β2-AR Protein Research Articles

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.

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.

Computational Study on the Different Ligands Induced Conformation Change of β2 Adrenergic Receptor-Gs Protein Complex

β2 adrenergic receptor (β2AR) regulated many key physiological processes by activation of a heterotrimeric GTP binding protein (Gs protein). This process could be modulated by different types of ligands. But the details about this modulation process were still not depicted. Here, we performed molecular dynamics (MD) simulations on the structures of β2AR-Gs protein in complex with different types of ligands. The simulation results demonstrated that the agonist BI-167107 could form hydrogen bonds with Ser2035.42, Ser2075.46 and Asn2936.55 more than the inverse agonist ICI 118,551. The different binding modes of ligands further affected the conformation of β2AR. The energy landscape profiled the energy contour map of the stable and dissociated conformation of Gαs and Gβγ when different types of ligands bound to β2AR. It also showed the minimum energy pathway about the conformational change of Gαs and Gβγ along the reaction coordinates. By using interactive essential dynamics analysis, we found that Gαs and Gβγ domain of Gs protein had the tendency to separate when the inverse agonist ICI 118,551 bound to β2AR. The α5-helix had a relatively quick movement with respect to transmembrane segments of β2AR when the inverse agonist ICI 118,551 bound to β2AR. Besides, the analysis of the centroid distance of Gαs and Gβγ showed that the Gαs was separated from Gβγ during the MD simulations. Our results not only could provide details about the different types of ligands that induced conformational change of β2AR and Gs protein, but also supplied more information for different efficacies of drug design of β2AR.

β2-Adrenoceptor transfection enhances contractile reserve of isolated rat ventricular myocytes exposed to chronic isoprenaline stimulation by improving β1-adrenoceptor responsiveness

Context: Heart failure (HF) is a progressive deterioration in heart function associated with overactivity of the sympathetic nervous system. Elevated sympathetic nervous system activity down regulates the β-adrenergic signal system, suppressing β-adrenoceptors (β-ARs)-mediated contractile support in the failing heart.Objective: We investigated the effects of β2-AR gene transfer on shortening amplitude of isolated ventricular myocytes under chronic exposure to isoprenaline (ISO), and further determine the contributions of β1-AR and β2-AR to the contraction.Materials and methods: Cardiomyocytes were isolated from adult rat hearts and then transfected with β2-AR gene using an adenovirus vector. Four hours after the infection, cardiomyocytes were treated with ISO for another 24 hours to imitate high levels of circulating catecholamines in HF. Western blotting was performed to measure myocardial protein expression of β2-AR. Video-based edge-detection system was used to evaluate basal and ISO-stimulated shortening amplitudes of cardiomyocytes.Results: β2-AR gene transfer increased β2-AR protein content. Chronic ISO stimulation produced a negative inotropic response, whereas acute ISO stimulation showed a positive inotropic response. β2-AR gene transfer had no significant effects on shortening amplitude of cardiomyocytes under normal conditions, but enhanced the blunted contraction of cardiomyocytes under pathological conditions induced by chronic ISO stimulation, and the effect was inhibited by β1-AR antagonist, CGP 20712A, instead of β2-AR antagonist, ICI 118,551.Discussion and conclusions: We conclude that β2-AR gene transfer in isolated ventricular myocytes under chronic ISO stimulation improves cellular contraction, and the beneficial effects might be mediated by improving β1-adrenoceptor responsiveness.

