Quantitative Analysis of the Cardiac Phosphoproteome in Response to Acute ß-Adrenergic Receptor Stimulation In Vivo.

ß-adrenergic receptor (ß-AR) stimulation represents a major mechanism of modulating cardiac output. In spite of its fundamental importance, its molecular basis on the level of cell signalling has not been characterised in detail yet. We employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC (spike-in stable isotope labelling by amino acids in cell culture) standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic ß-AR agonist that targets both ß1-AR and ß2-AR subtypes. Our data describe 8597 quantitated phosphopeptides corresponding to 10,164 known and novel phospho-events from 2975 proteins. In total, 197 of these phospho-events showed significantly altered phosphorylation, indicating an intricate signalling network activated in response to ß-AR stimulation. In addition, we unexpectedly detected significant cardiac expression and ISO-induced fragmentation of junctophilin-1, a junctophilin isoform hitherto only thought to be expressed in skeletal muscle. Data are available via ProteomeXchange with identifier PXD025569.

Bucindolol Decreases Atrial Fibrillation Burden in Patients With Heart Failure and the ADRB1 Arg389Arg Genotype.

[Figure: see text].

Carvedilol induces biased ß1 adrenergic receptor-nitric oxide synthase 3-cyclic guanylyl monophosphate signalling to promote cardiac contractility.

AIMS: ß-blockers are widely used in therapy for heart failure and hypertension. ß-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by ß-blockers in animal hearts. METHODS AND RESULTS: Wild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure ß-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes ß1 adrenergic receptor (ß1AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote ß1AR coupling to a Gi-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust ß1AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced ß1AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts. CONCLUSION: These observations present a novel ß1AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

Compartmentalized ß1-adrenergic signalling synchronizes excitation-contraction coupling without modulating individual Ca2+ sparks in healthy and hypertrophied cardiomyocytes.

AIMS: ß-adrenergic receptors (ßARs) play pivotal roles in regulating cardiac excitation-contraction (E-C) coupling. Global signalling of ß1ARs up-regulates both the influx of Ca2+ through sarcolemmal L-type Ca2+ channels (LCCs) and the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptors (RyRs). However, we recently found that ß2AR stimulation meditates 'offside compartmentalization', confining ß1AR signalling into subsarcolemmal nanodomains without reaching SR proteins. In the present study, we aim to investigate the new question, whether and how compartmentalized ß1AR signalling regulates cardiac E-C coupling. METHODS AND RESULTS: By combining confocal Ca2+ imaging and patch-clamp techniques, we investigated the effects of compartmentalized ßAR signalling on E-C coupling at both cellular and molecular levels. We found that simultaneous activation of ß2 and ß1ARs, in contrast to global signalling of ß1ARs, modulated neither the amplitude and spatiotemporal properties of Ca2+ sparks nor the kinetics of the RyR response to LCC Ca2+ sparklets. Nevertheless, by up-regulating LCC current, compartmentalized ß1AR signalling synchronized RyR Ca2+ release and increased the functional reserve (stability margin) of E-C coupling. In circumstances of briefer excitation durations or lower RyR responsivity, compartmentalized ßAR signalling, by increasing the intensity of Ca2+ triggers, helped stabilize the performance of E-C coupling and enhanced the Ca2+ transient amplitude in failing heart cells. CONCLUSION: Given that compartmentalized ßAR signalling can be induced by stress-associated levels of catecholamines, our results revealed an important, yet unappreciated, heart regulation mechanism that is autoadaptive to varied stress conditions.

Mathematical model for ß1-adrenergic regulation of the mouse ventricular myocyte contraction.

The ß1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the ß1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of ß1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the ß1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of ß1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the ß1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases.NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the ß1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle.

Sex differences in vascular reactivity of coronary artery bypass graft conduits.

Females have increase in-hospital mortality and poorer outcomes following coronary artery bypass grafting (CABG). Biological differences in the reactivity of the graft conduits to circulating catecholamine may contribute to this sex difference. This study examined sex differences in the vasoconstrictor responses of internal mammary artery (IMA) and saphenous vein (SV) conduits to phenylephrine (PE) and endothelin-1 (ET-1). Functional IMA and SV were obtained from 78 male and 50 female patients undergoing CABG (67.7 ± 11 and 69 ± 10 years, respectively) and subjected to the following experimental conditions. (1) Concentration response curves for PE and ET-1 were generated in an intact IMA and SV and endothelium denuded IMA segments, (2) in the presence of the nitric oxide synthase inhibitor (L-NAME) or the cyclooxygenase inhibitor (indomethacin) in an endothelium-intact IMA and (3) the activity state (abundance and phosphorylation) of the α1-adrenergic receptor was investigated using Phos-tag™ western blot analysis. (1) Compared to male, female IMA and SV were hypersensitive to PE but not ET-1 (p < 0.05). The female IMA hypersensitivity response to PE was abolished following endothelial denudation, (2) persisted in the presence of L-NAME but was abolished in the presence of indomethacin and (3) there was no sex differences in the abundance and phosphorylation of the α1-adrenergic receptor in IMA. Female IMA and SV graft conduits are hypersensitive to α1-adrenergic stimuli. This endothelial cyclooxygenase pathway-mediated hypersensitivity may produce excessive IMA and SV graft constriction in females administered catecholamines and could contribute to their poorer CABG outcomes.

