Astrocyte ß-Adrenergic Receptor Activity Regulates NMDA Receptor Signaling of Medial Prefrontal Cortex Pyramidal Neurons.

Glutamate spillover from the synapse is tightly regulated by astrocytes, limiting the activation of extrasynaptically located NMDA receptors (NMDAR). The processes of astrocytes are dynamic and can modulate synaptic physiology. Though norepinephrine (NE) and ß-adrenergic receptor (ß-AR) activity can modify astrocyte volume, this has yet to be confirmed outside of sensory cortical areas, nor has the effect of noradrenergic signaling on glutamate spillover and neuronal NMDAR activity been explored. We monitored changes to astrocyte process volume in response to noradrenergic agonists in the medial prefrontal cortex of male and female mice. Both NE and the ß-AR agonist isoproterenol (ISO) increased process volume by ∼20%, significantly higher than changes seen when astrocytes had G-protein signaling blocked by GDPßS. We measured the effect of ß-AR signaling on evoked NMDAR currents. While ISO did not affect single stimulus excitatory currents of Layer 5 pyramidal neurons, ISO reduced NMDAR currents evoked by 10 stimuli at 50 Hz, which elicits glutamate spillover, by 18%. After isolating extrasynaptic NMDARs by blocking synaptic NMDARs with the activity-dependent NMDAR blocker MK-801, ISO similarly reduced extrasynaptic NMDAR currents in response to 10 stimuli by 18%. Finally, blocking ß-AR signaling in the astrocyte network by loading them with GDPßS reversed the ISO effect on 10 stimuli-evoked NMDAR currents. These results demonstrate that astrocyte ß-AR activity reduces extrasynaptic NMDAR recruitment, suggesting that glutamate spillover is reduced.

Stimulating ß-adrenergic receptors promotes synaptic potentiation by switching CaMKII movement from LTD to LTP mode.

Learning, memory, and cognition are thought to require synaptic plasticity, specifically including hippocampal long-term potentiation and depression (LTP and LTD). LTP versus LTD is induced by high-frequency stimulation versus low-frequency, but stimulating ß-adrenergic receptors (ßARs) enables LTP induction also by low-frequency stimulation (1 Hz) or theta frequencies (∼5 Hz) that do not cause plasticity by themselves. In contrast to high-frequency stimulation-LTP, such ßAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate-type glutamate receptors. Surprisingly, we found that ßAR-LTP still required a nonionotropic scaffolding function of the N-methyl-D-aspartate-type glutamate receptor: the stimulus-induced binding of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to its GluN2B subunit that mediates CaMKII movement to excitatory synapses. In hippocampal neurons, ß-adrenergic stimulation with isoproterenol (Iso) transformed LTD-type CaMKII movement to LTP-type movement, resulting in CaMKII movement to excitatory instead of inhibitory synapses. Additionally, Iso enabled induction of a major cell-biological feature of LTP in response to LTD stimuli: increased surface expression of GluA1 fused with super-ecliptic pHluorein. Like for ßAR-LTP in hippocampal slices, the Iso effects on CaMKII movement and surface expression of GluA1 fused with super-ecliptic pHluorein involved L-type Ca2+-channels and specifically required ß2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.

Visualization of ß-adrenergic receptor dynamics and differential localization in cardiomyocytes.

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

Leveraging ß-Adrenergic Receptor Signaling Blockade for Improved Cancer Immunotherapy Through Biomimetic Nanovaccine.

The establishment of effective antitumor immune responses of vaccines is mainly limited by insufficient priming tumor infiltration of T cells and immunosuppressive tumor microenvironment (TME). Targeting ß-adrenergic receptor (ß-AR) signaling exerts promising benefits on reversing the suppressive effects directly on T cells, but it appears to have considerably limited antitumor performance when combined with vaccine-based immunotherapies. Herein, a tumor membrane-coated nanoplatform for codelivery of adjuvant CpG and propranolol (Pro), a ß-AR inhibitor is designed. The biomimetic nanovaccine displayed an improved accumulation in lymph nodes and sufficient drug release, thereby inducing dendritic cell maturation and antigen presentation. Meanwhile, the integration of vaccination and blockade of ß-AR signaling not only promoted the priming of the naive CD8+ T cells and effector T cell egress from lymph nodes, but also alleviated the immunosuppressive TME by decreasing the frequency of immunosuppressive cells and increasing the tumor infiltration of B cells and NK cells. Consequently, the biomimetic nanovaccines outperformed greater prophylactic and therapeutic efficacy than nanovaccines without Pro encapsulation in B16-F10 melanoma mice. Taken together, the work explored a biomimetic nanovaccine for priming tumor infiltration of T cells and immunosuppressive TME regulation, offering tremendous potential for a combined ß-AR signaling-targeting strategy in cancer immunotherapy.

