Adrenoceptor Desensitization: Current Understanding of Mechanisms.

G-protein coupled receptors (GPCRs) transduce a wide range of extracellular signals. They are key players in the majority of biologic functions including vision, olfaction, chemotaxis, and immunity. However, as essential as most of them are to body function and homeostasis, overactivation of GPCRs has been implicated in many pathologic diseases such as cancer, asthma, and heart failure (HF). Therefore, an important feature of G protein signaling systems is the ability to control GPCR responsiveness, and one key process to control overstimulation involves initiating receptor desensitization. A number of steps are appreciated in the desensitization process, including cell surface receptor phosphorylation, internalization, and downregulation. Rapid or short-term desensitization occurs within minutes and involves receptor phosphorylation via the action of intracellular protein kinases, the binding of ß-arrestins, and the consequent uncoupling of GPCRs from their cognate heterotrimeric G proteins. On the other hand, long-term desensitization occurs over hours to days and involves receptor downregulation or a decrease in cell surface receptor protein level. Of the proteins involved in this biologic phenomenon, ß-arrestins play a particularly significant role in both short- and long-term desensitization mechanisms. In addition, ß-arrestins are involved in the phenomenon of biased agonism, where the biased ligand preferentially activates one of several downstream signaling pathways, leading to altered cellular responses. In this context, this review discusses the different patterns of desensitization of the α 1-, α 2- and the ß adrenoceptors and highlights the role of ß-arrestins in regulating physiologic responsiveness through desensitization and biased agonism. SIGNIFICANCE STATEMENT: A sophisticated network of proteins orchestrates the molecular regulation of GPCR activity. Adrenoceptors are GPCRs that play vast roles in many physiological processes. Without tightly controlled desensitization of these receptors, homeostatic imbalance may ensue, thus precipitating various diseases. Here, we critically appraise the mechanisms implicated in adrenoceptor desensitization. A better understanding of these mechanisms helps identify new druggable targets within the GPCR desensitization machinery and opens exciting therapeutic fronts in the treatment of several pathologies.

[Activation of α7 nAChR improves white fat homeostasis and promotes beige adipogenesis and thermogenesis in obese mice].

OBJECTIVE: To investigate the effects of α7 nicotinic acetylcholine receptor (nAChR) agonist on ß3-adrenoceptor agonist-induced impairment of white fat homeostasis and beige adipose formation and heat production in obese mice. METHODS: Forty obese C57BL/6J mice were randomized into high-fat feeding group, ß3-adrenoceptor agonist-treated model group, α7 nAChR agonist group, and α7 nAChR inhibitor group (n=10), with another 10 mice with normal feeding as the blank control group. White adipose tissue from the epididymis of the mice were sampled for HE staining of the adipocytes. The expression levels of TNF-α, IL-1ß, IL-10 and TGF-ß in the white adipose tissue were determined by ELISA, and the mRNA levels of iNOS, Arg1, UCP-1, PRDM-16 and PGC-1α were detected using RT-qPCR. Western blotting was performed to detect the expression levels of NF-κB P65, p-JAK2, p-STAT3 in the white adipose tissue. RESULTS: Compared with those in the blank control group, the mice with high-fat feeding showed significantly increased body weight, more fat vacuoles in the white adipose tissue, increased volume of lipid droplets in the adipocytes, upregulated iNOS mRNA expression and protein expression of TNF-α and IL-1ß, and lowered expression of Arg-1 mRNA and IL-10 and TGF-ß proteins (P < 0.01). Treatment with α7 nAChR significantly reduced mRNA levels of PRDM-16, PGC-1α and UCP-1, lowered TNF-α and IL-1ß expressions, increased IL-10 and TGF-ß expressions, and reduced M1/M2 macrophage ratio in the white adipose tissues (P < 0.05 or 0.01). CONCLUSION: Activation of α7 nAchR improves white adipose tissue homeostasis impairment induced by ß3 agonist, promotes transformation of M1 to M2 macrophages, reduces inflammatory response in white adipose tissue, and promote beige adipogenesis and thermogenesis in obese mice.