Association of Genetic Polymorphisms in the Beta-1 Adrenergic Receptor with Recovery of Left Ventricular Ejection Fraction in Patients with Heart Failure.

Two common genetic polymorphisms in the beta-1 adrenergic receptor (ADRB1 Ser49Gly [rs1801252] and Arg389Gly [rs1801253]) significantly affect receptor function in vitro. The objective of this study was to determine whether ADRB1 Ser49Gly and Arg389Gly are associated with recovery of left ventricular ejection fraction (LVEF) in patients with heart failure. Patients with heart failure and baseline LVEF ≤ 40% were genotyped (n = 98), and retrospective chart review assessed the primary outcome of LVEF recovery to ≥ 40%. Un/adjusted logistic regression models revealed that Ser49Gly, but not Arg389Gly, was significantly associated with LVEF recovery in a dominant genetic model. The adjusted odds ratio for Ser49 was 8.2 (95% CI = 2.1-32.9; p = 0.003), and it was the strongest predictor of LVEF recovery among multiple clinical variables. In conclusion, patients with heart failure and reduced ejection fraction that are homozygous for ADRB1 Ser49 were significantly more likely to experience LVEF recovery than Gly49 carriers.

Cannabinoid CB1 and CB2 receptors antagonists AM251 and AM630 differentially modulate the chronotropic and inotropic effects of isoprenaline in isolated rat atria.

BACKGROUND: Drugs targeting CB1 and CB2 receptors have been suggested to possess therapeutic benefit in cardiovascular disorders associated with elevated sympathetic tone. Limited data suggest cannabinoid ligands interact with postsynaptic ß-adrenoceptors. The aim of this study was to examine the effects of CB1 and CB2 antagonists, AM251 and AM630, respectively, at functional cardiac ß-adrenoceptors. METHODS: Experiments were carried out in isolated spontaneously beating right atria and paced left atria where inotropic and chronotropic increases were induced by isoprenaline and selective agonists of ß1 and ß2-adrenergic receptors. RESULTS: We found four different effects of AM251 and AM630 on the cardiostimulatory action of isoprenaline: (1) both CB receptor antagonists 1 µM enhanced the isoprenaline-induced increase in atrial rate, and AM630 1 µM enhanced the inotropic effect of isoprenaline; (2) AM251 1 µM decreased the efficacy of the inotropic effect of isoprenaline; (3) AM251 0.1 and 3 µM and AM630 3 µM reduced the isoprenaline-induced increases in atrial rate; (4) AM630 0.1 and 3 µM enhanced the inotropic effect of isoprenaline, which was not changed by the same concentrations of AM251. CONCLUSIONS: Our results show that the CB1 and CB2 receptor antagonists AM251 and AM630 have bidirectional effects on the cardiostimulatory action of isoprenaline, most likely related to an interaction with ß1-adrenoceptors. Provided that the results translate to human heart, caution should be taken when using CB1 and CB2 receptor antagonists, as an enhanced sympathetic tone accompanies many cardiovascular disorders.

Modulation of serotonin and noradrenaline in the BLA by pindolol reduces long-term ethanol intake.

Repeated cycles of binge-like alcohol consumption and abstinence change the activity of several neurotransmitter systems. Some of these changes are consolidated following prolonged alcohol use and are thought to play an important role in the development of dependence. We have previously shown that systemic administration of the dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist pindolol selectively reduces long-term but not short-term binge-like consumption of ethanol and alters excitatory postsynaptic currents in basolateral amygdala (BLA) principal neurons. The aim of this study was to investigate the effects of pindolol microinfusions in the BLA on long-term ethanol intake using the drinking-in-the-dark paradigm in mice. We also microinfused RU24969 (5-HT1A/1B receptor partial agonist) and CGP12177 (ß1/2 adrenergic antagonist) following long-term ethanol intake and determined the densities of 5-HT1A/1B receptors and ß1/2 adrenergic in the BLA following short-term (4 weeks) and long-term ethanol (12 weeks) consumption. We show that intra-BLA infusion of pindolol (1000 pmol/0.5 µl), RU24969 (0.3 and 3 pmol/0.5 µl) and CGP12177 (500 pmol/0.5 µl) produce robust decreases in long-term ethanol consumption. Additionally, we identified reduced ß1/2 adrenergic receptor expression and no change in 5-HT1A/1B receptor density in the BLA of long-term ethanol-consuming mice. Collectively, our data highlight the effects of pindolol on voluntary, binge-like ethanol consumption behavior following long-term intake.

