexpression. The corresponding affinity-purified anti-β1-AR peptide IgG mimicked these effects. Both effects were prevented by inhibition of cyclo-oxygenase. Conclusion: This article supports the participation of humoral immune alterations in chronic periodontal disease resulting in postsynaptic functional deregulation. Overproduction of proinflammatory mediators (PGE2 and CD40 expression) is induced as a consequence of antibody–β1-AR interaction. The PGE2–CD40–IgG axis may play a part in the pathophysiological mechanisms underlying the inflammatory process in chronic periodontal disease. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the role of nitric oxide (NO) in relaxations to β-adrenoceptor agonists in mesenteric artery from wild-type (WT) and NO synthase-3 knockout (NOS-3-KO) mice. Isoprenaline, formoterol and BRL 37344 ((R ⁎,R ⁎)-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid) were chosen as non-selective and β2- and β3-adrenoceptor agonists, respectively. Atenolol, ICI 118,551 ((±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) and SR59230A (1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride) were chosen as selective β1-, β2- and β3-adrenoceptor antagonists, respectively. Experiments employing isoprenaline were carried out in the presence of prazosin (0.1 µM). Isoprenaline produced relaxations with a potency of 5.68±0.36 (−log M, n =6) in WT mice. Relaxations to isoprenaline were blocked by atenolol (10 µM) and were absent in vessels from NOS-3-KO animals. Formoterol produced relaxations with two components. ICI 118,551 (1 µM) abolished relaxations to low concentrations of formoterol (0.1–10 µM), but failed to affect relaxations to formoterol (100 µM). In NOS-3-KO mice only the highest concentration of formoterol (100 µM) produced relaxations: the relaxation was resistant to all of the β-adrenoceptor antagonists employed. BRL 37344 (5.75±0.28, n =9) was approximately equipotent with isoprenaline but produced a smaller degree of relaxation, in WT mice. SR59230A (1 µM) abolished relaxations to BRL 37344 in WT mice. In NOS-3-KO mice, BRL 37344 produced concentration-dependent relaxations which were abolished by SR59230A. It is concluded that the predominant β-adrenoceptor mediating relaxations in mouse mesenteric artery is β1, and relaxations involve NOS-3. In addition, β3-adrenoceptors mediate smaller relaxations at least partly independent of NOS-3, and β2-adrenoceptors may mediate smaller relaxations dependent on NOS-3. [Copyright &y& Elsevier]

Abstract: There are evidence suggesting that the function of adrenergic receptor is affected in the amygdala of animals with anxiety-like behavior. However, β-adrenoceptor (β-AR) subtypes, consisting of three subtypes, exert different effects on anxiety regulation. In order to determine the function of the β1-AR subtype in anxiety-like behavior, we investigated the change of β1-AR expression by immunostaining in the basolateral amygdala (BLA) of rats treated by conditional fear training. The results indicated that the level of β1-AR was significantly increased in the BLA of fear-conditioned animals as compared that of controls. In animal behavioral tests, animals treated with selective β1-AR antagonist metoprolol before conditional fear training exhibited a significant attenuation of anxiety-like behavior characterized by increased percentage of time spent and percentage of entries in the open arms, and increased number of head-dips in the elevated plus-maze (EPM) test compared with the animals treated with only saline. Furthermore, the rats pretreated with metoprolol in the conditional fear training significantly decreased the freezing behavior in the test compared with the controls. The results suggested that the β1-AR played an important role in anxiety-like behavior, and inhibition of the β1-AR in the BLA could produce anxiolytic effect. [Copyright &y& Elsevier]

1. The acute effect of amiodarone on haemodynamics (mean arterial pressure and heart rate) and ventricular function (+dP/dtmax and –dP/dtmax) was investigated in conscious rats. In addition, the effects of amiodarone on dobutamine stress were determined. 2. Catheters were inserted in rats into the left ventricle and femoral artery and vein. Three groups of rats received 25 or 50 mg/kg, i.v., amiodarone or vehicle (a 1 : 1 : 8 mixture of Tween 80 : 99.5% ethanol : distilled water), followed by dobutamine (10 µg/kg). 3. The hypotensive effect of 50 mg/kg amiodarone was combined with marked bradycardia and attenuation of +dP/dtmax and –dP/dtmax. A slight, but significant, hypotension was caused by 25 mg/kg amiodarone, without affecting heart rate, +dP/dtmax and –dP/dtmax. However, although both doses of amiodarone attenuated the tachycardia caused by dobutamine, neither 25 nor 50 mg/kg amiodarone affected the increase in mean arterial pressure or the enhanced response of +dP/dtmax and –dP/dtmax. 4. In conclusion, amiodarone caused hypotension, bradycardia, negative inotropic (+dP/dtmax) and lusitropic (–dP/dtmax) effects in conscious rats. In addition, amiodarone attenuated the tachycardia without affecting the hypertensive, contractile (+dP/dtmax) and lusitropic (–dP/dtmax) responses to dobutamine stress. [ABSTRACT FROM AUTHOR]

