Human β3-AR Research Articles

Cardiac effects of long-term active immunization with the second extracellular loop of human β1- and/or β3-adrenoceptors in Lewis rats

β1- and β3-adrenoceptor (AR) auto-antibodies were detected in patients with dilated cardiomyopathy. Many studies have shown that β1-AR auto-antibodies with partial agonist-like effect play an important role in the pathogenesis of this disease. Moreover, a recent study carried out in our laboratory has shown that β3-AR antibodies (β3-ABs), produced in rats, were able to reduce cardiomyocyte contractility via β3-AR activation. The aims of this study were (1) to investigate, in isolated cardiomyocytes from rabbit, the role of Gi proteins in the β3-ABs-induced cardiac negative inotropy, (2) to determine whether β3-ABs may exhibit β3-AR antagonistic property which is characteristic of partial agonists, and (3) to determine whether long-term active immunization producing both β1-ABs and/or β3-ABs leads to the development of cardiac dysfunction in Lewis rats.Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of human β3-AR and/or β1-AR. Agonistic effect of β3-ABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Echocardiography and ex vivo isolated perfused heart studies were conducted on immunized rats. Finally, β-AR expression was quantified by immunofluorescence and RT-qPCR.SR58611A (10nM), a preferential β3-AR agonist, and purified β3-ABs (25μg/ml) induced a decrease in cell shortening (−39.71±4.9% (n=10) and −17.06±3.9% (n=10) respectively). This effect was significantly inhibited when the cardiomyocytes were preincubated with pertussis toxin (0.3μg/ml), a Gi protein inhibitor (p<0.05). In addition, SR58611A-mediated negative inotropic effect was decreased when cardiomyocytes were preincubated with β3-ABs (p<0.0001). Echocardiography revealed a decrease in the fractional shortening and ejection fraction in rats immunized against β1-AR and both β1- and β3-AR. However, the study on isolated heart showed a decrease of the isoproterenol-induced lusitropic and inotropic effects in the 3 groups of immunized rats. These systolic and diastolic dysfunctions are correlated with a decrease in the expression of β1-ARs and an increase of β3-ARs in rats immunized against the β1-AR and an increase of both β3-AR and β1-AR in rats immunized against the β3-AR. For the first time, these results showed that β3-ABs had a β3-AR partial agonist-like activity which might play a role in the pathogenesis of cardiac dysfunction.

β3-Adrenergic Regulation of L-Type Ca2+ Current and Force of Contraction in Human Ventricle

β3-Adrenergic receptor (β3-AR) is expressed in human atrial and ventricular tissues. Recently, we have demonstrated that it was involved in the activation of L-type Ca(2+) current (I(Ca,L)) in human atrial myocytes and the force of contraction of human atrial trabeculae. In the present study, we examined the effect of β3-AR agonist CGP12177 which also is a β1-AR/β2-AR antagonist on I(Ca,L) in human ventricular myocytes (HVMs) and the force of contraction of human ventricular trabeculae. CGP12177 stimulated I(Ca,L) in HVMs with high potency but much lower efficacy than isoprenaline. The β3-AR antagonist L-748,337 inhibited the effect of CGP12177. CGP12177 and L748,337 competed selectively on β3-ARs because L748,337 had no effect on isoprenaline-induced stimulation of I(Ca,L), while CGP12177 completely blocked the effect of isoprenaline. The activation of β3-ARs by CGP12177 does not involve the activation of Gi proteins because CGP12177 had no effect on forskolin-induced stimulation of I(Ca,L). CGP12177 had no effect on the force of contraction of human ventricular trabeculae. L-NMMA, an inhibitor of NO synthase, and IBMX, a nonselective inhibitor of phosphodiesterases, did not potentiate the effect of CGP12177 either on contraction of human ventricular trabeculae or on I(Ca,L) in HVMs. We conclude that in human ventricles β3-AR activation has no inotropic effect, while it slightly increases I(Ca,L). In contrast to human atrium, the activation of β3-ARs in human ventricle is not accompanied by increased activity of phosphodiesterases.