Adrenergic ß receptor activation in the basolateral amygdala, which is intracellular Zn2+-dependent, rescues amyloid ß1-42-induced attenuation of dentate gyrus LTP.

On the basis of the evidence that the basolateral amygdala (BLA) modulates hippocampal memory processes via synaptic plasticity, here we report that adrenergic ß receptor activation in the BLA rescues amyloid ß1-42 (Aß1-42)-induced attenuation of long-term potentiation (LTP) at perforant pathway-dentate granule cell (DGC) synapses. When 500 µM isoproterenol (2 µl), an adrenergic ß receptor agonist, was injected into the BLA 20 min before LTP induction, LTP was enhanced. Isoproterenol-mediated enhancement of LTP was blocked by co-injection with 100 µM ZnAF-2DA, an intracellular Zn2+ chelator, suggesting that intracellular Zn2+ is required for the intracellular signaling cascade after adrenergic ß receptor activation in the BLA. Aß1-42-induced attenuation of LTP, which was induced by Aß1-42 injection into the dentate gyrus 60 min before LTP induction, was rescued by isoproterenol injection into the BLA 20 min before LTP induction, but not by 500 µM phenylephrine (2 µl), an adrenergic α1 receptor agonist, injection into the BLA, which did not enhance LTP unlike the case of isoproterenol injection. Interestingly, Aß1-42-induced attenuation of LTP was also rescued by 100 µM isoproterenol injection into the BLA 20 min before LTP induction, which did not enhance LTP. The present study demonstrates that adrenergic ß receptor activation in the BLA, which is linked with intracellular Zn2+ signaling, rescues Aß1-42-induced attenuation of dentate gyrus LTP. It is likely that adrenergic ß receptor activation in the BLA is a strategy for rescuing Aß1-42-induced cognitive decline that is associated with hippocampal synaptic plasticity.

Inhibition of prostatic smooth muscle contraction by the inhibitor of G protein-coupled receptor kinase 2/3, CMPD101.

Alpha1-adrenoceptors induce prostate smooth muscle contraction, and hold a prominent role for pathophysiology and therapy of lower urinary tract symptoms in benign prostatic hyperplasia. G protein-coupled receptors are regulated by posttranslational regulation, including phosphorylation by G protein-coupled receptor kinases 2 and 3 (GRK2/3). Although posttranslational adrenoceptor regulation has been recently suggested to occur in the prostate, this is still marginally understood. With the newly developed CMPD101, a small molecule inhibitor with assumed specificity for GRK2/3 is now available. Here, we studied effects of CMPD101 on smooth muscle contraction of human prostate tissue. Electric field stimulation caused frequency-dependent contractions, which were inhibited concentration-dependently by CMPD101 (5 µM, 50 µM). 50 µM of CMPD101 did not affect myosin light chain (MCL) phosphorylation or Rho kinase activity, and did not alter contractions induced by highmolar KCl. Noradrenaline, the α1-adrenoceptor agonist phenylephrine, endothelin-1, and the thromboxane A2 analogue U46619 induced concentration-dependent contractions, which were inhibited by CMPD101 (50 µM). CMPD101 (50 µM) did not change phosphorylation of ß2-adrenoceptors or ß2-adrenergic relaxation of prostate strips. Molecular detection by Western blot and peroxidase staining suggested expression of GRK2 and GRK3 in human prostates. Double labeling in fluorescence staining confirmed that immunoreactivity for GRK2 and GRK3 was located to smooth muscle cells in the prostate stroma. In conclusion, CMPD101 inhibits adrenergic, neurogenic, and non-adrenergic smooth muscle contractions in the human prostate. Underlying mechanisms may be independent from GRK inhibition, and from inhibition of MLC kinase and Rho kinase. This may point to unknown properties of CMPD101.

Glucagon-Like Peptide 1 Receptor Activation Augments Cardiac Output and Improves Cardiac Efficiency in Obese Swine After Myocardial Infarction.