Effects of chronic stress on cancer development and the therapeutic prospects of adrenergic signaling regulation.

Long-term chronic stress is an important factor in the poor prognosis of cancer patients. Chronic stress reduces the tissue infiltration of immune cells in the tumor microenvironment (TME) by continuously activating the adrenergic signaling, inhibits antitumor immune response and tumor cell apoptosis while also inducing epithelial-mesenchymal transition (EMT) and tumor angiogenesis, promoting tumor invasion and metastasis. This review first summarizes how adrenergic signaling activates intracellular signaling by binding different adrenergic receptor (AR) heterodimers. Then, we focused on reviewing adrenergic signaling to regulate multiple functions of immune cells, including cell differentiation, migration, and cytokine secretion. In addition, the article discusses the mechanisms by which adrenergic signaling exerts pro-tumorigenic effects by acting directly on the tumor itself. It also highlights the use of adrenergic receptor modulators in cancer therapy, with particular emphasis on their potential role in immunotherapy. Finally, the article reviews the beneficial effects of stress intervention measures on cancer treatment. We think that enhancing the body's antitumor response by adjusting adrenergic signaling can enhance the efficacy of cancer treatment.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

α1 and ß3 adrenergic receptor-mediated excitatory effects of adrenaline on the caudal neurosecretory system (CNSS) in olive flounder, Paralichthys olivaceus.

Adrenaline is one of the most important neurotransmitters in the central nervous system and is produced during stress. In this study, we investigated the modulatory role of adrenaline and adrenergic receptors on the neuroendocrine Dahlgren cells in the caudal neurosecretory system (CNSS) of olive flounder. Ex vivo electrophysiological recordings revealed that adrenaline significantly increased the firing frequency and altered the firing pattern of Dahlgren cells. Moreover, treatment with adrenaline led to a significant upregulation of ion channels and major hormone secretion genes in CNSS at the mRNA levels. Additionally, treatment with adrenaline resulted in a significantly elevation in the expression levels of α1- and ß3-adrenergic receptors. Furthermore, the ß3-adrenergic receptor antagonist exerts a significant inhibitory effect on adrenaline-induced enhancement firing activities of Dahlgren cells, whereas the α1-adrenergic receptor antagonist displays a comparatively weaker inhibitory effect. Additionally, the enhanced firing activity induced by adrenaline could be effectively suppressed by both α1- and ß3-adrenergic receptor antagonists. Taken together, these findings provide strong evidence in favor of the excitatory effects of adrenaline through α1 and ß3 adrenergic receptors in CNSS to stimulate the secretion of stress-related hormones, ß3-adrenergic receptor plays a more dominant role in the modulation of firing activities of Dahlgren cells by adrenaline and thereby regulates the stress response in olive flounder.

G protein-coupled estrogen receptor (GPER)/GPR30 forms a complex with the ß1-adrenergic receptor, a membrane-associated guanylate kinase (MAGUK) scaffold protein, and protein kinase A anchoring protein (AKAP) 5 in MCF7 breast cancer cells.

G protein-coupled receptor 30 (GPR30), also named G protein-coupled estrogen receptor (GPER), and the ß1-adrenergic receptor (ß1AR) are G protein-coupled receptors (GPCR) that are implicated in breast cancer progression. Both receptors contain PSD-95/Discs-large/ZO-1 homology (PDZ) motifs in their C-terminal tails through which they interact in the plasma membrane with membrane-associated guanylate kinase (MAGUK) scaffold proteins, and in turn protein kinase A anchoring protein (AKAP) 5. GPR30 constitutively and PDZ-dependently inhibits ß1AR-mediated cAMP production. We hypothesized that this inhibition is a consequence of a plasma membrane complex of these receptors. Using co-immunoprecipitation, confocal immunofluorescence microscopy, and bioluminescence resonance energy transfer (BRET), we show that GPR30 and ß1AR reside in close proximity in a plasma membrane complex when transiently expressed in HEK293. Deleting the GPR30 C-terminal PDZ motif (-SSAV) does not interfere with the receptor complex, indicating that the complex is not PDZ-dependent. MCF7 breast cancer cells express GPR30, ß1AR, MAGUKs, and AKAP5 in the plasma membrane, and co-immunoprecipitation revealed that these proteins exist in close proximity also under native conditions. Furthermore, expression of GPR30 in MCF7 cells constitutively and PDZ-dependently inhibits ß1AR-mediated cAMP production. AKAP5 also inhibits ß1AR-mediated cAMP production, which is not additive with GPR30-promoted inhibition. These results argue that GPR30 and ß1AR form a PDZ-independent complex in MCF7 cells through which GPR30 constitutively and PDZ-dependently inhibits ß1AR signaling via receptor interaction with MAGUKs and AKAP5.