Antibodies against the second extracellular loop of β1-adrenergic receptors induce endothelial dysfunction in conductance and resistance arteries of the Wistar rat

Autoantibodies against β₁-adrenoceptors (β₁-ARs) have been detected in the serum of patients with various cardiac diseases; however, the pathological impact of these autoantibodies (β₁-AABs) has only been evaluated in cardiac tissue. The purpose of the present study was to evaluate whether β₁-AABs have deleterious effects on vascular reactivity in rats. An enzyme-linked immunosorbent assay was used to detect β₁-AABs in sera from immunized rats over a period of 1-3 months using the peptidic sequence of the second extracellular loop of human β₁-AR. Functional studies were performed in thoracic aortic (TA) and small mesenteric artery (SMA) rings from immunized rats. Following pre-contraction with phenylephrine (0.3 μM and 3 μM for the TA and SMA respectively), cumulative concentration-response curves (CCRCs) to various β-AR agonists (isoproterenol, dobutamine, salbutamol, SR 58611A), acetylcholine, A23187, and sodium nitroprusside (SNP) were then plotted. The relaxations induced by dobutamine, SR 58611A, and acetylcholine were significantly impaired, but salbutamol-induced relaxations were not affected, in both vessels from immunized rats. A significant impairment of isoproterenol-induced relaxation was only observed in SMA. CCRCs to SNP were not modified in either of the vessels. A23187-induced relaxation was impaired in immunized rats. Following pretreatment with L-arginine, vasorelaxation to acetylcholine and SR 58611A was restored in immunized rats. This study demonstrates that immunization against the second extracellular loop of β₁-ARs has a deleterious impact on vasorelaxations in the TA and SMA of rats, involving alterations in endothelium-dependent NO signaling pathways.

Myogenic effect of SP-1f and SP-1h two novel β3-adrenoceptor (β3-AR) agonists in human colonic circular smooth muscle

The effect of two novel β3-adrenoceptor (β3-AR) agonists SP-1f and SP-1h on human colon circular smooth muscle contractility and β3-AR mRNA expression have been determined. β3-AR is ascertained co-participates to the control of the gut motility. Isometric tension on human colon muscle strips was measured in response to increasing concentrations of SP-1f, SP-1h and (−)-isoprenaline, alone and in the presence of Betaxolol, ICI 11,855 and SR 59230A (β1-, β2- and β3-AR antagonists, respectively). (−)-Isoprenaline concentration-dependently relaxed circular muscle strips with an EC50=0.32±0.06μM. Such an effect was antagonized either by the contemporaneously presence of Betaxolol and ICI 11,855 [(−)-isoprenaline EC50=1.75±0.35μM, pKB=7.88±0.10] or by Betaxolol, ICI 11,855 and SR 59230A [(−)-isoprenaline EC50=3.49±0.38μM, pKB=8.51±0.14]. Besides, SP-1f and SP-1h concentration-dependently relaxed circular muscle strips with an EC50=0.35±0.07μM and 0.45±0.12μM, respectively. These values remained unchanged by blocking the β1- and β2-AR. The presence of SR 59230A antagonized the relaxing effect of SP-1f (EC50=3.51±0.94μM, pKB=8.93±0.16) and did not modify the SP-1h relaxing potency. In colon circular smooth muscle and in mucosa, β3-AR mRNA expression levels were found to be 0.39±0.70 and 0.26±0.12 (P<0.05), respectively. Such results provide further evidence of the β3-adrenoceptor functional role in the human colon and the crucial contribution of SP-1f to the control of the gut dysmotility.