Casted-immobilization downregulates glucocorticoid receptor expression in rat slow-twitch soleus muscle

AimsGlucocorticoids bind to the glucocorticoid receptor (GR) and increase catabolism of muscle proteins via the ubiquitin-proteasome pathway. Activation of β2-adrenergic receptor (β2-AR) in skeletal muscle has been shown to induce muscle hypertrophy by promoting muscle protein synthesis and/or attenuating protein degradation. The aim of this study was to investigate the correlation between disuse-induced muscle atrophy, and expression of GR and β2-AR. MethodsRats were subjected to casted-immobilization (knee and foot arthrodesis), a model for muscle disuse, for 10days. Fast-twitch (extensor digitorum longus: EDL) and slow-twitch (soleus: SOL) muscles were isolated and subsequently used for analysis. The expression of GR and β2-AR was analyzed by real-time RT-PCR and western blotting. In addition, we analyzed plasma catecholamine and corticosterone concentrations by ELISA. Key findingsCasted-immobilization-induced muscle atrophy was much greater in the SOL muscle than in the EDL muscle. Casted-immobilization decreased the expression of GR mRNA and protein in the SOL muscle but not in the EDL muscle. Although the expression of β2-AR protein in the cytosol and membrane-rich fractions was not changed by casted-immobilization in either muscle, casted-immobilization decreased the expression of β2-AR mRNA in the SOL muscle. Plasma catecholamine and corticosterone concentrations, however, were largely unaffected by casted-immobilization during the experimental period. SignificanceThis study provides evidence that casted-immobilization-induced muscle disuse downregulates GR expression in slow-twitch muscle. These results suggest that muscle disuse suppresses glucocorticoid signals, such as muscle protein breakdown and transcription of the β2-AR gene, via downregulation of GR expression in slow-twitch muscle.

Norepinephrine Stimulates Pancreatic Cancer Cell Proliferation, Migration and Invasion Via ß-Adrenergic Receptor-Dependent Activation of P38/MAPK Pathway

There is growing evidence that chronic stress is associated with cancer pathogenesis and progression, but the mechanisms involved in this association are poorly understood. This study aims to examine the effect of stress hormone norepinephrine (NE) on proliferation, migration and invasion of human pancreatic cancer cells and the molecular pathway involved. PANC-1 cells were examined for the expression of β-Adrenergic receptor (β-AR) subtypes, β1-AR, β2-AR by RT-PCR and western blotting, respectively. The PANC-1 cells proliferation, cell cycle distribution, cell migration and invasion were assessed by CCK-8, flow cytometry, scratch wound healing and transwell Matrigel, respectively. PANC- 1 cells expressed both β1-AR and β2-AR protein. Exposure of PANC-1 cells to increasing concentrations of NE stimulated cell proliferation in a dose-dependent fashion. In addition, NE increased S-phase population and decreased G1 and G2-phase population of PANC- 1 cells. Furthermore, NE significantly enhanced migration and invasion of PANC-1 cells. More strikingly, we observed that NE elevated P38/MAPK phosphorylation (Phos-p38) level. These stimulatory effects were completely abolished by β-AR antagonist propranolol (PRO) or P38/MAPK inhibitor SB203580. NE could stimulate pancreatic cancer cell proliferation, migration and invasion through a β-AR-dependent activation of P38/MAPK pathway involved.

Amyloid β Peptide-(1–42) Induces Internalization and Degradation of β2 Adrenergic Receptors in Prefrontal Cortical Neurons

Emerging evidence indicates that amyloid β peptide (Aβ) initially induces subtle alterations in synaptic function in Alzheimer disease. We have recently shown that Aβ binds to β(2) adrenergic receptor (β(2)AR) and activates protein kinase A (PKA) signaling for glutamatergic regulation of synaptic activities. Here we show that in the cerebrums of mice expressing human familial mutant presenilin 1 and amyloid precursor protein genes, the levels of β(2)AR are drastically reduced. Moreover, Aβ induces internalization of transfected human β(2)AR in fibroblasts and endogenous β(2)AR in primary prefrontal cortical neurons. In fibroblasts, Aβ treatment also induces transportation of β(2)AR into lysosome, and prolonged Aβ treatment causes β(2)AR degradation. The Aβ-induced β(2)AR internalization requires the N terminus of the receptor containing the peptide binding sites and phosphorylation of β(2)AR by G protein-coupled receptor kinase, not by PKA. However, the G protein-coupled receptor kinase phosphorylation of β(2)AR and the receptor internalization are much slower than that induced by βAR agonist isoproterenol. The Aβ-induced β(2)AR internalization is also dependent on adaptor protein arrestin 3 and GTPase dynamin, but not arrestin 2. Functionally, pretreatment of primary prefrontal cortical neurons with Aβ induces desensitization of β(2)AR, which leads to attenuated response to subsequent stimulation with isoproterenol, including decreased cAMP levels, PKA activities, PKA phosphorylation of serine 845 on α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor subunit 1 (GluR1), and AMPA receptor-mediated miniature excitatory postsynaptic currents. This study indicates that Aβ induces β(2)AR internalization and degradation leading to impairment of adrenergic and glutamatergic activities.