Heightened Hippocampal ß-Adrenergic Receptor Function Drives Synaptic Potentiation and Supports Learning and Memory in the TgF344-AD Rat Model during Prodromal Alzheimer's Disease.

The central noradrenergic (NA) system is critical for the maintenance of attention, behavioral flexibility, spatial navigation, and learning and memory, those cognitive functions lost first in early Alzheimer's disease (AD). In fact, the locus coeruleus (LC), the sole source of norepinephrine (NE) for >90% of the brain, is the first site of pathologic tau accumulation in human AD with axon loss throughout forebrain, including hippocampus. The dentate gyrus is heavily innervated by LC-NA axons, where released NE acts on ß-adrenergic receptors (ARs) at excitatory synapses from entorhinal cortex to facilitate long-term synaptic plasticity and memory formation. These synapses experience dysfunction in early AD before cognitive impairment. In the TgF344-AD rat model of AD, degeneration of LC-NA axons in hippocampus recapitulates human AD, providing a preclinical model to investigate synaptic and behavioral consequences. Using immunohistochemistry, Western blot analysis, and brain slice electrophysiology in 6- to 9-month-old wild-type and TgF344-AD rats, we discovered that the loss of LC-NA axons coincides with the heightened ß-AR function at medial perforant path-dentate granule cell synapses that is responsible for the increase in LTP magnitude at these synapses. Furthermore, novel object recognition is facilitated in TgF344-AD rats that requires ß-ARs, and pharmacological blockade of ß-ARs unmasks a deficit in extinction learning only in TgF344-AD rats, indicating a greater reliance on ß-ARs in both behaviors. Thus, a compensatory increase in ß-AR function during prodromal AD in TgF344-AD rats heightens synaptic plasticity and preserves some forms of learning and memory.SIGNIFICANCE STATEMENT The locus coeruleus (LC), a brain region located in the brainstem which is responsible for attention and arousal, is damaged first by Alzheimer's disease (AD) pathology. The LC sends axons to hippocampus where released norepinephrine (NE) modulates synaptic function required for learning and memory. How degeneration of LC axons and loss of NE in hippocampus in early AD impacts synaptic function and learning and memory is not well understood despite the importance of LC in cognitive function. We used a transgenic AD rat model with LC axon degeneration mimicking human AD and found that heightened function of ß-adrenergic receptors in the dentate gyrus increased synaptic plasticity and preserved learning and memory in early stages of the disease.

ß-Blockers are associated with increased B-type natriuretic peptide levels differently in men and women in heart failure with preserved ejection fraction.

