β2-AR mRNA Research Articles

Agonist-induced β2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells

It is not clear whether increased asthma severity associated with long-term use of β2-adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o− cells grown in the presence and absence of β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Atorvastatin Prevents Sepsis-Induced Downregulation of Myocardial β1-Adrenoceptors and Decreased Camp Response in Mice

Impaired cardiac β-adrenoceptor signaling is an important cause of sepsis-induced myocardial depression in man and experimental animals. We examined the effect of atorvastatin (ATR) pretreatment on myocardial β1-adrenoceptor (β1-AR) expressions and post-receptor signaling in a mouse model of sepsis (cecal ligation and puncture [CLP]). After 20 ± 2 h of surgery, hearts were isolated for the measurement of left ventricular functions (left ventricular developed pressure, dp/dt(max) and dp/dt(min)) using Langendorff setup. Western blot was used to determine β1-AR and G protein-coupled receptor kinase 2 protein expressions. Real-time polymerase chain reaction was done to determine β1-AR mRNA expression. Atorvastatin prevented sepsis-induced decrease in left ventricular functions, such as left ventricular developed pressure (CLP 75.90 ± 0.53 vs. ATR 100.24 ± 1.64 mmHg), dp/dtmax (CLP 3,742 ± 71 vs. ATR 4,291 ± 88 mmHg/s), and dp/dt(min) (CLP -1,010 ± 24 vs. ATR -1,346 ± 84 mmHg/s). Associated with functional impairments, sepsis decreased both myocardial β1-AR protein and mRNA expressions by 52% ± 9% and 62% ± 7%, respectively. However, ATR treatment of CLP mice (ATR) preserved β1-AR protein (96% ± 11%) and mRNA (88% ± 14%) expressions comparable to sham-operated level. Furthermore, it not only attenuated sepsis-induced decrease in basal cardiac adenosine 3',5'-cyclic monophosphate content (CLP 1.30 ± 0.27 vs. ATR 6.30 ± 0.67 pmol/mg protein), but also prevented its refractoriness to dobutamine stimulation (CLP 1.72 ± 0.27 vs. ATR 10.83 ± 1.37 pmol/mg protein). Atorvastatin also inhibited sepsis-induced increase in cardiac G protein-coupled receptor kinase 2 protein expression (CLP 1.73 ± 0.18-fold vs. ATR 1.10 ± 0.18-fold), protein kinase A activity (CLP 1.12 ± 0.14 vs. ATR 0.66 ± 0.08 U/mg protein) and plasma catecholamines (CLP 138 ± 22 vs. ATR 59 ± 2 pg/mL). In conclusion, ATR seems to improve left ventricular functions in vitro through the preservation of β(1)-AR signaling in sepsis.

Let-7e replacement yields potent anti-arrhythmic efficacy via targeting beta 1-adrenergic receptor in rat heart.

Beta-adrenoceptor (β-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of β1-AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β1-AR expression was significantly up-regulated and let-7a, c, d, e and i were markedly down-regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let-7e suppressed β1-AR expression at the protein level, without affecting β1-AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced β1-AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited β1-AR expression in rats, whereas len-AMO-let-7e up-regulated β1-AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β1-AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on β1-AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of β1-AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective β-AR blocker and metoprolol, a selective β1-AR blocker. Down-regulation of let-7e contributes to the adverse increase in β1-AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse β1-AR up-regulation and treating AMI-induced arrhythmia.

Effects of chronic administration of β-blockers on airway responsiveness in a murine model of heart failure

