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Background: Increasing attention was focused on the β2-adrenergic receptor gene (ADRB2), whose genetic variability has been implicated as a risk factor for asthma-related phenotypes. However, only a few studies reported the associations by utilizing haplotypic approaches. We therefore examined the relationship of childhood wheezing illness with polymorphisms at codons 16 and 27, and evaluated the influence of polymorphisms individually and in combination as haplotypes. Methods: We conducted a genetic case–control study comprising 215 wheezing children and 183 nonwheezing controls, all of whom were selected from 2524 fourth- to ninth-grade schoolchildren in southern Taiwan. Results: All participants were homozygous at the ADRB2 Thr164 locus. After controlling for possible confounders, ADRB2 Glu27 allele was significantly associated with wheezing illness in all genetic models, but the risks on Arg16Gly genotypes were inconclusive. Estimated frequencies for the three main hyplotypes were Arg16/Gln27 57.2%, Gly16/Gln27 35.3%, Gly16/Glu27 7.4% in wheezing children, and Arg16/Gln27 56.3%, Gly16/Gln27 32.2%, Gly16/Glu27 10.4% in controls. The protective effect of Gly16/Glu27 haplotype remained relative to all other ADRB2 haplotypes [adjusted relative risk (aRR) = 0.58; 95% confidence interval (CI) 0.35–0.97]. As compared with children without Gly16/Glu27 haplotype, those with Gly16/Glu27 haplotype had a significantly lower risk for wheezing illness (aRR = 0.56; 95% CI 0.33–0.99). The copy numbers of Gly16/Glu27 haplotype also showed a clear dose-response relationship on the decreased risks. No significant association was found with the prevalence of wheezing illness for other haplotypes. Conclusion: We concluded that ADRB2 Glu27 allele and Gly16/Glu27 haplotype were significantly protective factors for wheezing illness in Taiwanese schoolchildren. [ABSTRACT FROM AUTHOR]

7TM receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM receptors. Such 15 TM concatameric homo- and heterodimers of the β2-adrenergic and the NK1 receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two receptors. It is concluded that covalently joined 7TM receptor dimers with surprisingly normal receptor properties can be constructed with use of an artificial transmembrane connector, which perhaps can be used to fuse other membrane proteins. [ABSTRACT FROM AUTHOR]

Abstract: High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor–ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for β2-adrenergic receptor–ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human β2-adrenergic receptors overexpressed in human embryonic kidney HEK293i cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC50 values for β2-adrenergic antagonist propranolol were 60 and 37nM, the Z′ values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands. [Copyright &y& Elsevier]

Adrenergic activation of electrogenic K+ secretion in isolated mucosa from guinea pig distal colon was desensitized by peptide-YY (PYY). Addition of PYY or neuropeptide-Y (NPY) to the bathing solution of mucosae in Ussing chambers suppressed the short-circuit current (Isc) corresponding to electrogenic Cl- secretion, whether stimulated by epinephrine (epi), prostaglandin-E2 (POE2), or carbachol (CCh). Neither peptide markedly inhibited the large transient component of synergistic secretion (POE2 + CCh). Sustained Cl- secretory Isc was inhibited ∼65% by PYY or NPY, with IC50s of 4.1 ± 0.9 nM and 9.4 ± 3.8 nM, respectively. This inhibition was eliminated by BIIE0246, an antagonist of the Y2-neuropeptide receptor (Y2-NpR), but not by Yl-NpR antagonist BVD10. Adrenergic sensitivity for activation of K+ secretion in the presence of Y2-NpR blockade by BIIE0246 was (EC505) 2.9 ± 1.2 nM for epi and 13.3 ± 1.0 nM for norepinephrine, approximately fourfold greater than in the presence of PYY. Expression of mRNA for both Yl-NpR and Y2-NpR was indicated by RT-PCR of RNA from colonic mucosa, and protein expression was indicated by immunoblot. Immunoreactivity (ir) for Yl-NpR and Y2-NpR was distinct in basolateral membranes of columnar epithelial cells in the crypts of LieberkUhn as well as intercrypt surface epithe- hum. Adrenergic nerves in proximity with crypts were detected by ir for dopamine-β-hydroxylase, and a portion of these nerves also contained NPYir. BIIE0246 addition increased secretagog-activated Isc, consistent with in vitro release of either PYY or NPY. Thus PYY and NPY were able to suppress Cl- secretory capacity and desensitize the adrenergic K+ secretory response, providing a direct inhibitory counterbalance against secretory activation. [ABSTRACT FROM AUTHOR]