ß-Blocker (BB) use is a mainstay for the treatment of heart failure (HF) with reduced ejection fraction (HFrEF), whereas its efficacy for heart failure with preserved ejection fraction (HFpEF) remains controversial. Women outnumber men in HFpEF, whereas men outnumber women in HFrEF. Plasma B-type natriuretic peptide (BNP) is established as a biomarker for HF. We examined whether BB use is associated with plasma BNP levels differently in men and women with HFpEF. The study subjects comprised 721 patients with HFpEF [left ventricular ejection fraction (LVEF) ≥ 50%] (184 men, mean age 78.2 ± 9.2 yr and 537 women, mean age 83.1 ± 8.8 yr), 179 on BB (66 men and 113 women) and 542 no BB (118 men and 424 women), 583 in sinus rhythm (SR) and 138 in atrial fibrillation (AF). A multivariable logistic regression test was used. Plasma BNP levels were higher (P = 0.0005), systolic blood pressure and LVEF lower (P = 0.0003, and P = 0.0059, respectively) on BBs than on no BBs in women, whereas in men, plasma BNP levels, systolic blood pressure, and LVEF were not altered significantly (P = 0.0849, P = 0.9129, and P = 0.4718, respectively) on BBs compared with no BBs in patients with SR. Multivariable logistic regression analysis revealed that BB use and women were a positive and a negative predictor for high BNP levels (P = 0.003 and P = 0.032, respectively) in SR but not in AF. BB use was associated with high-plasma BNP levels and lower LVEF in women but not in men with HFpEF and SR, suggesting that the pathogenesis and treatment of HFpEF may differ in men and women in SR.NEW & NOTEWORTHY Pathogenesis and treatment for heart failure with preserved ejection fraction (HFpEF) may differ in men and women in sinus rhythm (SR).

Fluorescence Spectroscopy of Low-Level Endogenous ß-Adrenergic Receptor Expression at the Plasma Membrane of Differentiating Human iPSC-Derived Cardiomyocytes.

The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the ß1- and ß2-adrenergic receptors (ß1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of ß-ARs in adult CMs.

Regulation of Cisplatin Resistance in Lung Cancer Cells by Nicotine, BDNF, and a ß-Adrenergic Receptor Blocker.

It is well-recognized that cigarette smoking is a primary risk factor in the development of non-small cell lung cancer (NSCLC), known to account for ~80% of all lung cancers with nicotine recognized as the major addictive component. In investigating the effect of nicotine, brain-derived neurotrophic factor (BDNF), and the ß-adrenergic receptor blocker, propranolol, on sensitivity of NSCLC cell lines, A549 and H1299, to cisplatin, we found increased cell viability, and enhanced cisplatin resistance with nicotine and/or BDNF treatment while opposite effects were found upon treatment with propranolol. Cell treatment with epinephrine or nicotine led to EGFR and IGF-1R activation, effects opposite to those found with propranolol. Blocking EGFR and IGF-1R activation increased cell sensitivity to cisplatin in both cell lines. PI3K and AKT activities were upregulated by nicotine or BDNF and downregulated by cell treatment with inhibitors against EGFR and IGF-1R and by propranolol. Apoptosis and cell sensitivity to cisplatin increased upon co-treatment of cells with cisplatin and inhibitors against PI3K or AKT. Our findings shed light on an interplay between nicotine, BDNF, and ß-Adrenergic receptor signaling in regulating survival of lung cancer cells and chemoresistance which can likely expand therapeutic opportunities that target this regulatory network in the future.

Intravenous Infusion of the ß3-Adrenergic Receptor Antagonist APD418 Improves Left Ventricular Systolic Function in Dogs With Systolic Heart Failure.

BACKGROUND: Unlike ß1- and ß2-adrenergic receptors (ARs), ß3-AR stimulation inhibits cardiac contractility and relaxation. In the failing left ventricular (LV) myocardium, ß3-ARs are upregulated, and can be maladaptive in the setting of decompensation by contributing to LV dysfunction. This study examined the effects of intravenous infusions of the ß3-AR antagonist APD418 on cardiovascular function and safety in dogs with systolic heart failure (HF). METHODS AND RESULTS: Three separate studies were performed in 21 dogs with coronary microembolization-induced HF (LV ejection fraction [LVEF] of approximately 35%). Studies 1 and 2 (n = 7 dogs each) were APD418 dose escalation studies (dosing range, 0.35-15.00 mg/kg/h) designed to identify an effective dose of APD418 to be used in study 3. Study 3, the sustained efficacy study, (n = 7 dogs) was a 6-hour constant intravenous infusion of APD418 at a dose of 4.224 mg/kg (0.70 mg/kg/h) measuring key hemodynamic endpoints (e.g., EF, cardiac output, the time velocity integral of the mitral inflow velocity waveform representing early filling to time-velocity integral representing left atrial contraction [Ei/Ai]). Studies 1 and 2 showed a dose-dependent increase of LVEF and Ei/Ai, the latter being an index of LV diastolic function. In study 3, infusion of APD418 over 6 hours increased LVEF from 31 ± 1% to 38 ± 1% (P < .05) and increased Ei/Ai from 3.4 ± 0.4 to 4.9 ± 0.5 (P < .05). Vehicle had no effect on the LVEF or Ei/Ai. In study 3, APD418 had no significant effects on the HR or the systemic blood pressure. CONCLUSIONS: Intravenous infusions of APD418 in dogs with systolic HF elicit significant positive inotropic and lusitropic effects. These findings support the development of APD418 for the in-hospital treatment of patients with an acute exacerbation of chronic HF.