Abstract: Our previous study showed that a cardioprotective effect was produced by pretreatment with acupuncture at bilateral Neiguan acupoints (PC6) and the effect of EA was diminished by propranolol, a nonspecific antagonist of β-adrenoceptors (β-ARs) which are the most powerful cardiac receptors, indicating an involvement of β-ARs. The present study explored further the signaling mechanism underlying the cardioprotective effect of acupuncture pretreatment in rats subjected to myocardial ischemia and reperfusion (MIR). Myocardial ischemia was achieved by ligating the left anterior descending coronary artery and reperfusion by releasing the ligation. Adult rats were divided into three groups, namely, a normal control (NC) group, a group subjected to ischemia and reperfusion (IR) only, and a group given electro-acupuncture (EA) before IR. For EA, bilateral Neiguan points (PC6) of the rats were stimulated for 30 min once a day for 3 consecutive days. The ST segment of ECG, the ratio of infarct size over risk zone, and the contents of β1-adrenoceptor (β1-AR), Gsα protein and cAMP in ischemic myocardium were compared among the three groups. IR increased the elevation of ECG ST segment, myocardial infarct size, contents of β1-AR, Gsα protein and cAMP. These effects were attenuated by EA pretreatment at bilateral Neiguan acupoints. In conclusion, the present results indicate that EA produces cardioprotective effect against IR which may be mediated via the β1-AR–Gs-protein–cAMP pathway. [Copyright &y& Elsevier]

Ischemic heart disease in diabetic patients might be linked to the accumulation of advanced-glycation end products (AGEs). In ischemic rat hearts, expression of receptor for AGEs and its ligands is significantly enhanced and involved in cardiac ischemia/reperfusion (I/R) injury even in the absence of diabetes. It has recently been reported that diabetic human myocardium cannot be protected by preconditioning. In this context, our hypothesis was that β1-adrenergic preconditioning might be altered in the presence of AGEs. Using an isolated non-working rat heart model, this study investigated the effect of AGEs on cardioprotection induced by transient β1-adrenoceptor ( β1-AR) stimulation with xamoterol (Xa). After 6-hydroxydopamine (6-OHDA) pre-treatment and a 20-min stabilization period, hearts were perfused at constant pressure for 20 min, then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R, Ctrl); and exposed to 0.01 μm Xa for 5 min framed with or without 15.2 μm albumin (Alb) or glycated albumin (Gly Alb). The main endpoints were the mean coronary flow (MCF), the left ventricular end-diastolic pressure (LVEDP), rate-pressure product (RPP) and creatine kinase (CK) release and necrosis area. XA induced an increase in the MCF after I/R ( t = 85 min), a protective effect on the LVEDP, an improvement in RPP, a decrease of CK release during reperfusion and a reduction of necrotic area. The beneficial effects induced by Xa during reperfusion were suppressed by the administration of Gly Alb during Xa infusion, whereas Alb did not hamper Xa-induced protection. These results suggest that AGEs suppress the cardioprotection resulting from the activation of β1-ARs and thus might contribute to cardiovascular damages seen in diabetic patients. [ABSTRACT FROM AUTHOR]

Abstract: To test the hypothesis that κ-opioid receptor stimulation inhibits cardiac hypertrophy induced by β1-adrenoceptor stimulation, we determined the effects of trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective β-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of β2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii) [3H]leucine incorporation; and iii) cell size. 10 μmol/l isoprenaline increased all three parameters. The effects were abolished by 2 μmol/l propranolol, a β-adrenergic receptor antagonist, or 300 nmol/l CGP20712A, a β1-adrenoceptor antagonist, but not by 100 nmol/l ICI118,551, a β2-adrenoceptor antagonist. The effects were also abolished by Rp-cAMPs 100 μmol/l, a protein kinase A inhibitor and not by pertussis toxin 5 mg/l. The effects of isoprenaline in the presence or absence of ICI118,551 were also abolished by 1 μmol/l U50,488H. The inhibitory effects of U50,488H were abolished by 1 μmol/l nor-binaltorphimine, a selective κ-opioid receptor antagonist. U50,488H also abolished the increases in the amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by 10 μmol/l isoprenaline in the presence or absence of ICI118,551, an effect also abolished by nor-binaltorphimine. In conclusion the results show that κ-opioid receptor stimulation abolished both the cardiac hypertrophy and enhanced amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by β1-adrenoceptor stimulation. [Copyright &y& Elsevier]