Lung function abnormalities, both at rest and during exercise, are frequently observed in patients with chronic heart failure (HF), also in absence of respiratory disease. It has been documented that, in HF, chronic adrenergic stimulation down-regulates β-adrenoceptors (β-ARs) and modifies airway relaxant responses. This study was designed to investigate in an animal model of HF whether a treatment with a β-AR blocker, metoprolol, could modify the altered airway hyperresponsiveness.In rats, randomly assigned to 3 experimental groups sham-operated rats (SH), rats with HF induced by left anterior descending coronaric occlusion (HF n = 10), and rats treated with metoprolol 100 mg/kg/die (MET = 10), HF was evaluated after 10 weeks and resulted in increases in plasma norepinephrine and epinephrine and left ventricular end diastolic pressure. β2-ARs and G-protein-βAR2-kinase (GRK2) mRNA levels were determined by real time reverse transcriptase PCR. Carbachol-precontracted isolated tracheal rings were used to functionally assess airway smooth muscle relaxation.In pulmonary tissues, β2-AR mRNA level was significantly decreased in HF groups (−48.73 ± 5.18%, P < 0.01); in the same groups the GRK2 mRNA-levels were significantly enhanced (+222.50 ± 6.13%, P < 0.001); in lung deriving from MET groups the levels of mRNA were significantly increased (+339.86 ± 11.26%, P < 0.001), while the GRK2 mRNA-levels unchanged (−59.02 ± 3.97%, P < 0.001), when compared to SH groups. Relaxation of tracheal strips in response to salbutamol was significantly reduced in HF groups; in tracheal rings, deriving from MET groups, the relaxant effects of salbutamol were significantly enhanced (SH, Emax: 34.87 ± 2.98%, pD2: 7.45 ± 0.27; HF, Emax: 34.87 ± 2.98%, pD2: 7.45 ± 0.27; MET, Emax: 85.43 ± 6.80%, pD2: 6.95 ± 0.59, P < 0.001).In HF, the down-regulation of pulmonary β-ARs results in a significant attenuation of airway relaxation. These effects have been reversed by a treatment with metoprolol, suggesting a potential role of β-AR blockers in the treatment of patients suffering from HF and chronic obstructive airway diseases.

Heart ventricles specific stress-induced changes in β-adrenoceptors and muscarinic receptors.

The left and right ventricles fulfill different role in heart function. Here we compare chamber specific changes in local catecholamine concentrations; gene expression and the receptor protein amount of all three β-adrenoceptors (β-AR) in rat right heart ventricles exposed to acute (1 session) and repeated (7 sessions) immobilization stress (IMMO) vs. previously observed changes in left ventricles. Density of muscarinic receptors as main cardio-inhibitive receptors was also measured. In the right ventricles, noradrenaline and adrenaline were increased. No β1-AR changes were observed, in spite of the increased sympathetic activity. On the other hand, we have found a decrease of β2-AR gene expression (reduction to 30%) after 7 IMMO and protein (to 59%) after 1 IMMO. β3-AR gene expression was increased after 7 IMMO. Muscarinic receptor density was not changed. When comparing correlation in left and right ventricles, there was strong correlation between adrenaline and β2-AR gene expression, protein and β3-AR gene expression in the left ventricles while only correlation between adrenaline and β2-AR mRNA and protein in the right ventricles was found. Our results show that maintenance of cardiac homeostasis under stress conditions are to a great extent achieved by a balance between different receptors and also by a balanced receptor changes in left vs. right ventricles. Taken together, decrease of cardio-stimulating β2-AR represents a new important mechanism by which β2-AR contributes to the heart physiology.

Heart-protective effect of n-3 PUFA demonstrated in a rat model of diabetic cardiomyopathy

This study was designed to examine in vivo functional changes of the heart in the early stages of streptozotocin (STZ)-induced diabetic cardiomyopathy and to evaluate the effects of n-3 PUFA intake. Moreover, we investigated whether modulation of diabetes-related abnormalities of myocardial connexin-43 (Cx43), β-myosin heavy chain (β-MHC), and β1-adrenergic receptors (β1-AR) might be implicated in the cardioprotective mechanism of n-3 PUFA. Our results showed significantly reduced cardiac output and ejection fraction (using the microtip pressure-volume catheter technique) as well as stroke volume and stroke work, 4weeks after STZ-induced diabetes, with improvement of these parameters due to n-3 PUFA consumption. Myocardial expression of Cx43 mRNA estimated by real-time polymerase chain reaction did not change in diabetic rats regardless of n-3 PUFA consumption (100mg/100gb.w./day). In contrast, the total and functional phosphorylated form of Cx43 protein increased significantly, and its cardiomyocyte-related distribution was disordered in the diabetic heart, but these changes normalized because of n-3 PUFA intake. Furthermore, acute diabetes was accompanied by decrease of myocardial β1-AR mRNA expression and mild yet nonsignificant increase of β-MHC mRNA. These alterations were not significantly affected by n-3 PUFA. In conclusion, the results point out that STZ-diabetic rats benefit from n-3 PUFA consumption particularly because of the attenuation of myocardial Cx43 abnormalities that most likely contributes to improvement of cardiac function.