Bisoprolol-based 18F-PET tracer: Synthesis and preliminary in vivo validation of ß1-blocker selectivity for ß1-adrenergic receptors in the heart.

The selectivity of a drug toward various isoforms of the target protein family is important in terms of toxicology. Typically, drug or candidate selectivity is assessed by in vitro assays, but in vivo investigations are currently lacking. Positron emission tomography (PET) allows the non-invasive determination of the in vivo distribution of a radiolabeled drug, which can provide in vivo data regarding drug selectivity. Since the discovery of propranolol, a non-selective ß-blocker inhibiting both ß1- and ß2-adrenoreceptors (ß-ARs), various selective ß1-blockers, including bisoprolol, have been developed to overcome disadvantages associated with ß2-AR inhibition. As a proof of concept, we performed an in vivo PET study to understand the selectivity and efficacy of bisoprolol as a selective ß-blocker toward ß1-AR, as the heart and peripheral smooth muscles demonstrate distinct populations of ß1- and ß2-ARs. Biodistribution of 18F-labeled bisoprolol (1, [18F]bisoprolol) showed the retention of its uptake in the heart compared with other ß-AR-rich organs at late time points post-injection. The competitive blocking assay using unlabeled bisoprolol exhibited no inhibition of [18F]bisoprolol uptake in any organ but exhibited significantly rapid loss of radioactivity between two different time points in ß1-AR-rich organs such as the heart and brain. Furthermore, the organ-to-blood ratio revealed the slow excretion and better accumulation of [18F]bisoprolol inside the heart. Collectively, the ex vivo biodistribution and blocking study presented insightful evidence to better comprehend the in vivo distribution pattern of bisoprolol as a selective inhibitor targeting ß1-ARs in the heart and provided the possibility of PET as an in vivo technique for evaluating drug selectivity.

Role of ß-Adrenergic Receptors and Estrogen in Cardiac Repair after Myocardial Infarction: An Overview.

Acute myocardial infarction (MI) is associated with an intense inflammatory response that is critical for cardiac repair but is also involved in the pathogenesis of adverse cardiac remodeling, i.e., the set of size, geometry, and structure changes that represent the structural substrate for the development of post-MI heart failure. Deciphering the pathophysiological mechanisms underlying cardiac repair after MI is, therefore, critical to favorably regulate cardiac wound repair and to prevent development of heart failure. Catecholamines and estrogen play an active role in regulating the inflammatory response in the infarcted area. For example, stress-induced catecholamines alter recruitment and trafficking of leukocytes to the heart. Additionally, estrogen affects rate of cardiac rupture during the acute phase of MI, as well as infarct size and survival in animal models of MI. In this review, we will summarize the role of ß-adrenergic receptors and estrogen in cardiac repair after infarction in preclinical studies.

Screening of ß1- and ß2-Adrenergic Receptor Modulators through Advanced Pharmacoinformatics and Machine Learning Approaches.

Cardiovascular diseases (CDs) are a major concern in the human race and one of the leading causes of death worldwide. ß-Adrenergic receptors (ß1-AR and ß2-AR) play a crucial role in the overall regulation of cardiac function. In the present study, structure-based virtual screening, machine learning (ML), and a ligand-based similarity search were conducted for the PubChem database against both ß1- and ß2-AR. Initially, all docked molecules were screened using the threshold binding energy value. Molecules with a better binding affinity were further used for segregation as active and inactive through ML. The pharmacokinetic assessment was carried out on molecules retained in the above step. Further, similarity searching of the ChEMBL and DrugBank databases was performed. From detailed analysis of the above data, four compounds for each of ß1- and ß2-AR were found to be promising in nature. A number of critical ligand-binding amino acids formed potential hydrogen bonds and hydrophobic interactions. Finally, a molecular dynamics (MD) simulation study of each molecule bound with the respective target was performed. A number of parameters obtained from the MD simulation trajectories were calculated and substantiated the stability between the protein-ligand complex. Hence, it can be postulated that the final molecules might be crucial for CDs subjected to experimental validation.