Biased Signaling in Mutated Variants of ß2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations.

The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of ß2-adrenergic receptors (ß2AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor toward the ß-arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of ß-arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigated the allosteric mechanism of receptor bias in ß2AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R1313.50 and Y3267.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. Moreover, partial dissociation of G protein from the receptor core is observed in the simulations of the triple mutant in contrast to the single mutant and wild-type receptor. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of ß2AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side-effects and superior efficacy.

Glucocorticoid and Adrenergic Receptor Distribution Across Human Organs and Tissues: A Map for Stress Transduction.

OBJECTIVE: Psychosocial stress is transduced into disease risk through energy-dependent release of hormones from the hypothalamic-pituitary-adrenal and sympathetic-adrenal-medullary axes. The levels of glucocorticoid and adrenergic hormones, together with the sensitivity of tissues to their signaling, define stress responses. To understand existing pathways responsible for the psychobiological transduction of stressful experiences, we provide a quantitative whole-body map of glucocorticoid and adrenergic receptor (AR) expression. METHODS: We systematically examined gene expression levels for the glucocorticoid receptor (GR), α- and ß-ARs (AR-α1B, AR-α2B AR-ß2, and AR-ß3), across 55 different organs using the Human Protein Atlas and Human Proteome Map datasets. Given that mitochondria produce the energy required to respond to stress, we leveraged the Human Protein Atlas and MitoCarta3.0 data to examine the link between stress hormone receptor density and mitochondrial gene expression. Finally, we tested the functional interplay between GR activation and AR expression in human fibroblast cells. RESULTS: The GR was expressed ubiquitously across all investigated organ systems, whereas AR subtypes showed lower and more localized expression patterns. Receptor co-regulation, meaning the correlated gene expression of multiple stress hormone receptors, was found between GR and AR-α1B, as well as between AR-α1B and AR-α2B. In cultured human fibroblasts, activating the GR selectively increased AR-ß2 and AR-α1B expression. Consistent with the known energetic cost of stress responses, GR and AR expressions were positively associated with the expression of specific mitochondrial pathways. CONCLUSIONS: Our results provide a cartography of GR and AR expression across the human body. Because stress-induced GR and AR signaling triggers energetically expensive cellular pathways involving energy-transforming mitochondria, the tissue-specific expression and co-expression patterns of hormone receptor subtypes may in part determine the resilience or vulnerability of different organ systems.

Blocking brown adipocyte ß3-adrenoceptor attenuates blood-spinal cord barrier impairment and chronic postsurgical pain in a rat model of preoperative stress.