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

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

Early postnatal maternal separation causes alterations in the expression of β3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity

The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague–Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved by separating the rat pups from their mothers for 3h each day during the 10–15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), β3-adrenergic receptor (β3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through β3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the β3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.

Antiobese function of platelet‐activating factor: increased adiposity in platelet‐activating factor receptor‐deficient mice with age

Platelet-activating factor receptor (PAFR)-deficient mice developed a more severe obese state characterized by higher body mass (~25%) and epididymal fat mass (~55%) with age than that of wild-type (WT) littermates. PAFR-deficient mice did not show changes in the expression of critical genes involved in anabolic and catabolic metabolism in adipose, liver, and muscle tissues between 6 and 36 wk. However, a 38-81% reduction in β3/β1-adrenergic receptor (AR) and uncoupling protein 1 (UCP1) mRNA and protein levels was observed in the interscapular brown adipose tissue (BAT) of PAFR-deficient mice. Whereas a single injection of the β3-adrenergic agonist, CL-316,243 (25 μg/kg) increased temperatures in the brown fat and rectums of WT mice, this increase in temperature was markedly suppressed in PAFR-deficient mice. Acetyl-CoA:lyso-platelet-activating factor (PAF) acetyltransferase, which is involved in PAF biosynthesis, and the PAF receptor were predominantly localized in BAT macrophages, whereas brown adipocytes possessed the enzyme and functional PAF receptors. The stimulation of brown adipocytes by PAF induced the expression of β3-AR mRNA and protein (1.5- and 1.9-fold, respectively), but not that of UCP1. These results indicate that obesity in PAFR-deficient mice resulted from impaired BAT activity and suggest that the antiobese function of PAF occurs through β3-AR/UCP1 expression in BAT.

Gonadal hormone dependent developmental plasticity of catecholamine:β2-adrenoceptor signaling complex in male rat thymus: Putative implications for thymopoiesis

The study was undertaken considering that: i) androgens affect β2-adrenoceptor (AR)-mediated catecholamine (CA) action in many tissues; and ii) peripubertal changes in both circulating androgen and thymic CA levels are implicated in rat thymic involution. Its aims were to: i) explore putative effects of the late prepubertal orchidectomy on thymic CA:β2-AR complex in young adult rats, and ii) delineate the direct effects of testicular hormone withdrawal on the CA:β2-AR complex from those elicited secondarily through altered influence of this complex components on each other's availability. Upon showing that prepubertal orchidectomy augmented the efficacy of thymopoiesis through increasing the thymocyte surface density of Thy-1, whose expression is negatively regulated by β2-AR-mediated signaling, we examined the effects of orchidectomy and 14-day-long propranolol (PROP) treatment in orchidectomized (ORX) and sham-ORX rats on thymic norepinephrine (NE) concentration and metabolism and β2-AR expression. Orchidectomy, despite an increase in the average NE amount per thymocyte and total thymocyte NE content, diminished thymic NE concentration. This decrease reflected the diminished density of CA-synthesizing nerve fibers, CD68+ macrophages, cortical (aminopeptidase A+), and medullary (UEA-1+) thymic epithelial cells (TECs) and their CA content (probably due to lessened TH expression accompanied by increased MAO-A expression). Moreover, orchidectomy decreased the surface β2-AR expression on thymocytes, CD68+ macrophages and OX-62+ dendritic cells, but increased its expression on the TECs. In sham-ORX rats, PROP reduced thymic NE concentration by diminishing TH expression in the thymic cells. Additionally, PROP in thymocytes and thymic stromal cells diminished and enhanced the β2-AR mRNA expression, respectively. However, in ORX rats PROP did not significantly affect CA(NE):β2-AR complex components. This indicated that prepubertal orchidectomy affects ability of young adult rats to respond to β-AR blockade by altering thymic NE and β2-AR availability. Collectively, the results showed that testicular hormones contribute to alterations in thymus/thymopoiesis during the critical peripubertal period by shaping modulatory sympathetic influence and CA autocrine/paracrine action within the organ.