Structural Insights into Ligand-Receptor Interactions Involved in Biased Agonism of G-Protein Coupled Receptors.

G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Ligand directed signaling is observed when agonists, upon binding to the same receptor, trigger significantly different configuration of intracellular events. The current work reviews the structurally defined ligand - receptor interactions that can be related to specific molecular mechanisms of ligand directed signaling across different receptors belonging to class A of GPCRs. Recent advances in GPCR structural biology allow for mapping receptors' binding sites with residues particularly important in recognition of ligands' structural features that are responsible for biased signaling. Various studies show particular role of specific residues lining the extended ligand binding domains, biased agonists may alternatively affect their interhelical interactions and flexibility what can be translated into intracellular loop rearrangements. Studies on opioid and angiotensin receptors indicate importance of residues located deeper within the binding cavity and direct interactions with receptor residues linking the ortosteric ligand binding site with the intracellular transducer binding domain. Collection of results across different receptors may suggest elements of common molecular mechanisms which are responsible for passing alternative signals from biased agonists.

Presence and function of ß-adrenergic receptors in primary equine bronchial epithelia cells.

The ß-adrenergic receptor (ß-AR) plays an important role in regulating a variety of cell and organ functions in different animal species and is an important target in asthma pathogenesis and therapy. The ß-AR expression and function in equine bronchial epithelial cells (EBEC) were not known but innervation and significant decrease in receptor level were reported in the equine bronchial tissues from asthmatic horses. 125I-iodocyanopindolol (ICYP) binding studies were undertaken in primary freshly isolated and cultured EBEC to identify the presence of the ß-ARs. The receptor distribution was assessed using subtype-selective ß-AR antagonists (ICI 118 551 (ß2) and CGP 20712A (ß1). The ß-AR function was confirmed by measuring the agonist-induced intracellular cAMP accumulation in freshly isolated and cultured EBEC. In both freshly isolated and cultured EBEC, the specific ICYP binding was saturable and of high affinity. The maximal receptor density (Bmax) was 9763 ± 140 binding sites/cell (mean ± SEM, n = 7) and 10575 ± 194 binding sites/cell (mean ± SEM, n = 5) in freshly isolated and cultured EBEC, respectively. The receptor affinity to the ligand (KD) was also not different between the two cell conditions. ICI 118.551 displaced ICYP with 25 000-fold higher affinity than CGP 20712A. Moreover, in both fresh isolated and cultured EBEC, cAMP-accumulation was stimulated with a rank-order of potency of isoproterenol > adrenaline > noradrenaline. These results highlight the ß2-AR to be a key subtype in both freshly isolated and cultured primary EBEC.

Ganoderic acid A protects neural cells against NO stress injury in vitro via stimulating ß adrenergic receptors.

Excessive nitric oxide (NO) causes extensive damage to the nervous system, and the adrenergic system is disordered in many neuropsychiatric diseases. However, the role of the adrenergic system in protection of the nervous system against sodium nitroprusside (SNP) injury remains unclear. In this study, we investigated the effect of ganoderic acid A (GA A) against SNP injury in neural cells and the role of adrenergic receptors in GA A neuroprotection. We found that SNP (0.125-2 mM) dose-dependently decreased the viability of both SH-SY5Y and PC12 cells and markedly increased NO contents. Pretreatment with GA A (10 µM) significantly attenuated SNP-induced cytotoxicity and NO increase in SH-SY5Y cells, but not in PC12 cells. Furthermore, pretreatment with GA A caused significantly higher adrenaline content in SH-SY5Y cells than in PC12 cells. In order to elucidate the mechanism of GA A-protecting SH-SY5Y cells, we added adrenaline, phentolamine, metoprolol, or ICI 118551 1 h before GA A was added to the culture medium. We found that addition of adrenaline (10 µM) significantly improved GA A protection in PC12 cells. The addition of ß1-adrenergic receptor antagonist metoprolol (10 µM) or ß2-adrenergic receptor antagonist ICI 118551 (0.1 µM) blocked the protective effect of GA A, whereas the addition of α-adrenergic receptor antagonist phentolamine (0.1 µM) did not affect GA A protection in SH-SY5Y cells. These results suggest that ß-adrenergic receptors play an important role in the protection of GA A in SH-SY5Y cells against SNP injuries, and excessive adrenaline system activation caused great damage to the nervous system.