Preoperative stress has been recognized as an independent risk factor for chronic postsurgical pain (CPSP). However, the underlying mechanisms of CPSP influenced by preoperative stress remain elusive. Previous studies indicated that excessive stress could induce disruption of the blood-spinal cord barrier (BSCB). We wondered whether and how BSCB involves in CPSP by using a single prolonged stress (SPS) combining plantar incision model in male rats to mimic preoperative stress-related postsurgical pain. Here, we observed that preoperative SPS-exposed rats exhibited relentless incisional pain, which was accompanied by impairment of BSCB and persistent elevation of serum IL-6. Intraperitoneal injections of Tocilizumab (an IL-6 receptor monoclonal antibody) not only mitigated BSCB breakdown but also alleviated pain behaviors. In addition, intervening ß3-adrenoceptor (ADRB3) signaling in brown adipocytes by SR59230a (a specific ADRB3 antagonist) treatment or removal of brown adipose tissues could effectively decrease serum IL-6 levels, ameliorate BSCB disruption, and alleviate incisional pain. Further results displayed that SI-exposed rats also showed markedly spinal microglia activation. And exogenous His-tagged IL-6 could pass through the disrupted BSCB, which might contribute to microglia activation. Injection of SR59230a or ablation of brown adipose tissues could effectively reduce the activation of spinal microglia. Thus, our findings suggest that serum IL-6 induced by brown adipocyte ADRB3 signaling contributed to BSCB disruption and spinal microglia activation, which might be involved in preoperative stress mediated CPSP. This work indicates a promising treatment strategy for preoperative stress induced CPSP by blocking ADRB3.

Phenylacetyl glutamine (PAGln) enhances cardiomyocyte death after myocardial infarction through ß1 adrenergic receptor.

This study aims to explore the roles of phenylacetyl glutamine (PAGln) on myocardial infarction (MI) pathogenesis. Here, using targeted metabolomics analysis, it was found that the plasma metabolite PAGln was upregulated in coronary artery disease (CAD) patients and MI mice and could be an independent risk factor for CAD. In vivo and in vitro functional experiments revealed that PAGln pretreatment enhanced MI-induced myocardial injury and cardiac fibrosis, as evident by the increased infarct size, cardiomyocyte death, and the upregulated expression of cardiac fibrosis markers (Col1a1 and α-SMA). Combined with RNA-sequencing analysis and G protein-coupled receptor (GPCR) inhibitor, we found that the GPCR signaling activation is essential for PAGln-mediated effects on cardiomyocyte death. Furthermore, drug affinity responsive target stability and cellular thermal shift assay demonstrated that PAGln could interact with ß1-adrenergic receptor (AR). Moreover, ß1-AR blocker treatment indeed extended the cardiac remodeling after PAGln-enhanced MI. These results suggest that PAGln might be a potential therapeutic target for extending the cardiac remodeling window in MI patients that signals via ß1-AR.

Roles of the α1B-Adrenergic Receptor Phosphorylation Domains in Signaling and Internalization.

The function of the α1B-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with non-phosphorylatable amino acids. Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration-response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α1B-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.

Redox and proteolytic regulation of cardiomyocyte ß1-adrenergic receptors - a novel paradigm for the regulation of catecholamine responsiveness in the heart.

Conventional models view ß1-adrenergic receptors (ß1ARs) as full-length proteins that activate signaling pathways that influence contractile function and ventricular remodeling - and are susceptible to agonist-dependent desensitization. This perspective summarizes recent studies from my laboratory showing that post-translational processing of the ß1-adrenergic receptor N-terminus results in the accumulation of both full-length and N-terminally truncated forms of the ß1AR that differ in their signaling properties. We also implicate oxidative stress and ß1AR cleavage by elastase as two novel mechanisms that would (in the setting of cardiac injury or inflammation) lead to altered or decreased ß1AR responsiveness.

Inhibiting Intracellular α2C-Adrenoceptor Surface Translocation Using Decoy Peptides: Identification of an Essential Role of the C-Terminus in Receptor Trafficking.

The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet-hands, Raynaud's phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein-protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy.

Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema.