Effects of Exercise on Stress-Induced Changes of Norepinephrine and Serotonin in Rat Hippocampus

Exercise is beneficial to brain and can attenuate stress-induced hippocampal damages. However, the details involved monoamine neurotransmitter in exercise to counteract stress have not been well elucidated. The aim of this study was to examine exercise-induced responses of the norepinephrine (NE) and serotonin (5-HT) systems in counteracting stress-induced hippocampal damages. Rats were divided into exercise (four weeks of voluntary wheel running), stress (three weeks of restraint stress), exercise-stress (three weeks of stress following four weeks of exercise), and control groups. Levels of NE and 5-HT were detected with high-performance liquid chromatography (HPLC), mRNA expression was detected with real-time fluorescence quantitative reverse transcription polymerase reaction (FQ-RT-PCR) and proteins associated with 5-HT₁Α receptors (5-HT₁Α-R) and β₂-adrenergic receptors (β₂-AR) were analyzed by western blotting. 5-HT levels were highest (P < 0.01) in the exercised group, lowest (P < 0.05) in the stressed rats, and were similar (P = 0.065) in stressed and exercise-stressed rats. NE levels were highest (P < 0.01) in the exercised group, and higher in the exercise-stressed than the stressed rats (P < 0.01). 5-HT₁A-R mRNA expression was highest (P < 0.01) in the exercised group, lowest in the stressed group. The 5-HT₁Α-R protein expression changed in the same tendency as its mRNA levels. The β₂-AR mRNA was highest in exercised rats (P < 0.05), and its protein expression was higher in the exercised and exercise-stress rats than in the control and stress rats (P < 0.05). In conclusion, norepinephrine may represent endophenotypic features of exercise states. Serotonin levels may be more susceptible to stress and responsible for deleterious stress-induced effects. Norepinephrine and serotonin may both contribute to counteraction of stress-induced hippocampal damages of physical exercises.

Construction of a cell model of β2-adrenoceptor downregulation.

To establish a cell model of β2-adrenergic receptor (β2AR) downregulation of murine airway smooth muscle induced by salbutamol to elucidate the molecular and biological mechanisms of β2AR downregulation. Airway smooth muscle cells (ASMCs) derived from Balb/c mice were primary cultured. Passage 2-5 cells were characterized by cell morphology and indirect immunofluorescence. More than 95% pure cells at passage 3 or 4 were randomly divided into two groups: control and salbutamol-treated groups. β2AR mRNA and protein expression levels were then detected by RT-PCR and western blot analyses. Primary cultured cells demonstrated a typical "peak and valley"-like growth characteristic. Smooth muscle α-actin filaments paralleled the cell longitudinal axis in the cytoplasm. The origin of the ASMCs was validated and consistent with their morphology and biological characteristics. β2AR mRNA expression in the salbutamol-treated group was lower than that in the control group (P<0.05), and β2AR protein expression was also markedly lower than that in the control group (P<0.05). We successfully established a cell model of β2AR downregulation in ASMCs, which may provide the foundation for further study of the mechanism of β2AR downregulation in asthmatic patients.

Relationship between the Autoantibody and Expression of β3-Adrenoceptor in Lung and Heart