Enhanced Expression of β3-Adrenoceptors in Cardiac Myocytes Attenuates Neurohormone-Induced Hypertrophic Remodeling Through Nitric Oxide Synthase

β1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. β3-AR expression is enhanced in diseased human myocardium, but its impact on remodeling is unknown. Mice with cardiac myocyte-specific expression of human β3-AR (β3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). β3-TG and WT had similar morphometric and hemodynamic parameters at baseline. β3-AR colocalized with caveolin-3, endothelial nitric oxide synthase (NOS) and neuronal NOS in adult transgenic myocytes, which constitutively produced more cyclic GMP, detected with a new transgenic FRET sensor. Iso and Ang II produced hypertrophy and fibrosis in WT mice, but not in β3-TG mice, which also had less re-expression of fetal genes and transforming growth factor β1. Protection from Iso-induced hypertrophy was reversed by nonspecific NOS inhibition at low dose Iso, and by preferential neuronal NOS inhibition at high-dose Iso. Adenoviral overexpression of β3-AR in isolated cardiac myocytes also increased NO production and attenuated hypertrophy to Iso and phenylephrine. Hypertrophy was restored on NOS or protein kinase G inhibition. Mechanistically, β3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. Cardiac-specific overexpression of β3-AR does not affect cardiac morphology at baseline but inhibits the hypertrophic response to neurohormonal stimulation in vivo and in vitro, through a NOS-mediated mechanism. Activation of the cardiac β3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.

Implications of a Vasodilatory Human Monoclonal Autoantibody in Postural Hypotension

Functional autoantibodies to the autonomic receptors are increasingly recognized in the pathophysiology of cardiovascular diseases. To date, no human activating monoclonal autoantibodies to these receptors have been available. In this study, we describe for the first time a β2-adrenergic receptor (β2AR)-activating monoclonal autoantibody (C5F2) produced from the lymphocytes of a patient with idiopathic postural hypotension. C5F2, an IgG3 isotype, recognizes an epitope in the N terminus of the second extracellular loop (ECL2) of β2AR. Surface plasmon resonance analysis revealed high binding affinity for the β2AR ECL2 peptide. Immunoblotting and immunofluorescence demonstrated specific binding to β2AR in H9c2 cardiomyocytes, CHO cells expressing human β2AR, and rat aorta. C5F2 stimulated cyclic AMP production in β2AR-transfected CHO cells and induced potent dilation of isolated rat cremaster arterioles, both of which were specifically blocked by the β2AR-selective antagonist ICI-118551 and by the β2AR ECL2 peptide. This monoclonal antibody demonstrated sufficient activity to produce postural hypotension in its host. Its availability provides a unique opportunity to identify previously unrecognized causes and new pharmacological management of postural hypotension and other cardiovascular diseases.

Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody

G protein-coupled receptors (GPCRs) are integral membrane proteins that play an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. Thus far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists1,2 or receptor stabilization by mutagenesis3-5. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here, we present structures of active-state β2AR bound to three chemically distinct agonists: the ultra high-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ~80 pM. The adrenaline-bound receptor structure is overall similar to the others, but shows substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

Molecular interactions between fenoterol stereoisomers and derivatives and the β2-adrenergic receptor binding site studied by docking and molecular dynamics simulations

The β2 adrenergic receptor (β2-AR) has become a model system for studying the ligand recognition process and mechanism of the G protein coupled receptors activation. In the present study stereoisomers of fenoterol and some of its derivatives (N = 94 molecules) were used as molecular probes to identify differences in stereo-recognition interactions between β2-AR and structurally similar agonists. The present study aimed at determining the 3D molecular models of the fenoterol derivative-β2-AR complexes. Molecular models of β2-AR have been developed by using the crystal structure of the human β2-AR T4 lysozyme fusion protein with bound (S)-carazolol (PDB ID: 2RH1) and more recently reported structure of a nanobody-stabilized active state of the β2-AR with the bound full agonist BI-167107 (PDB ID: 3P0G). The docking procedure allowed us to study the similarities and differences in the recognition binding site(s) for tested ligands. The agonist molecules occupied the same binding region, between TM III, TM V, TM VI and TM VII. The residues identified by us during docking procedure (Ser203, Ser207, Asp113, Lys305, Asn312, Tyr308, Asp192) were experimentally indicated in functional and biophysical studies as being very important for the agonist-receptor interactions. Moreover, the additional space, an extension of the orthosteric pocket, was identified and described. Furthermore, the molecular dynamics simulations were used to study the molecular mechanism of interaction between ligands ((R,R’)- and (S,S’)-fenoterol) and β2-AR. Our research offers new insights into the ligand stereoselective interaction with one of the most important GPCR member. This study may also facilitate the design of improved selective medications, which can be used to treat, prevent and control heart failure symptoms.Electronic supplementary materialThe online version of this article (doi:10.1007/s00894-013-1981-y) contains supplementary material, which is available to authorized users.