Cerebral oedema is associated with morbidity and mortality after traumatic brain injury (TBI)1. Noradrenaline levels are increased after TBI2-4, and the amplitude of the increase in noradrenaline predicts both the extent of injury5 and the likelihood of mortality6. Glymphatic impairment is both a feature of and a contributor to brain injury7,8, but its relationship with the injury-associated surge in noradrenaline is unclear. Here we report that acute post-traumatic oedema results from a suppression of glymphatic and lymphatic fluid flow that occurs in response to excessive systemic release of noradrenaline. This post-TBI adrenergic storm was associated with reduced contractility of cervical lymphatic vessels, consistent with diminished return of glymphatic and lymphatic fluid to the systemic circulation. Accordingly, pan-adrenergic receptor inhibition normalized central venous pressure and partly restored glymphatic and cervical lymphatic flow in a mouse model of TBI, and these actions led to substantially reduced brain oedema and improved functional outcomes. Furthermore, post-traumatic inhibition of adrenergic signalling boosted lymphatic export of cellular debris from the traumatic lesion, substantially reducing secondary inflammation and accumulation of phosphorylated tau. These observations suggest that targeting the noradrenergic control of central glymphatic flow may offer a therapeutic approach for treating acute TBI.

Crystal structures of QseE and QseG: elements of a three-component system from Escherichia coli.

Bacteria regulate virulence by using two-component systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). TCSs respond to environmental signals and change gene-expression levels. The HK QseE and the RR QseF regulate the virulence of Enterobacteriaceae bacteria such as enterohemorrhagic Escherichia coli. The operon encoding QseE/QseF also contains a gene encoding an outer membrane lipoprotein, qseG. The protein product QseG interacts with QseE in the periplasmic space to control the activity of QseE and constitutes a unique QseE/F/G three-component system. However, the structural bases of their functions are unknown. Here, crystal structures of the periplasmic regions of QseE and QseG were determined with the help of AlphaFold models. The periplasmic region of QseE has a helix-bundle structure as found in some HKs. The QseG structure is composed of an N-terminal globular domain and a long C-terminal helix forming a coiled-coil-like structure that contributes to dimerization. Comparison of QseG structures obtained from several crystallization conditions shows that QseG has structural polymorphisms at the C-terminus of the coiled-coil structure, indicating that the C-terminus is flexible. The C-terminal flexibility is derived from conserved hydrophilic residues that reduce the hydrophobic interaction at the coiled-coil interface. Electrostatic surface analysis suggests that the C-terminal coiled-coil region can interact with QseE. The observed structural fluctuation of the C-terminus of QseG is probably important for interaction with QseE.

Evolution and divergence of teleost adrenergic receptors: why sometimes 'the drugs don't work' in fish.

Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and ß-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in 'omics' technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts.

Improved Cardiac Performance and Decreased Arrhythmia in Hypertrophic Cardiomyopathy With Non-ß-Blocking R-Enantiomer Carvedilol.

BACKGROUND: Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. ß-Adrenergic receptor antagonists (ß-blockers) are the first-line therapy for HCM. However, ß-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown. METHODS: We screened 21 ß-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM (MYH7R403Q/+). RESULTS: We identified that carvedilol, a ß-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a ß1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of ß-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ iPSC-derived cardiomyocytes. CONCLUSIONS: R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.

Agonists and hydrogen peroxide mediate hyperoxidation of ß2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to ß2-agonists in constrictive airway disorders.

Asthma and other airway obstructive disorders are characterized by heightened inflammation and excessive airway epithelial cell reactive oxygen species (ROS), which give rise to a highly oxidative environment. After decades of use, ß2-adrenergic receptor (ß2AR) agonists remain at the forefront of treatment options for asthma, however, chronic use of ß2-agonists leads to tachyphylaxis to the bronchorelaxant effects, a phenomenon that remains mechanistically unexplained. We have previously demonstrated that ß2AR agonism increases ROS generation in airway epithelial cells, which upholds proper receptor function via feedback oxidation of ß2AR cysteine thiolates to Cys-S-sulfenic acids (Cys-SOH). Our previous results also demonstrate that prevention of normal redox cycling of this post-translational oxi-modification back to the thiol prevents proper receptor function. Given that Cys-S-sulfenic acids can be irreversibly overoxidized to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participation in redox reactions, we hypothesized that ß2-agonist tachyphylaxis may be explained by hyperoxidation of ß2AR to S-sulfinic acids. Here, using airway epithelial cell lines and primary small airway epithelial cells from healthy and asthma-diseased donors, we show that ß2AR agonism generates H2O2 in a receptor and NAPDH oxidase-dependent manner. We also demonstrate that acute and chronic receptor agonism can facilitate ß2AR S-sulfination, and that millimolar H2O2 concentrations are deleterious to ß2AR-mediated cAMP formation, an effect that can be rescued to a degree in the presence of the cysteine-donating antioxidant N-acetyl-L-cysteine. Our results reveal that the oxidative state of ß2AR may contribute to receptor functionality and may, at least in part, explain ß2-agonist tachyphylaxis.