ß-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

KEY POINTS: Prevailing dogma holds that activation of the ß-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling. ABSTRACT: Voltage-dependent L-type CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the ß-adrenergic receptor (ßAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether ßAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the ßAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of ßAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.

BDNF downregulates ß-adrenergic receptor-mediated hypotensive mechanisms in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) is upregulated in the paraventricular nucleus of the hypothalamus (PVN) in response to hypertensive stimuli such as stress and hyperosmolality, and BDNF acting in the PVN plays a key role in elevating sympathetic activity and blood pressure. However, downstream mechanisms mediating these effects remain unclear. We tested the hypothesis that BDNF increases blood pressure, in part by diminishing inhibitory hypotensive input from nucleus of the solitary tract (NTS) catecholaminergic neurons projecting to the PVN. Male Sprague-Dawley rats received bilateral PVN injections of viral vectors expressing either green fluorescent protein (GFP) or BDNF and bilateral NTS injections of vehicle or anti-dopamine-ß-hydroxylase-conjugated saporin (DSAP), a neurotoxin that selectively lesions noradrenergic and adrenergic neurons. BDNF overexpression in the PVN without NTS lesioning significantly increased mean arterial pressure (MAP) in awake animals by 18.7 ± 1.8 mmHg. DSAP treatment also increased MAP in the GFP group, by 9.8 ± 3.2 mmHg, but failed to affect MAP in the BDNF group, indicating a BDNF-induced loss of NTS catecholaminergic hypotensive effects. In addition, in α-chloralose-urethane-anesthetized rats, hypotensive responses to PVN injections of the ß-adrenergic agonist isoprenaline were significantly attenuated by BDNF overexpression, whereas PVN injections of phenylephrine had no effect on blood pressure. BDNF treatment was also found to significantly reduce ß1-adrenergic receptor mRNA expression in the PVN, whereas expression of other adrenergic receptors was unaffected. In summary, increased BDNF expression in the PVN elevates blood pressure, in part by downregulating ß-receptor signaling and diminishing hypotensive catecholaminergic input from the NTS to the PVN.NEW & NOTEWORTHY We have shown that BDNF, a key hypothalamic regulator of blood pressure, disrupts catecholaminergic signaling between the NTS and the PVN by reducing the responsiveness of PVN neurons to inhibitory hypotensive ß-adrenergic input from the NTS. This may be occurring partly via BDNF-mediated downregulation of ß1-adrenergic receptor expression in the PVN and results in an increase in blood pressure.

Cardiovascular variability and ß-ARs gene expression at two stages of doxorubicin - Induced cardiomyopathy.