AMP-activated protein kinase dependent signaling pathways and modulation of autophagy are involved in the anti-hypertrophic effect of the human beta 3 adrenoreceptor

Background: The human heart contains β3-adrenoreceptors (β3AR) which are coupled to nitric oxide (NO) synthesis; however, their role in cardiac remodeling is unclear Methods: Mice with cardiomyocyte-specific overexpression of human β3AR (TG) & wild-type littermates (WT) were treated with isoproterenol (Iso30mg/kg/d), angiotensin II (AngII 2mg/kg/d), or saline by osmotic minipump, with/without L-NAME (2mg/mL). In vitro hypertrophic response, signaling & NO production (EPR) were analyzed in neonatal rat ventricular myocytes infected with an adenovirus expressing the human β3AR (hβ3-NRVM). Subcellular localization of β3AR was studied in adult ventricular myocytes using the proximity ligation assay (PLA). Results: PLA showed colocalization of the β3AR & eNOS with caveolin-3, which co-segregated with AMP-activated protein kinase (AMPK) & eNOS after sucrose gradient fractionation. Following 10 day Iso or AngII, cardiac myocyte hypertrophy & an upregulation of βMHC mRNA expression were observed in WT (μm2: 682±18 Sal vs 914±54 Iso; p<0.001; n=9), but not TG mice (697±16 Sal vs 783±39 Iso; p=ns; n=9). L-NAME treatment abrogated the protection from Iso-induced hypertrophy in TG mice (728±30 L-NAME vs 963±60 L-NAME+Iso; p<0.001; n = 9). In vitro, when compared to GFP-NRVM, hβ3-NRVM showed higher NO production (GFP 2108±245; hβ3 3631±341; n =4; p<0.01) & failed to show a cardiac hypertrophic response (assessed by measurement of cell size, protein synthesis & NFAT activation) following PE stimulation. Treatment of hβ3-NRVM with L-NAME abolished the protection from PE-induced hypertrophy. In GFP-NRVM, PE inhibited AMPK (phospho-Thr172 AMPK), an inhibitor of protein synthesis & cardiac hypertrophy, yet in hβ3-NRVM AMPK phosphorylation was preserved after PE. Transfection of hβ3-NRVM with siRNA against AMPK abolished the protection from PE-induced hypertrophy (μm2: Scr: hβ3-veh 1088±64; hβ3-PE 1139±56, p=ns; siRNA: hβ3-veh 1143±57; hβ3- PE 1594±101, n=3; p<0.01). AMPK is also known to activate autophagy. In GFP-NRVM, PE inhibited autophagy (LC3 II/I ratio and p62 abundance), yet in hβ3-NRVM autophagy was preserved after PE. Conclusion: Cardiac-specific overexpression of β3AR inhibits hypertrophy following neurohormone stimulation in vivo & in vitro, partly through NO-dependent & AMPK-dependent mechanisms, consistent with colocalization of hβ3AR with NOS and AMPK in caveolae. Protection from PE-induced hypertrophy may involve AMPK-mediated maintenance of autophagy. Cardiac β3ARs may thus provide a therapeutic target in the treatment of cardiac remodeling associated with elevated sympathetic tone.