BackgroundEvidences suggest that β3 -adrenoceptor (β3-AR) plays an important role in heart failure (HF), although no data is reported indicating how these effects may change with the increasing age. Pulmonary congestion and edema are the major life-threatening complications associated with HF. The purpose of this study is to explore the relationship between the anti-β3-AR autoantibody and the expression of β3-AR in the lungs and heart for both aged patients and rats with HF.MethodsSynthetic β3-AR peptides served as the target antigens in ELISA were used to screen the anti-β3-AR autoantibody in aged patients and rats. Two aged rat models were constructed based on aortic banding and sham-operation. The expression of β3-AR mRNA and protein in the lung and heart was measured in intervention and non-intervention groups by Western blot analysis at the baseline, 5th, 7th, 9th and 11th week, respectively.ResultsThe frequency and titer of anti-β3-AR autoantibody in aged patients and rats with HF were higher than those in the control group (p<0.05). The expression of β3-AR mRNA and protein in pulmonary tissues decreased continually from the 7th week (p<0.05), followed by HF observed during the 9th week. The expression of β3-AR in myocardial tissues continued to increase after the 9th week (p<0.05), and the expression of both β3-AR mRNA and protein in the BRL group [HF group with BRL37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino] phenoxyacetic acid) (a β3-AR agonist) injection] was positively correlated with BRL37344 when compared with non-BRL group (HF group without BRL37344 injection) (p<0.05).ConclusionAnti-β3-AR autoantibody was detected in aged patients and rats with HF. The expression of β3-AR mRNA and protein in pulmonary tissues decreased continually, and began earlier than in the heart, but its expression in myocardial tissues increased continually and could be further promoted by β3-AR agonist.

Abstract 19478: The Protective Effect of Beta3-Adrenergic Signaling Occurs via Differential Phosphorylation of nNOS in Cardiac Myocytes

Rationale: Stimulation of β3-adrenoreceptor (β3-AR) signaling improves left ventricular function via a nNOS/NO-dependent manner in hypertrophied and failing animal hearts. nNOS, which is expressed in cardiac myocytes, is regulated by modification of stimulatory phosphorylation residue Ser1412 and inhibitory residue Ser847. However, how differential phosphorylation of these residues contribute to cardiomyocyte β3-AR-mediated NOS activation and signaling has yet to be explored. Objective: The aim of this study is to identify the molecular mechanisms involved in β3-AR-dependent regulation of nNOS activation and its downstream signaling in cardiomyocytes in the setting of hypertrophy. Methods and Results: In neonatal rat ventricular cardiomyocytes isolated from 4 day old pups, 48 hours of ET-1or 72 hours of norepinephrine lead to increased cell size and β3-AR mRNA. In hypertrophied cells, specific β3-AR agonist BRL-37433 induced NOS activity, nNOS phosphorylation at site Ser1412, dephosphorylation of site Ser847, and ROS suppression. BRL-dependent NOS activity and ROS suppression were attenuated by the nNOS inhibitor L-VNIO. NOS activity was also attenuated in phosphomimetic mutants Ser1412A (constitutively dephosphorylated) and Ser847D (constitutively phosphorylated). However, BRL failed to reduce ROS in the presence of Ser847D. In addition, Akt inhibition decreased BRL-induced Ser1412 phosphorylation and NOS activity, while Gi/o blockade altered BRL-regulation of both phosphorylation sites, NOS activity, and ROS reduction. In vivo, BRL administration completely dephosphorylated Ser847 and moderately increased Ser1412 phosphorylation in mice hearts under pressure overload. Conclusion: Our data suggest that β3-AR regulates myocardial NOS activity and ROS via alterations in nNOS phosphorylation in hypertrophied myocytes and failing hearts. This is the first study to demonstrate a role for nNOS phosphorylation as a key factor in cardiomyocyte and β3-AR signaling. These results contribute significantly to our understanding the protective properties of β3-AR signaling during cardiac sympathetic overdrive, and will ultimately aid in drug discoveries that target molecular mechanisms to treat heart failure.

P 23: The possible contribution of thyroid hormones to insulin effect on beta adrenoceptor mediated cardiac responses in diabetic rats