This study tested the hypothesis that glucagon-like peptide 1 (GLP-1) therapies improve cardiac contractile function at rest and in response to adrenergic stimulation in obese swine after myocardial infarction. Obese Ossabaw swine were subjected to gradually developing regional coronary occlusion using an ameroid occluder placed around the left anterior descending coronary artery. Animals received subcutaneous injections of saline or liraglutide (0.005-0.015 mg/kg/day) for 30 days after ameroid placement. Cardiac performance was assessed at rest and in response to sympathomimetic challenge (dobutamine 0.3-10 µg/kg/min) using a left ventricular pressure/volume catheter. Liraglutide increased diastolic relaxation (dP/dt; Tau 1/2; Tau 1/e) during dobutamine stimulation (P < 0.01) despite having no influence on the magnitude of myocardial infarction. The slope of the end-systolic pressure volume relationship (i.e., contractility) increased with dobutamine after liraglutide (P < 0.001) but not saline administration (P = 0.63). Liraglutide enhanced the slope of the relationship between cardiac power and pressure volume area (i.e., cardiac efficiency) with dobutamine (P = 0.017). Hearts from animals treated with liraglutide demonstrated decreased ß1-adrenoreceptor expression. These data support that GLP-1 agonism augments cardiac efficiency via attenuation of maladaptive sympathetic signaling in the setting of obesity and myocardial infarction.

Blockade of ß2-adrenoceptor, rather than ß1-adrenoceptor, deteriorates cardiac anaphylaxis in isolated blood-perfused rat hearts.

BACKGROUND: Cardiac anaphylaxis is one of the features of anaphylactic hypotension. Patients treated with propranolol, a nonselective ß-adrenoceptor (AR) antagonist, develop severe anaphylaxis, but the mechanism remains unknown. Under examination were the effects of ß1- and ß2-AR antagonist on anaphylaxis-induced coronary vasoconstriction and cardiac dysfunction in isolated blood-perfused rat hearts. METHODS: Isolated hearts from ovalbumin-sensitized Wistar rats were subjected to coronary perfusion with blood at a constant pressure and measurements were made of coronary blood flow and left ventricu-lar (LV) pressure. Following pretreatment with selective ß2-AR antagonist ICI118,551 or selective ß1-AR antagonist atenolol, cardiac anaphylaxis was induced by intracoronary injections of ovalbumin antigen. LV contractility was evaluated by the maximum increasing rate of systolic LV pressure (dP/dtmax). RESULTS: In response to antigen administrations, ICI118,551 pretreated hearts showed a greater de-crease in coronary blood flow and consequently a greater increase in coronary vascular resistance than the atenolol pretreated hearts. Pretreatment with ICI118,551 caused a greater decrease in dP/dtmax than those with atenolol. CONCLUSIONS: Cardiac anaphylaxis-induced contractile dysfunction and coronary spasm are severe in b2-, rather than ß1-AR antagonist, pretreated isolated blood-perfused rat hearts.

ß-Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α-PP2A-Mediated Dephosphorylation.

BACKGROUND: Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. ß-Adrenoceptor (ß-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of ß-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. METHODS AND RESULTS: A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the ß-AR agonist isoproterenol to induce ß1-AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. CONCLUSIONS: ß-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A-mediated phosphorylation of Ser279.

Heterologous desensitization of cardiac ß-adrenergic signal via hormone-induced ßAR/arrestin/PDE4 complexes.

AIMS: Cardiac ß-adrenergic receptor (ßAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between ßARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. METHODS AND RESULTS: Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of ßARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of ß1AR and ß2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote ß2AR phosphorylation effectively inhibit ßAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced ß2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated ß2AR in a ß-arrestin 2 dependent manner without promoting ß2AR endocytosis. The increased binding between ß2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in ß2AR, inhibition of PDE4, or genetic ablation of PDE4D or ß-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of ß-arrestin 2 or PDE4D gene also rescues ß-adrenergic stimuli-induced myocyte contractile function. CONCLUSIONS: These data reveal essential roles of ß-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac ßARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.

Stimulation of ß1- and ß2-adrenoceptors dilates retinal blood vessels in rats.