β2-Adrenergic Receptor Signaling in Osteoblasts Contributes to the Catabolic Effect of Glucocorticoids on Bone

The sympathetic nervous system is a physiological regulator of bone homeostasis. Autonomic nerves are indeed present in bone, bone cells express the β2-adrenergic receptors (β2AR), and pharmacological or genetic disruption of sympathetic outflow to bone induces bone gain in rodents. These recent findings implied that conditions that affect β2AR signaling in osteoblasts and/or sympathetic drive to bone may contribute to bone diseases. In this study, we show that dexamethasone stimulates the expression of the β2AR in differentiated primary calvarial osteoblasts, as measured by an increase in Adrβ2 mRNA and β2AR protein level after short-term dexamethasone treatment. Isoproterenol-induced cAMP accumulation and the expression of the β2AR target gene Rankl were also significantly increased after dexamethasone pretreatment, indicating that dexamethasone promotes the responsiveness of differentiated osteoblasts to adrenergic stimulation. These in vitro results led to the hypothesis that glucocorticoid-induced bone loss, provoked by increased endogenous or high-dose exogenous glucocorticoids given for the treatment of inflammatory diseases, might, at least in part, be mediated by increased sensitivity of bone-forming cells to the tonic inhibitory effect of sympathetic nerves on bone formation or their stimulatory effect on bone resorption. Supporting this hypothesis, both pharmacological and genetic β2AR blockade in mice significantly reduced the bone catabolic effect of high-dose prednisolone in vivo. This study emphasizes the importance of sympathetic nerves in the regulation of bone homeostasis and indicates that this neuroskeletal signaling axis can be modulated by hormones or drugs and contribute to enhance pathological bone loss.

Protective Effects of Glycyrrhizin against .BETA.2-Adrenergic Receptor Agonist-Induced Receptor Internalization and Cell Apoptosis

It has been reported that treatment with β₂ adrenergic receptor (β₂AR) agonist bronchodilators may result in airway β₂ARs internalization and cardiac muscle cells apoptosis. This could lead to the loss of pharmacological effect of β₂AR agonists and increase adverse cardiovascular events in asthma patients receiving β₂AR agonist therapy. Glycyrrhizin, the major bioactive component of licorice root extract, has been reported to exhibit protective effect on respiratory system. Here, we investigate the effects of glycyrrhizin against β₂AR agonist salbutamol-induced receptor internalization and cell apoptosis. In our study, the live cell confocal imaging and fixed-cell enzyme-linked immunosorbent assay (ELISA) assay revealed that glycyrrhizin significantly inhibited salbutamol-induced surface β₂AR internalization. The underlying mechanisms were then identified to be that glycyrrhizin could reduce the association of β₂ARs with β-arrestins and clathrin heavy chain as well as the level of G protein-coupled receptor kinase (GRK) mediated phosphorylation of β₂ARs. The inhibition of receptor internalization by glycyrrhizin further lead to stabilization of the β₂AR mRNA and protein expression, thus amplified the transmembrane signaling via the β₂ARs. We also proved that glycyrrhizin could profoundly attenuate salbutamol-induced early cellular apoptosis by regulating the expressions of B-cell lymphoma 2 (Bcl-2) family genes. Taken together, our results suggest that glycyrrhizin exhibits protective effects against β₂AR agonist-induced receptor internalization and cell apoptosis. These findings might have practical implications for future strategies of combined application of glycyrrhizin with β₂AR receptor agonists to improve the efficacy of bronchodilators in patients with asthma and chronic obstructive pulmonary disease (COPD).