As is well known, diabetes-induced cardiovascular complications are leading factors of morbidity and mortality. Among them atherosclerosis, hypertension and heart failure are most important ones. The very first problem seen in diabetic heart is the decrease of beta adrenoceptor mediated cardiac responses. The decrease of catecholamine stimulated beta adrenoceptor responsiveness is mostly associated with diminished receptor sensitivity and density. Insulin treatment of diabetic rats has been demonstrated to correct these parameters. However, the improving effect of insulin is shown to be abolished in tyhroidectomized diabetic rats. Although the effect of the relationship between these two hormones on some pathways has already been studied, their exact role on beta adrenoceptor mediated cardiac responses is uncertain. Thus in the present study, we aimed to determine the possible relationship between insulin and thyroid hormones to normalization of diabetes induced cardiac abnormalities. Male Sprague-Dawley rats were used for this study. Ten days after surgical thyroidectomy, diabetes was induced with 38mg/kg streptozotocin by a single intravenous tail-vein injection. After 6-weeks of-diabetes induction, some groups (D-I, TxD-I) were treated with insulin (5–20U/kg/day, subcutan), some other groups (TxD-I-T2.5, TxD-I-T5) were administered insulin + thyroid hormone combination (2,5 or 5ug/kg/day T3 via osmotic minipump) for 2 weeks. Cardiac function was determined by the assessment of in vivo pressure-volume (PV) analysis and in vitro left ventricular papillary muscle experiments (isoprenaline, 0,0001uM-3uM; forskoline, 3uM). mRNA expressions of β1-AR and SERCA were evaluated by QPCR. Heart rate and end systolic pressure were markedly reduced in diabetic (D) and thyroidectomized diabetic (TxD) groups; however, they were improved with insulin treatment in D group. On the other hand, in TxD group, only insulin and 5ug/kg T3 combination (TxD-I-T5) ameliorated these parameters. End diastolic pressure and ejection fraction were found to be unchanged between the groups. Rate of contraction and relaxation, time constant of left ventricle pressure decay were reduced in D and TxD groups. These parameters were corrected significantly in D-I and TxD-I-T5 groups. Cardiac index was reduced in D and TxD groups. This parameter was not increased markedly in any of the treated groups. End systolic volume index was raised in D and TxD groups, and reduced only in D-I group. End diastolic pressure index was also increased in D and TxD groups. It was significantly decreased in D-I and TxD-I-T5 group. Isoprenaline, a nonselective β-AR agonist, induced concentrationdependent positive inotropic effects on papillary muscles of control rats (C). Maximum response (Emax) was markedly diminished in D and TxD groups. These parameters were ameliorated in D-I group significantly. Furthermore, in TxD-I-T5 group, the contractile response was enhanced compared to TxD group. Forskolin induced positive inotropic effect in all groups. The response was decreased in D and TxD groups. Insulin treatment improved this decrease in D-I and TxD-I-T5 groups. β1-AR mRNA levels were decreased both in D and TxD groups. Insulin treatment increased β1-AR mRNA levels. In TxD-I group insulin did not increase β1-AR mRNA levels. On the other hand, in TxD-I-T5 group β1-AR mRNA levels were significantly increased compared to TxD, TXD-I and TxD-I-T2.5 groups. SERCA mRNA levels were also found to be reduced in D and TxD groups. Insulin treatment did not correct the decrease in mRNA levels of SERCA of D and TxD groups. In TxD-I-T5 group, SERCA mRNA levels were significantly increased compared to TxD, TXD-I and TxD-I-T2.5 groups. Both in vivo and in vitro cardiac experiments showed that heart function was impaired in D and TxD groups. Insulin exerted beneficial effects only in D-I and TxD-I-T5 groups. As no improvement was seen in TxD-I group, it could be thought that insulin could not reverse beta adrenoceptor mediated cardiac responses in thyroid deficiency. The slight but still insignificant recovery that we observed in TxD-I-T2.5 group could be interpreted with inadequacy of T3 dose.

Expression of β-adrenergic receptors in pediatric malignant brain tumors.

β-adrenergic receptors (β-ARs) are G protein-coupled receptors that activate signal transduction pathways involved in angiogenesis, resulting in enhanced tumor vascularization and more aggressive growth. In this study, we evaluated the expression of β-ARs in a population of 12 children affected by malignant primary brain tumors. We found a significant expression of β1- and β2-ARs in all 12 samples as well as the 3 cell lines tested (U87MG, T98G and DAOY). The mean absolute β1-AR mRNA level standardized to GAPDH was 5.81 (range, -7.91 to 11.29) for brain tumors and 8.59 (range, 6.046 to 12.59) for cell lines (U87MG, DAOY and T98G), respectively. The mean absolute β2-AR mRNA level was 4.74 (range, -9.30 to 8.45) for tumor specimens and 7.64 (range, 5.85 to 8.88) for cell lines. These real-time quantitative (qRT)-PCR expression data were confirmed by immunohistochemical analysis. Our study evaluated the presence of β1- and β2-ARs in malignant pediatric brain tumors and brain tumor cell lines.