Localization and neurochemical identity of alpha1-adrenergic receptor-containing elements in the mouse locus coeruleus.

The locus coeruleus (LC) is the major source for norepinephrine (NE) in the brain and projects to areas involved in learning and memory, reward, arousal, attention, and autonomic functions related to stress. There are three types of adrenergic receptors that respond to NE: alpha1-, alpha2-, and beta-adrenergic receptors. Previous behavioral studies have shown the alpha1-adrenergic receptor (α1AR) to be present in the LC, however, with conflicting results. For example, it was shown that α1ARs in the LC are involved in some of the motivational effects of stimulation of the medial forebrain bundle, which was reduced by α1AR antagonist terazosin. Another study showed that during novelty-induced behavioral activation, the α1AR antagonist prazosin reduced c-fos expression in brain regions known to contain motoric α1ARs, except for the LC, where c-fos expression was enhanced. Despite new research delineating more specific connectivity of the neurons in the LC, and some roles of the adrenergic receptors, the α1ARs have not been localized at the subcellular level. Therefore, in order to gain a greater understanding of the aforementioned studies, we used immunohistochemistry at the electron microscopic (EM) level to determine which neuronal or glial elements in the LC express the α1AR. We hypothesized, based on previous work in the ventral periaqueductal gray area, that the α1AR would be found mainly presynaptically in axon terminals, and possibly in glial elements. Single labeling immunohistochemistry at the EM revealed that about 40% of labeled elements that contained the α1AR were glial elements, while approximately 50% of the labeled neuronal elements were axon terminals or small unmyelinated axons in the LC. Double labeling immunohistochemistry found the α1AR expressed in GFAP-labeled astrocytes, in both GABAergic and glutamatergic axon terminals, and in a portion of the α1AR dendrites, colocalized with tyrosine hydroxylase (TH, a marker for noradrenergic neurons). This study sheds light on the neuroanatomical framework underlying the effects of NE and pharmaceuticals acting directly or indirectly on α1ARs in the LC.

Thyroid hormone induces restrictive cardiomyopathy in ß1-adrenoceptor knockout mice.

The purpose of this study was to characterize the role of ß1-AR signaling and its cross-talk between cardiac renin-angiotensin system and thyroid-hormone-induced cardiac hypertrophy. T3 was administered at 0.5 mg·kg-1·day-1 for 10 days in ß1-KOT3 and WTT3 groups, while control groups received vehicle alone. Echocardiography and myocardial histology was performed; cardiac and serum ANGI/ANGII and ANP and cardiac levels of p-PKA, p-ERK1/2, p-p38-MAPK, p-AKT, p-4EBP1, and ACE were measured. WTT3 showed decreased IVSTd and increased LVEDD versus WTsal (p < 0.05). ß1-KOT3 exhibited lower LVEDD and higher relative IVSTd versus ß1-KOsal, the lowest levels of ejection fraction, and the highest levels of cardiomyocyte diameter (p < 0.05). Cardiac ANP levels decreased in WTT3 versus ß1-KOT3 (p < 0.05). Cardiac ACE expression was increased in T3-treated groups (p < 0.05). Phosphorylated-p38 MAPK levels were higher in WTT3 versus WTsal or ß1-KOT3, p-4EBP1 was elevated in ß1-KO animals, and p-ERK1/2 was up-regulated in ß1-KOT3. These findings suggest that ß1-AR signaling is crucial for TiCH.