Our previous studies have demonstrated that adrenaline dilates rat retinal arterioles by stimulating propranolol-sensitive ß-adrenoceptors and ß3-adrenoceptors, and selective stimulation of ß2- or ß3-adrenoceptors causes retinal vasodilator responses. In the present study, we compared the effects of ß1- and ß2-adrenoceptor stimulation on rat retinal arterioles in vivo. Rat ocular fundus images were captured using an original high-resolution digital fundus camera. Diameters of retinal arterioles contained in the images were measured. Systemic blood pressure and heart rate were recorded continuously. Denopamine, a ß1-adrenoceptor agonist, increased the diameter of retinal arterioles and heart rate, and produced a small but statistically insignificant decrease in mean arterial pressure. CGP20712A, a ß1-adrenoceptor antagonist, but not ICI118551, a ß2-adrenoceptor antagonist, significantly prevented denopamine-induced retinal vasodilator and heart rate responses. Salbutamol, a ß2-adrenoceptor agonist, increased the diameter of retinal arterioles and decreased mean arterial pressure without significantly changing heart rate. The effects of salbutamol were significantly prevented by ICI118551, but not by CGP20712A. These results suggest that stimulation of ß1- and ß2-adrenoceptors dilates retinal blood vessels and indicate that all three ß-adrenoceptor subtypes (ß1, ß2, and ß3) may be involved in the retinal vasodilator response to adrenaline in rats.

Use of non-selective ß-blockers is associated with decreased tumor proliferative indices in early stage breast cancer.

Previous studies suggest beta-adrenergic receptor (ß-AR) antagonists (ß-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of ß1-AR and ß3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of ß-blocker usage on tumor proliferation. Our analysis revealed that non-selective ß-blockers, but not selective ß-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of ß-blockers were measured in a panel of breast cancer lines, demonstrating that mammary epithelial cells were resistant to propranolol, and that most breast cancer cell lines displayed dose dependent viability decreases following treatment. Selective ß-blockers alone or in combination were not as effective as propranolol at reducing breast cancer cell proliferation. Molecular analysis revealed that propranolol treatment of the SK-BR-3 breast cancer line, which showed high sensitivity to beta blockade, led to a reduction in Ki67 protein expression, decreased phosphorylation of the mitogenic signaling regulators p44/42 MAPK, p38 MAPK, JNK, and CREB, increased phosphorylation of the cell survival/apoptosis regulators AKT, p53, and GSK3ß. In conclusion, use of non-selective ß-blockers in patients with early stage breast cancer may lead to decreased tumor proliferation.

Antiadrenergic autoimmunity in postural tachycardia syndrome.

AIMS: Postural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and ß1/2-adrenergic receptors (ß1/2AR). METHODS AND RESULTS: Immunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and ß1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and ß1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, ß1AR and ß2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and ß1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the ß1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated ß1AR and α1AR activity but not with ß2AR activity. CONCLUSION: These data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and ß1AR responsiveness are important in the pathophysiology of postural tachycardia.

Selective α1-adrenergic blockade disturbs the regional distribution of cerebral blood flow during static handgrip exercise.

Handgrip-induced increases in blood flow through the contralateral artery that supplies the cortical representation of the arm have been hypothesized as a consequence of neurovascular coupling and a resultant metabolic attenuation of sympathetic cerebral vasoconstriction. In contrast, sympathetic restraint, in theory, inhibits changes in perfusion of the cerebral ipsilateral blood vessels. To confirm whether sympathetic nerve activity modulates cerebral blood flow distribution during static handgrip (SHG) exercise, beat-to-beat contra- and ipsilateral internal carotid artery blood flow (ICA; Doppler) and mean arterial pressure (MAP; Finometer) were simultaneously assessed in nine healthy men (27 ± 5 yr), both at rest and during a 2-min SHG bout (30% maximal voluntary contraction), under two experimental conditions: 1) control and 2) α1-adrenergic receptor blockade. End-tidal carbon dioxide (rebreathing system) was clamped throughout the study. SHG induced increases in MAP (+31.4 ± 10.7 mmHg, P < 0.05) and contralateral ICA blood flow (+80.9 ± 62.5 ml/min, P < 0.05), while no changes were observed in the ipsilateral vessel (-9.8 ± 39.3 ml/min, P > 0.05). The reduction in ipsilateral ICA vascular conductance (VC) was greater compared with contralateral ICA (contralateral: -0.8 ± 0.8 vs. ipsilateral: -2.6 ± 1.3 ml·min(-1)·mmHg(-1), P < 0.05). Prazosin was effective to induce α1-blockade since phenylephrine-induced increases in MAP were greatly reduced (P < 0.05). Under α1-adrenergic receptor blockade, SHG evoked smaller MAP responses (+19.4 ± 9.2, P < 0.05) but similar increases in ICAs blood flow (contralateral: +58.4 ± 21.5 vs. ipsilateral: +54.3 ± 46.2 ml/min, P > 0.05) and decreases in VC (contralateral: -0.4 ± 0.7 vs. ipsilateral: -0.4 ± 1.0 ml·min(-1)·mmHg(-1), P > 0.05). These findings indicate a role of sympathetic nerve activity in the regulation of cerebral blood flow distribution during SHG.