Upregulation of β3-Adrenergic Receptor Expression in the Atrium of Rats With Chronic Heart Failure

To investigate the expression of β(3)-adrenergic receptor (β(3)-AR) in the atrium of rats with chronic heart failure (CHF). The heart failure rat model was established by aortic constriction. Thirty-six male Wistar rats were divided into Sham group (n = 10) and heart failure model group (n = 26), which were further divided into CHF control (CHF group) and BRL group. The rats in the BRL group were treated with a selective β(3)-AR agonist, BRL-37344 (4.0 nmol/kg, twice weekly) for 4 weeks. In the BRL group, the left ventricular end-systolic pressure (83.21 ± 13.0 vs 101.50 ± 12.12 mm Hg) and the absolute values of the maximal rate of rise and fall of left ventricular pressure ([±dP/dtmax] 2.81 ± 0.04 vs 0.35 ± 0.04 and -2.72 ± 0.06 vs -3.33 ± 0.06) were lower than in the CHF group (P < .01). The left atrial mass index (LAMI) in the BRL group (0.4132 ± 0.0306) was higher than that in the CHF (0.3212 ± 0.0136) or Sham group (0.2683 ± 0.0145; P < .01). The levels of the left atrial β(3)-AR messenger RNA (mRNA) expression in the BRL group (0.932 ± 0.055) was higher than that in the CHF (0.706 ± 0.043) or Sham group (0.310 ± 0.020; P < .01). In all animals, there was a positive correlation between the level of β(3)-AR mRNA expression and the left or right atrial mass index (correlation coefficient ranged from 0.744 to 0.937). There is a significant increase in the β(3)-AR mRNA expression in the atrium of rats with heart failure. The level of β(3)-AR mRNA expression was associated with the AMI and was enhanced by a selective β(3)-AR agonist, BRL-37344.

Abstract 652: Regulation of Cardiac MicroRNA miR-208a by Angiotensin II and the Beta-Adrenergic Receptor Blocker Nebivolol

The microRNA miR-208a is a pathologic myo-miR required for hemodynamic pressure overload induced cardiac fibrosis. It is unclear how cardio-deleterious hormones and cardioprotective drugs regulate expression of miR-208a. We hypothesized that the pro-inflammatory hormone angiotensin II (Ang II) and anti-inflammatory drugs rapamycin (Rap: inhibitor of the nutrient sensor kinase mTOR) and nebivolol (Neb: a 3 rd generation β1 adrenergic receptor [β1-AR] blocker that exhibits β3-AR agonism) modulate miR-208a expression in cardiomyocytes. Methods and Results: In mouse atrial cardiomyocyte HL-1 cells, 12 hour treatment with Ang II (100nM) increased miR-208a by 6 fold as determined by real-time RT-PCR analysis (P&lt;0.05; n=5). AT1 receptor (AT1R) blocker losartan (10μM), but not AT2 receptor (AT2R) blocker PD123319 (10μM), inhibited this effect. Pre-treatment (1hr) with Rap (10nM) and Neb (1μM), but not apocynin (500μM: NADPH oxidase inhibitor) or U73122 ((10μM: Phospholipase C inhibitor) attenuated Ang II-induced increases in miR-208a levels (P&lt;0.05). Thus, the AT1R-mediated activation of mTOR up-regulates miR-208a in cardiomyocytes. Immunoblotting revealed that Ang II increased phosphorylation (p) of mTOR (pSer 2448 ), S6K1 (pThr 389 ) and RPS6 (pSer 235/236 ) by 1.5 to 2 fold in HL-1 cells and Rap and Neb suppressed this effect (P&lt;0.05). Neb-induced β3-AR agonism was not involved in miR-208a suppression since β3-AR antagonist SR52930 (1μM) did not reverse Neb-induced suppression of miR-208a and β3-AR mRNA was not detected in HL-1 cells by real time RT-PCR. Interestingly, a 12 hour treatment of HL-1 cardiomyocytes with 1μM Neb attenuated 125 I-Ang II binding to the AT1R by (70%: P&lt;0.05); however a 15 minute pre-incubation with 1μM Neb did not prevent 125 I-Ang II binding to HL-1 cells. Thus, chronic Neb treatment down-regulates AT1R function in cardiomyocytes. Conclusions: We show for the first time that Ang II increases miR-208a expression in cardiomyocytes via activation of mTOR-S6K1 signaling pathway. Nebivolol, a β-blocker, inhibits Ang II-induced miR-208a expression and mTOR signaling. Moreover, chronic Neb treatment down-regulates AT1R in cardiomyocytes. Regulation of AT1R-mTOR-miR-208a axis by Neb is a novel cardioprotective mechanism.