We have compared the ability of three radioligands, [125I]-cyanopindolol, [3H]-CGP 12,177 and [3H]-dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [3H]-CGP 12,177 had very similar affinity for β1- and β2-adrenoceptors (about 40 pM), [125I]-cyanopindolol and [3H]-dihydroalprenolol had 4- to 6-fold higher affinity for β2- as compared to β1-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [125I]-cyanopindolol at β3-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β3-adrenoceptor affinity of [3H]-CGP 12,177 and [3H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [3H]-CGP 12,177 and [3H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β3-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [125I]-cyanopindolol appears the least unsuitable to label β3-adrenoceptors. There is a need for high-affinity radioligands which are either selective for β3-adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Immunoadsorption has been shown to improve cardiac performance and reduce mortality in patients with dilated cardiomyopathy. In this study, the underlying mechanism for these beneficial effects was investigated in cultured rat cardiomyocytes. Methods and results: Immunoadsorption was performed in patients with dilated cardiomyopathy (n =7). Antibody-induced complement-dependent cytotoxicity was investigated by colorimetric MTT. Autoantibodies against the β1-adrenoceptor were detected by ELISA and purified. Column eluent from six patients exhibited a cytotoxic effect, three patients were positive for the β1-adrenoceptor autoantibodies. The purified autoantibodies were able to visualize the β1-adrenoceptors by immunocytofluorescence on rat cardiomyocytes, and also displayed partial agonist properties and induced a positive chronotropic effect, which were blocked by the β1-selective antagonist bisoprolol and the peptide corresponding to the β1-adrenoceptor. Column eluent from one patient induced apoptosis in nick end labelling test (8.1±1.7% vs. 2.9±1.2% in control, p <0.05). Conclusion: Autoantibodies removed by immunoadsorption from patients with dilated cardiomyopathy have a pathophysiological role, as shown by the complement-dependent cytotoxicity and chronotropic action on rat cardiomyocytes. This implies that removal of circulating autoantibodies might be part of the underlying mechanism for improved cardiac function. [Copyright &y& Elsevier]

Abstract: Objective.: Growing evidence suggests that the β1-adrenoceptor-directed autoimmune mechanism may play an important role in the pathogenesis of idiopathic dilated cardiomyopathy. The aim of this study is to further study the effect of specific immunoabsorption of anti-β1-adrenoceptor autoantibodies on cardiac structure and function in autoimmune cardiomyopathy in rabbits. Methods and results.: Twenty-four male rabbits were divided into 2 groups: (1) one immunized with β1-adrenoceptor peptide (β1 group, n = 16), and (2) the other receiving saline injection as a control (control group, n = 8). Immunization was performed once a month for 8 months. A high concentration of anti-β1-adrenoceptor autoantibodies was exhibited throughout the immunization period. Rabbits in the β1 group showed increased left ventricular end-diastolic diameter (LVDd), decreased left ventricular ejection fraction (LVEF) and increased LV mass/body weight ratio after the 8th month. Immunoabsorption with β1-adrenoceptor peptide column was able to remove up to 35% of anti-β1-adrenoceptor autoantibodies in 2 h, resulting in decreased LVDd and increased LVEF 3 months after. Conclusions.: Specific removal of anti-β1-adrenoceptor autoantibodies improved cardiac structure and function in experimental autoimmune cardiomyopathy. These results suggest that anti-β1-adrenoceptor autoantibodies are of pathogenic importance in the induction of cardiomyopathy, and that specific immunoabsorption as an emerging therapy may be considered when anti-β1-adrenoceptor autoantibodies are pathophysiologically involved. [Copyright &y& Elsevier]

To clarify whether apoptosis is involved in the injury processes induced by autoantibody against cardiac β1-adrenoceptor, we investigated the biological and apoptotic effects of antibodies on cultured neonatal rat cardiomyocytes. Wistar rats were immunized with peptides corresponding to the second extracellular loop of the β1-adrenoceptor to induce the production of anti-β1-adrenoceptor antibodies in the sera. Immunoglobulin (Ig) G in the sera was detected using synthetic antigen enzyme-linked immunosorbent assay and purified using the diethylaminoethyl cellulose ion exchange technique. Apoptosis of cardiomyocytes was evaluated using agarose gel electrophoresis and flow cytometry. Our results showed that the positive serum IgG greatly increased the beating rates of cardiomyocytes and showed an “agonist-like” activity. Furthermore, positive serum IgG induced cardiomyocyte apoptosis after treatment with β1-adrenoceptor overstimulation for 48 h. The effects of monoclonal antibody against β1-adrenoceptor were also found to be similar to those of positive serum IgG. It was suggested that the autoantibody could induce cardiomyocyte apoptosis by excessive stimulation of β1-adrenoceptor. Edited by Satoru TANAKA [ABSTRACT FROM AUTHOR]