Development of an enzyme‐linked immunosorbent assay and a beta‐1 adrenergic receptor–based assay for monitoring the drug atenolol

Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive-binding assay, based on the interaction between ATL and its target receptor, β1 adrenergic receptor (β1AR). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme-linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 ± 0.048 and 0.032 ± 0.016 ng/ml, respectively, and the Abs did not cross-react with any of the tested beta-blocker drugs. Furthermore, a human β1AR (h-β1AR) was stably expressed in Spodoptera frugiperda cells (Sf9). The receptor was employed to develop a competitive-binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h-β1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose-dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost-effective large-scale, high-throughput monitoring of ATL in environmental, agricultural, and biological samples.

High‐throughput flow cytometry compatible biosensor based on fluorogen activating protein technology

Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.

Histamine Modulates Isoproterenol Efficacy at the β2 Adrenoceptor: Inferences Regarding Allosteric Modulation by Imidazole-Containing Compounds

In the last few years, evidence has been mounting of the possible allosteric modulation of second messenger formation on β2-adrenoceptors (β2ARs) induced by non-arylethylamine compounds. We herein addressed this issue by carrying out functional and molecular modeling studies to explore the possibility that imidazole-containing compounds (ICCs) affect the formation and accumulation of cAMP by mediating β2AR activation. Results show that histamine (an ICC) had no effect on basal cAMP accumulation in COS-7 cells transfected with the human β2AR (10.4 ± 1.1 pmol/mg protein), but significantly augmented the receptor response to the agonist isoproterenol (137 ± 7% of controls, EC50 10.5 μM, pEC50 5.87 ± 0.06). Moreover, histamine (10 μM) did not affect the isoproterenol inhibition of 3H]-dihydroalprenolol binding to hβ2ARs in membranes of transfected COS-7 cells. Q-site Finder program and molecular docking studies identified possible sites of interaction for ICCs on the β2AR, but the estimated affinities were lower than those reported for well-known β2AR ligands on the orthosteric site. On the basis of the present experimental and theoretical data, we postulate that direct ICC-hβ2AR interactions can allosterically modulate agonist-induced receptor activation. Implications of these findings for drug design are discussed.

Attenuated Desensitization of β-Adrenergic Receptor by Water-Soluble N-Nitrosamines That Induce S-Nitrosylation Without NO Release

The clinical problem of loss of β-adrenergic receptor (β-AR) response, both in the pathogenesis of heart failure and during therapeutic application of β-agonists, is attributable, at least in part, to desensitization, internalization, and downregulation of the receptors. In the regulation of β-AR signaling, G protein-coupled receptor kinase 2 (GRK2) primarily phosphorylates agonist-occupied β-ARs, and this modification promotes desensitization, internalization, and downregulation of β-ARs. It has been demonstrated that GRK2 is inhibited by its S-nitrosylation. However, compounds that induce S-nitrosylation, such as S-nitrosoglutathione, simultaneously generate NO, which has been demonstrated to operate for cardiovascular protection. We examine whether S-nitrosylation without NO generation inhibits desensitization of β(2)-AR by GRK2. We thus aim to synthesize compounds that specifically induce S-nitrosylation. We have developed water-soluble N-nitrosamines that have S-nitrosylating activity but lack NO-generating activity. These compounds, at least partly, rescue β-AR from desensitization in HEK 293 cells expressing FLAG-tagged human β(2)-AR and in rat cardiac myocytes. They inhibit isoproterenol-dependent phosphorylation and internalization of β(2)-AR. Indeed, they nitrosylate GRK2 in vitro and in cells, and their S-nitrosylation of GRK2 likely underlies their inhibition of β(2)-AR desensitization. Compounds that induce S-nitrosylation without NO release inhibit GRK2 and attenuate β(2)-AR desensitization. Developing water-soluble drugs that specifically induce S-nitrosylation may be a promising therapeutic strategy for heart failure.