Synthesized glucocorticoid, dexamethasone regulates the expressions of .BETA.2-adrenoceptor and glucocorticoid receptor mRNAs but not proteins in slow-twitch soleus muscle of rats

Effects of synthesized glucocorticoid, dexamethasone (DEX, dose = 1.0 mg/kg body weight/day for 10 days) on the expressions of β₂-adrenoceptor (AR) and glucocorticoid receptor (GR) were studied in fast-twitch (extensor digitorum longus (EDL)) and slow-twitch fiber-rich (soleus(SOL)) muscles of rats. DEX decreased the expression of β₂-AR mRNA in SOL muscle without changing that in EDL muscle. The expression of β₂-AR protein in EDL and SOL muscles was not affected by DEX. DEX-induced decreased action of the expression of GR mRNA was much greater in SOL muscle than in EDL muscle. However, there were no differences in the expression of GR protein in EDL and SOL muscles. DEX also decreased mRNA expression of cAMP response element binding protein (CREB, transcription factor of β₂-AR mRNA) in SOL muscle, whereas increased that in EDL muscle. Further, DEX tended to increase mRNA expressions of post-transcription factors of β₂-AR mRNA in EDL muscle without changing those in SOL muscle. These results demonstrated that the expressions of β₂-AR and GR are regulated at mRNA levels but not protein levels by DEX. Further, these results also suggest that DEX-induced decrease in the expression of β₂-AR mRNA in slow-twitch fiber-rich SOL muscle is associated with the transcriptional regulations.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

BackgroundThe β2-adrenergic receptor (β2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.Methodology/Principal FindingsWe constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5′UTR, coding, and 3′UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that β2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. “Whole-gene” transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface β2AR protein expression compared to the others. Agonist-promoted downregulation of β2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Conclusions/SignificanceThus for obstructive lung disease, the initial bronchodilator response from intermittent administration of β-agonist may be influenced by certain β2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where β2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies.

Folic Acid Supplementation Modifies β-Adrenoceptor–MediatedIn VitroLipolysis of Obese/Diabetic (+db/+db) Mice

The effects of folic acid (5.7 and 71 microg/kg, 4 weeks) consumption on the beta-adrenoceptors (beta-ARs)-elicited lipolysis in vitro of the abdominal adipocytes of lean/control (+m/+db) and obese/diabetic (+db/+db) mice (female) were investigated. beta-AR agonists (salbutamol, a beta(2)-AR agonist; BRL 37344 and CGP 12177, beta(3)-AR agonists; adrenaline, a beta-AR agonist)-mediated lipolysis, beta(2)-, and beta(3)-ARs protein expression of the adipose tissues after folic acid consumption were evaluated. Our results demonstrate that a smaller magnitude of the basal (spontaneous) and the beta-AR agonists-triggered lipolysis was observed in +db/+db mice, and folic acid supplementation (71 microg/kg) resulted in an improvement of both the baseline and the beta-ARs-mediated lipolysis. In controls, a lower beta(2)-and beta(3)-ARs protein expression of the adipose tissues was detected in +db/+db mice, compared to +m/+db mice. In both strains fed with folic acid (71 microg/kg), a reduction of beta(2)-AR protein expression was observed compared to the respective controls. In +db/+db mice, folic acid (5.7 and 71 microg/kg) consumption caused a dose-dependent increase of beta(3)-AR protein expression compared to controls. We demonstrate that lipolysis elicited by beta-AR (beta(2)- and beta(3)-ARs) agonists was blunted in +db/+db mice. Folic acid consumption has significant modulatory effects on beta-ARs protein expression and lipolysis.

Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Variable-length poly-C tract polymorphisms of the β2-adrenergic receptor 3′-UTR alter expression and agonist regulation

Beta(2)-adrenergic receptors (beta(2)-AR) expressed on airway epithelial and smooth muscle cells regulate mucociliary clearance and relaxation and are the targets for beta-agonists in the treatment of obstructive lung disease. However, the clinical responses display extensive interindividual variability, which is not adequately explained by genetic variability in the 5'-flanking or coding region of the intronless beta(2)-AR gene. The nonsynonymous coding polymorphism most often associated with a bronchodilator phenotype (Arg16) is found within three haplotypes that differ by the number of Cs (11, 12, or 13) within a 3'-untranslated region (UTR) poly-C tract. To examine potential effects of this variability on receptor expression, BEAS-2B cells were transfected with constructs containing the beta(2)-AR (Arg16) coding sequence followed by its 3'-UTR with the various polymorphic poly-C tracts. beta(2)Arg16-11C had 25% lower mRNA expression and 33% lower beta(2)-AR protein expression compared with the other two haplotypes. Consistent with this lower steady-state expression, beta(2)Arg16-11C mRNA displayed more rapid and extensive degradation after actinomycin D treatment compared with beta(2)Arg16-12C and -13C. However, beta(2)Arg16-12C underwent 50% less downregulation of receptor expression during beta-agonist exposure compared with the other two haplotypes. Thus these haplotypes direct a potential low-response phenotype due to decreased steady-state receptor expression combined with wild-type agonist-promoted downregulation (beta(2)Arg16-11C) and a high-response phenotype due to increased baseline expression combined with decreased agonist-promoted downregulation (beta(2)Arg16-12C). This heterogeneity may contribute to the variability of clinical responses to beta-agonist, and genotyping to identify these 3'-UTR polymorphisms may improve predictive power within the context of beta(2)-AR haplotypes in pharmacogenetic studies.

Exercise training normalizes β-adrenoceptor expression in dogs susceptible to ventricular fibrillation

Previous studies demonstrated an enhanced beta(2)-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta(1)-AR and beta(2)-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta(2)-ARs are located in caveolae, caveolin-3 was also quantified. beta(1)-AR gene expression decreased ( approximately 5-fold), beta(2)-AR gene expression was not changed, and the ratio of beta(2)-AR to beta(1)-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta(1)-AR protein decreased ( approximately 50%) and beta(2)-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta(1)-AR gene expression to levels seen in resistant animals but did not alter beta(2)-AR protein levels in susceptible dogs. These data suggest that beta(1)-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta(1)-AR gene expression, thereby restoring a more normal beta-AR balance.

Transcriptional and posttranscriptional regulation of beta(2)-adrenergic receptor gene in rat liver during sepsis.

Changes in beta(2)-adrenergic receptor (beta(2)-AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic (9 h after CLP; early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [(3)H]dihydroalprenolol binding studies show that the density of beta(2)-AR was decreased by 12 and 35% during the early and late phases of sepsis, respectively. Western blot analyses depict that the beta(2)-AR protein level was reduced by 37 and 72% during early and late sepsis, respectively. The reverse transcription polymerase chain reaction and Southern blot analyses reveal that the steady-state level of beta(2)-AR mRNA was decreased by 37% during early phase and 77% during late phase of sepsis. Nuclear run-off assays show that the rate of transcription of beta(2)-AR mRNA was reduced by 36% during early sepsis and 64% during late sepsis. The stability assays indicate that the half-life of beta(2)-AR mRNA was shortened by 21 and 50% during the early and late phases of sepsis, respectively, indicating that the rate of degradation of beta(2)-AR mRNA was progressively enhanced during sepsis. These findings demonstrate that the beta(2)-AR gene was underexpressed in the liver during the progression of sepsis, and, furthermore, the underexpression of the beta(2)-AR gene was the result of a reduction in the rate of transcription coupled with an enhancement in the rate of degradation of beta(2)-AR gene transcripts. Thus our findings that the transcriptional and posttranscriptional regulation of beta(2)-AR gene associated with decreases in beta(2)-AR number and its protein expression may provide a molecular mechanistic explanation for the development of hypoglycemia during the late stage of sepsis.