Background: Growing evidence suggests that autoimmune mechanism plays an important role in the pathogenesis of cardiomyopathy. The purpose of this study was to investigate whether passive transfer of IgG and/or lymphocytes from rabbits with autoimmune cardiomyopathy is able to reproduce cardiomyopathic changes in severe combined immunodeficiency (SCID) mice. Methods and results: SCID mice were injected intraperitoneally with IgG and/or peripheral blood lymphocytes (PBL) from either rabbits immunized with both β1-adrenoceptor peptide and M2-muscarinic receptor peptide (β1+M2 group) or rabbits with adjuvant (N group). Thirty five SCID mice were divided into seven groups; N-IgG, N-PBL, N-IgG & PBL, (β1+M2)-IgG, (β1+M2)-PBL, (β1+M2)-IgG & PBL and control groups. Heart weight in three (β1+M2) groups were significantly increased. All mice in three (β1+M2) groups showed high titer of rabbit anti-β1 adrenocepter autoantibodies, and 4 mice in the (β1+M2)-PBL group and 3 mice in the (β1+M2)-IgG & PBL group showed a significant increase in titer of rabbit anti-M2-muscarinic receptor autoantibodies. Focal infiltration of inflammatory cells in the myocardium was observed in the (β1+M2)-IgG & PBL group. In the (β1+M2)-PBL group and (β1+M2)-IgG & PBL group, cardiomyocyte diameters were significantly increased. Some myocytes of the (β1+M2)-IgG & PBL group exhibited intracellular edema, clumps of Z-band and increased numbers of mitochondria by using electron microscopy. Conclusion: Transfer of IgG and PBL from rabbits immunized with combined β1 and M2 peptides was able to reproduce the early stage of cardiomyopathic changes in SCID mice [ABSTRACT FROM AUTHOR]

Aim: Genetic polymorphisms causing Ser49Gly and Gly389Arg mutants of β1- adrenoceptor may result in significant changes in the function of this receptor. The aim of the present study was to investigate the frequencies of the Ser49Gly and Gly389Arg mutant alleles in healthy Chinese populations and to investigate the differences between 2 Chinese ethnic groups (Han and Dai populations) with respect to the frequencies of these alleles. Methods: A total of 225 Han Chinese and 175 Dai Chinese unrelated healthy volunteers were recruited for this study. Genomic DNA was extracted from peripheral blood leukocytes by using a standard manual chloroform-phenol extraction. Fragments spanning the 2 polymorphisms were amplified by using polymerase chain reaction with template genomic DNA and relevant primers. The DNA products including the polymorphic loci were subjected to restriction endonuclease digestion with Eco0109I and BcgI. Digested fragments were detected with an ultraviolet detector after electrophoresis (100 V for approximately 1.5 h). Results: The frequencies of the Gly49 and Arg389 alleles were, respectively, 16.2% and 76.4% in the Han population and 14.6% and 75.7% in the Dai population. Conclusion: The polymorphisms causing the Ser49Gly and Gly389Arg mutations of the β1-adrenoceptor existed in both healthy Han and Dai Chinese populations. The frequencies of the Ser49Gly and Gly389Arg mutant alleles were not significantly different in the Han and Dai populations. However, the frequency of the Gly389 variant seems to be significantly lower in these 2 populations than in an African-American population. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated whether the responses of right atria from sinoaortic denervated rats to CGP12177 (4(3-t-butylamino-2-hydroxypropoxy benzidimidazole-2 one, hydrochloride)), isoprenaline and norepinephrine desensitized in parallel and whether CGP12177 interacted with distinct conformations of β-adrenoceptors. Right atria from rats 48 h after sinoaortic denervation were subsensitive to isoprenaline, norepinephrine and CGP12177. One week after sinoaortic denervation, the sensitivity to CGP12177 had recovered whereas the responses to isoprenaline and norepinephrine were still subsensitive, suggesting that the binding sites for these molecules showed independent behavior. In atria from 48 h sinoaortic-denervated rats, propranolol or 3 μM CGP20712A (2-hydroxy-5(2-((2-hydroxy-3-(4-((methyl-4-trifluormethyl)1H imidazole-2-yl)-phenoxypropyl) amino) ethoxy)-benzamide monomethane sulphonate)) blocked the responses to 10 nM–1 μM CGP12177 and steepened the curves. The concentration–response curves to CGP12177 in the presence of ICI118,551 (erythro-dl-1(-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol) were biphasic, suggesting that CGP12177 interacted with at least two conformations of β-adrenoceptors that showed negative cooperativism, one acting through β2-adrenoceptor-Gi and the other via β1-adrenoceptor-Gs. This hypothesis was confirmed in right atria from sinoaortic-denervated rats treated with pertussis toxin. [Copyright &y& Elsevier]