Effects of the β‐agonist, isoprenaline, on the down‐regulation, functional responsiveness and trafficking of β2‐adrenergic receptors with N‐terminal polymorphisms

The β2-AR (β2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of β2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2-AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2-AR (designated β2-AR-RE, β2-AR-GE, β2-AR-RQ and β2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg16 (β2-AR-RE and β2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (β2-AR-GE and β2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β2-AR-RE relative to β2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of β2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of β2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.

Abstract 215: An Agonistic Human Monoclonal Autoantibody to the β2-adrenergic Receptor: Implications in Postural Hypotension

Functional autoantibodies directed to the autonomic receptors have been increasingly recognized to contribute to the pathophysiology of various cardiovascular diseases. However, to date, no human monoclonal autoantibodies to these receptors with agonist activity have been available. We have recently reported a subgroup of patients with idiopathic postural hypotension who have no known etiology are associated with vasodilatory autoantibodies to the β2-adrenergic receptor (β2AR). Using standard hybridoma techniques, we produced a β2AR-activating monoclonal autoantibody C5F2 from the peripheral blood lymphocytes of a patient with idiopathic postural hypotension and high ELISA antibody titers to a peptide corresponding to the second extracellular loop (ECL2) of β2AR. C5F2 was of the IgG3 isotype and recognized an epitope located at the N-terminus of β2AR ECL2 as determined by epitope mapping using multipin peptide synthesis. Confocal immunofluorescence revealed C5F2 binding to the β2AR in Chinese hamster ovary (CHO) cells transfected with human β2AR and rat cremaster arterioles, both of which were inhibited by presabsorption with the β2AR ECL2 peptide. Functionally, the C5F2 IgG stimulated cyclic AMP production in β2AR-expressing CHO cells in a dose-dependent manner (0.005-5 mg/ml). C5F2-induced cyclic AMP activation was specifically blocked by both the β2AR ECL2 peptide and β2AR selective antagonist ICI-118551. In an isolated rat cremaster arteriole assay, infusion of the C5F2 IgG produced a potent vasodilation which was inhibited by the β2AR ECL2 peptide and β2AR blocker ICI-118551 (from 67.8±5.8% to 9.3±5.1% and 4.4±2.1% of maximal dilatory response, respectively), indicating that antibody activation of vascular β2AR may contribute to systemic vasodilation. These data support the concept that circulating agonistic autoantibodies serve as vasodilators and may cause or exacerbate orthostasis by altering the compensatory postural vascular response. The availability of this first human monoclonal autoantibody to the β2AR provides opportunities to identify previously unrecognized causes and new pharmacological management of postural hypotension.

Detection of Anti–β1-AR Autoantibodies in Heart Failure by a Cell-Based Competition ELISA

Autoantibodies directed against the second extracellular loop of the cardiac β1-adrenergic receptor (β1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. We analyzed sera from 167 DCM patients (ejection fraction<45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human β1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-β1-AR antibody (β1-AR MAb) directed against the functionally relevant β1-AR epitope. Anti-β1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-β1-AR antibody titers (defined as inhibition of β1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular β1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. We established a simple and efficient screening assay detecting disease-relevant β1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.

Les anticorps dirigés contre la deuxième boucle extracellulaire des récepteurs adrénergiques β1 induisent une dysfonction endothéliale dans l’aorte de rat Wistar

PurposeTo 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. MethodsNine-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. ResultsCell 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. ConclusionsThe 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.