Using comprehensive analysis of heart rate (HRV) and blood pressure (BPV) short-term variability we estimated the time course of changes of autonomic nervous system remodeling in two stages of doxorubicin-induced cardiomyopathy (DCM). We also investigated the level of gene expression of cardiac ß-1 (ß-1AR) and ß-2 (ß-2AR) adrenoceptors. Experiments were performed in adult male Wistar rats equipped with indwelling catheters for BP recording and blood withdrawal. A 15 mg/kg total cumulative dose of doxorubicin was injected i.p. to rats to induce DCM or saline for control (n=18). Rats were assessed for general toxicity, cardiovascular hemodynamic and echocardiography before treatment (n=6), 35 days (DOX35; n=6) and 70 days (DOX70; n=6) post-treatment. HRV was evaluated by spectral analysis, Poincaré plots, sample and approximate entropy. Expression of ß-1AR and ß-2AR mRNA was evaluated by RT-qPCR. Doxorubicin-treated rats exhibited poor general condition and lower survival than saline-treated rats. In DOX35 rats, there were no echocardiography signs of decompensation, no increase in serum cardiac troponins, but there was an increase of HRV and decrease of HR complexity. In these rats typical microscopic signs of cardiotoxicity were seen along with over-expression of ß-1AR mRNA. 70 days post-treatment echocardiography revealed signs of decompensation and serum cardiac troponin T was increased. At this stage BPV decreased. In conclusion, HRV increase matches transient over-expression of cardiac ß-1AR mRNA in compensate stage of DCM while decompensate stage of DCM is characterized by a decrease of BPV and no changes in ß-1AR and ß-2AR gene expression.

In rats with estradiol valerate-induced polycystic ovary syndrome, the acute blockade of ovarian ß-adrenoreceptors improve ovulation.

BACKGROUND: Polycystic ovary syndrome is characterized by hyperactivity of the ovarian sympathetic nervous system, increases in the content and release of norepinephrine, as well as decreases in the number of ß-adrenoreceptors. In the present study, ß-adrenoreceptors in the ovaries of rats with polycystic ovary syndrome were blocked and analyzed the resultant effects on ovulation, hormone secretion and the enzymes responsible for the synthesis of catecholamines. METHODS: At 60 days of age, vehicle or estradiol valerate-treated rats were injected with propranolol [10- 4 M] into the ovarian bursas on oestrus day. The animals were sacrificed on the next day of oestrus, and the ovulation response, the steroid hormone levels in the serum and the immunoreactivity of tyrosine hydroxylase and dopamine ß-hydroxylase in the ovaries were measured. RESULTS: In animals with the induction of polycystic ovary syndrome and ß-adrenoreceptor blocking, ovulation was restored in more than half of the animals and resulted in decreased hyperandrogenism with respect to the levels observed in the estradiol valerate-treated group. Tyrosine hydroxylase and dopamine ß-hydroxylase were present in the theca cells of the growing follicles and the interstitial gland. Injection of propranolol restored the tyrosine hydroxylase and ovarian dopamine ß-hydroxylase levels in rats with polycystic ovary syndrome induction. CONCLUSIONS: The results suggest that a single injection into the ovarian bursas of propranolol, a nonselective antagonist of ß-adrenoreceptor receptors, decreases the serum testosterone concentration and the formation of ovarian cysts, improving the ovulation rate that accompanies lower levels of tyrosine hydroxylase and dopamine ß-hydroxylase in the ovary.

Isoproterenol induces an increase in muscle fiber size by the proliferation of Pax7-positive cells and in a mTOR-independent mechanism.

ß-Adrenergic signaling regulates many physiological processes in skeletal muscles. A wealth of evidence has shown that ß-agonists can increase skeletal muscle mass in vertebrates. Nevertheless, to date, the specific role of ß-adrenergic receptors in different cell phenotypes (myoblasts, fibroblasts, and myotubes) and during the different steps of embryonic skeletal muscle differentiation has not been studied. Therefore, here we address this question through the analysis of embryonic chick primary cultures of skeletal muscle cells during the formation of multinucleated myotubes. We used isoproterenol (ISO), a ß-adrenergic receptor agonist, to activate the ß-adrenergic signaling and quantified several aspects of muscle differentiation. ISO induced an increase in myoblast proliferation, in the percentage of Pax7-positive myoblasts and in the size of skeletal muscle fibers, suggesting that ISO activates a hyperplasic and hypertrophic muscle response. Interestingly, treatment with ISO did not alter the number of fibroblast cells, suggesting that ISO effects are specific to muscle cells in the case of chick myogenic cell culture. We also show that rapamycin, an inhibitor of the mammalian target of rapamycin signaling pathway, did not prevent the effects of ISO on chick muscle fiber size. The collection of these results provides new insights into the role of ß-adrenergic signaling during skeletal muscle proliferation and differentiation and specifically in the regulation of skeletal muscle hyperplasia and hypertrophy.