To determine the role played by Tyr356 (7.43) in the rat β1-adrenoceptor in binding the antagonists (+/–)cyanopindolol (4-[3-(t-butylamino]-3-(2′-cyano-indoloxy)-2-propanolol) and its iodinated analogue (+/–)[125Iodo]cyanopindolol (1-(t-butylamino]-3-(2′-cyano-3′-iodo-indoloxy)-2-propanolol), Tyr356 (7.43) was mutated to either Phe or Ala and binding affinities determined for wild type and mutant rat β1-adrenoceptors. Our results indicate that Tyr356 (7.43) is important for (+/–)cyanopindolol, but not (+/–)[125Iodo]cyanopindolol, binding and that (+/–)cyanopindolol adopts a “reverse” binding orientation whereas (+/–)[125Iodo]cyanopindolol cannot be accommodated in this binding mode. We define a “reverse” antagonist binding mode as one where the aryloxy moiety interacts with residues on transmembrane helices 1, 2, 3 and 7. The β1-adrenoceptor site-directed mutagenesis results are the first to support a “reverse” antagonist binding orientation and the involvement of Tyr356 (7.43) in this binding mode. [Copyright &y& Elsevier]

Cardiac effects of catecholamines on the L-type calcium channel depend on β-adrenoceptor subtype (β1- vs. β2-adrenoceptor). Chronic overexpression of these receptors leads to hypertrophy and early death at moderate (β1) or excessive (β2) levels of overexpression respectively. In order to examine the role of L-type calcium channels in altered cardiomyocyte calcium homeostasis found with β1-adrenoceptor overexpression, and to understand the quantitative differences between β-adrenoceptor subtypes regarding calcium channel regulation, we examined single channels in myocytes obtained from β1- and β2-adrenoceptor transgenic mice. The effects of the agonist isoproterenol were investigated and compared with acute receptor stimulation in the respective non-transgenic littermates. Channels from β1-adrenoceptor transgenic mice have normal baseline activity, and channel number is not reduced. This contrasts to previous findings with β2-adrenoceptor transgenic mice, where channel activity is depressed. Isoproterenol is unable to stimulate channel activity in both transgenic models. In conclusion, the L-type calcium channel is not likely to be involved in alterations of calcium handling of β1-adrenoceptor transgenic myocytes. Furthermore, chronic β1-adrenoceptor overexpression does not depress channel activity, giving another example of the difference between β1- and β2-adrenoceptor signal transduction. [ABSTRACT FROM AUTHOR]

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A substantial body of evidence suggests involvement of the human β1-adrenoceptor (β1-AR) gene in the pathophysiology of dilated cardiomyopathy (DCM), a severe heart disease of significant public health impact. β1-AR-mediated signal transduction is dramatically altered due to downregulation, resulting in an impairment of myocardial response. The important role of genetic factors in idiopathic dilated cardiomyopathy (IDCM) recently recognized, we analyzed this prime candidate gene for genetic variation in carefully selected patients and controls. In this preliminary study, 18 single nucleotide polymorphisms were observed, 17 of which were located in the N-terminal and C-terminal region of the coding exon, resulting in 7 amino acid exchanges: Ser-49–Gly, Ala-59–Ser, Gly-389–Arg, Arg-399–Cys, His-402–Arg, Thr-404–Ala, and Pro-418–Ala. These mutations resulted in 11 different β1-AR genotypes. Importantly, the genotypes carrying the Ser-49–Gly mutation in the N-terminus of the molecule in a heterozygous or homozygous form were observed significantly more frequently in the group of IDCM patients. The present results may provide a clue on the molecular mechanisms involved in IDCM, and add moreover interesting information on nature, distribution, and evolutionary aspects of sequence variation in human adrenergic receptor genes. [ABSTRACT FROM AUTHOR]