Interplay of microRNAs and RNA binding proteins

We have demonstrated previously that β‐adrenergic receptor (AR) mRNAs are targets for RNA‐binding proteins (BPs) that bind to A+U‐rich elements (AREs) within their 3′untranslated regions (UTRs). Our hypothesis is that ARE‐BPs and miRNAs interact affecting each other's ability to modulate gene expression. Herein, we focus on miR let‐7 and the ARE‐BP, HuR.ResultsThe human β1‐AR 3′UTR was placed down‐stream of a dual luciferase reporter and cells were treated with miR mimics/inhibitors. Compared to control (1.00), let‐7 mimic down‐regulated LUC/β1‐AR expression (0.61 ± 0.04). Conversely, let‐7 inhibitor increased β1‐AR expression (1.80 ± 0.21). Mutagenesis of the let‐7 site, (−)let‐7, completely abrogated the effects of let‐7 mimic/inhibitor. HuR knockdown (~75%) also resulted in decreased LUC/β1‐AR expression (0.62 ± 0.03). In cells expressing LUC/β1‐AR(−)let‐7, β‐AR down‐regulation 2o to HuR knockdown was attenuated (0.85 ± 0.06). The effects of let‐7 mimic/inhibitor were also examined in cells expressing LUC/β1‐AR(−)ARE. Absence of the ARE completely abrogated the effects of both let‐7 mimic/inhibitor.ConclusionThe current data supports the conclusion that modulation of β‐AR expression by HuR is let‐7 dependent. Reciprocally, let‐7 function appears dependent on the presence of an ARE and on the proteins that bind to it.

Autoantibodies against cardiac β1-adrenoceptor do not affect the low-affinity state β1-adrenoceptor-mediated inotropy in rat cardiomyocytes

Circulating autoantibodies directed against the 2nd extracellular loop (EL-2) of β(1)-adrenoceptors (β(1)-AABs) have been detected in the serum of patients with various cardiovascular pathologies. β(1)-AABs induce agonistic, positive inotropic effects via β(1)-adrenoceptors (β(1)ARs). In the mammalian heart, β(1)-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 β(1)-AAB is dependent on the affinity state of β(1)AR in isolated ventricular cardiomyocytes of adult Wistar rats. Immunoglobulin G (IgG) containing β(1)-AAB obtained from animals immunized with a peptide corresponding to the EL-2 of human β(1)-AR, caused a dose-dependent increase in cell shortening. Isoproterenol-induced inotropy was significantly reduced in cardiomyocytes that had been preincubated with IgG containing β(1)-AAB and in cardiomyocytes isolated from immunized rats. The negative effects of preincubation with IgG containing β(1)-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 β(1)-AAB in the presence of propranolol and 3-isobutyl-1-methylxanthine. The present study demonstrates that β(1)-AABs have no agonist/antagonist-like effects upon low-affinity state β(1)-ARs. This result indicates that β(1)-AABs recognize and stabilize the high-affinity state, but are unable to stabilize and (or) induce the low-affinity state receptor.

Age-Related Impairment in Insulin Release

In this study, we investigated the significance of β2-adrenergic receptor (β2AR) in age-related impaired insulin secretion and glucose homeostasis. We characterized the metabolic phenotype of β2AR-null C57Bl/6N mice (β2AR−/−) by performing in vivo and ex vivo experiments. In vitro assays in cultured INS-1E β-cells were carried out in order to clarify the mechanism by which β2AR deficiency affects glucose metabolism. Adult β2AR−/− mice featured glucose intolerance, and pancreatic islets isolated from these animals displayed impaired glucose-induced insulin release, accompanied by reduced expression of peroxisome proliferator–activated receptor (PPAR)γ, pancreatic duodenal homeobox-1 (PDX-1), and GLUT2. Adenovirus-mediated gene transfer of human β2AR rescued these defects. Consistent effects were evoked in vitro both upon β2AR knockdown and pharmacologic treatment. Interestingly, with aging, wild-type (β2AR+/+) littermates developed impaired insulin secretion and glucose tolerance. Moreover, islets from 20-month-old β2AR+/+ mice exhibited reduced density of β2AR compared with those from younger animals, paralleled by decreased levels of PPARγ, PDX-1, and GLUT2. Overexpression of β2AR in aged mice rescued glucose intolerance and insulin release both in vivo and ex vivo, restoring PPARγ/PDX-1/GLUT2 levels. Our data indicate that reduced β2AR expression contributes to the age-related decline of glucose tolerance in mice.