Abstract 92: The Protective Effect of β3-Adrenergic Signaling Occurs via Differential Phosphorylation of nNOS in Cardiac Myocytes

Objective: The aim of this study is to identify the mechanisms involved in β3-adrenoreceptor (β3-AR)-dependent cardiac protection via nNOS signaling in cardiomyocytes in the setting of hypertrophy. Background: β3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike β1- and β2-ARs, β3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, it serves as a “brake” to protect the heart from catecholamine overstimulation. We previously showed that β3-agonism restored left ventricular function, generated nitric oxide, and suppressed reactive oxygen species in mouse myocardium after pressure overload. Interestingly, cardioprotection was lost after acute nNOS inhibition and in nNOS -/- animals. Methods: Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 2-4 day old Sprague-Dawley pups. Cells were treated with hypertrophic agents (angiotensin II, endothelin-1, and norepinephrine), the specific β3-AR agonist (BRL-37433),and phosphometic Sindbis viruses for nNOS. Results: Forty-eight hours of ET-1 (100nM) or 72 hours of NE (100μ M) treatment increased cell size and β3-AR mRNA expression vs. untreated cells. In hypertrophied cardiomyocytes, BRL (75nM) reduced cell size and induced NOS activity, nNOS phosphorylation at stimulatory site Ser1412, dephosphorylation of deactivation site Ser847, and ROS suppression. BRL-dependent NOS activity and ROS suppression were both attenuated by the nNOS inhibitor L-VNIO. NOS activity was also attenuated by phosphomemetic mutants Ser1412A (constitutively dephosphorylated) and Ser847D (constitutively phosphorylated). In addition, G i/o and Akt inhibition suppressed BRL-induced nNOS-Ser1412 phosphorylation and NOS activity. Conclusion: Our data suggest that BRL regulates β3-specific myocardial NOS activity via alterations in nNOS phosphorylation in isolated hypertrophied myocytes and failing hearts of murine animals. This is the first study to demonstrate a role for nNOS phosphorylation as a key factor in cardiac myocyte and β3-AR signaling. These results contribute significantly to our understanding of the cardiac protection.

Relationship between expression of β3‐adrenoceptor mRNA in bladder mucosa and urodynamic findings in men with lower urinary tract symptoms

To investigate the relationship between urinary bladder mucosal expression of β3-adrenoceptor (AR) mRNA and urodynamic findings in patients with lower urinary tract symptoms and benign prostatic obstruction (BPO). During surgical prostate resection of 32 BPO patients, mucosal biopsies were collected and analyzed by reverse transcriptase polymerase chain reaction to determine the expression level of β3-AR mRNA. First desire to void (FDV) and strong desire to void (SDV), detrusor overactivity (DO), and bladder outlet obstruction (BOO) were measured pre-operatively. Patients with FDVs < 200 ml or SDVs < 300 ml were assigned to the small capacity group (n = 19). Patients with FDVs > 201 ml and SDVs > 301 ml were assigned to the large capacity group (n = 13). The same patients with positive DO were also assigned to the DO+ group (n = 11), and those with negative DO were assigned to the DO- group (n = 21). Finally, patients whose position on the Schäfer nomogram was greater than degree V were assigned to the severe BOO group (n = 17), while those with less than degree IV were assigned to the mild BOO group (n = 15). The expression level of β3-AR mRNA was similar in both bladder capacity groups and both DO groups. However, the expression level in the severe BOO group was significantly less than in the mild BOO group (P = 0.043). The expression of bladder mucosal β3-AR mRNA was significantly decreased in patients with severe BOO, suggesting that β3-ARs might be affected by the degree of BOO.