Abstract: G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25–50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993–6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021–3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven α-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734–40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1–12]. [Copyright &y& Elsevier]

Background: It has been speculated that chemokines and neurotransmitters might be involved in the organ-specific development of metastases because cancer metastasis is similar to the regulation of migratory activity in leukocytes. Here, we aimed to examine the expression of β2-adrenergic receptor (β2-AR) in oral squamous cell carcinoma (OSCC), and to investigate its correlation with tumor development and metastasis. Methods: Expression of β2-AR was examined in 65 cases of OSCC specimens, 10 cases of normal oral mucosa, and two cell lines using immunohistochemistry, Western blot and RT-PCR. The differences in β2-AR expression between various groups were evaluated using SPSS 13.0 Statistical Software. Cell proliferation assays were assayed by β-adrenergic receptors agonists (norepinephrine) and antagonists (propranolol). Norepinephrine-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. Results: β2-AR was highly expressed on OSCC compared to normal controls. In OSCC, positive β2-AR expression was significantly correlated with cervical lymph node metastasis ( P = 0.001), age ( P = 0.003), tumor size ( P = 0.001) and clinical stage ( P = 0.001), but not with gender. RT-PCR and Western blot also confirmed positive β2-AR expression in OSCC and TCa8113 cell line, and negative β2-AR expression in normal oral mucosa and ACC cell line. β-adrenoreceptor agonist (norepinephrine) was a potent mitogen for TCa8113 and ACC cell lines, and completely inhibited by the selective antagonist of β-adrenergic receptors (propranolol). Norepinephrine induced migratory activity of OSCC cells in a dose-dependent manner. Conclusion:  Increased expression of β2-AR may play an important role in the formation and metastasis of OSCC. [ABSTRACT FROM AUTHOR]

Abstract: Stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) triggers myeloid differentiation factor 88 (MyD88)-dependent early-phase NF-κB activation and Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF)-dependent late-phase NF-κB activation. In a previous study, we have shown that β2-adrenergic receptor (β2AR) functions as a negative regulator of NF-κB activation through β-arrestin 2 in the macrophage cell line RAW264 and that down-regulation of β2AR expression in response to LPS is essential for NF-κB activation and expression of its target gene, inducible nitric oxide synthase (NOS II). Here, we demonstrate that β2AR plays an important role in TRIF-dependent late-phase NF-κB activation. LPS-stimulated down-regulation was induced in MyD88-knockdown cells, but not in TRIF-knockdown cells, suggesting that β2AR expression was down-regulated by the TRIF-dependent pathway. On the other hand, depletion of β2AR or β-arrestin 2 expression by siRNA decreased cytoplasmic IκBα and abrogated late-phase IκBα degradation and NF-κB activation in response to LPS. Inducible nitric oxide synthase (NOS II) expression was increased continuously during 24h of LPS stimulation in control cells, but decreased in β2AR or β-arrestin 2-knockdown cells after 6h of LPS stimulation. These findings suggest that β2AR functions not only as a negative regulator of NF-κB activation, but also as a stabilizing factor of the NF-κB/IκBα complex through cytoplasmic β-arrestin 2, and that TRIF-dependent down-regulation of β2AR expression increases the level of cytoplasmic NF-κB/IκBα complex free from β-arrestin 2, leading to continuous late-phase NF-κB activation. [Copyright &y& Elsevier]

Abstract: Sustained complex cross-talk between the immune system and the nervous system plays a vital role in retaining homeostasis in a healthy individual. One of the central regulatory mechanisms involved is the existence and functions of β2-adrenergic receptors (β2AR) on T lymphocytes. This article reviews research progress made recently, including the expression of adrenergic receptors on T lymphocytes, the structure and intracellular pathways of β2AR, the activation of β2AR by either endogenous or exogenous agonists, and the effect of β2AR stimulation on T cells which alters T cell proliferation, differentiation, cytokine production and T-helper-mediated antibody production. Furthermore, we discuss the roles of β2AR played in the pathogenesis and treatment of autoimmune diseases. [Copyright &y& Elsevier]

Abstract: A-kinase Anchoring Proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAP5 and AKAP12 are multivalent (with respect to protein kinases and phosphatases) and display the ability to associate with the prototypic member of G protein-coupled receptors, the β2-adrenergic receptor. We probed the relative abundance, subcellular distribution and localization of AKAP5 and AKAP12 in human embryonic kidney HEK293 and epidermoid carcinoma A431 cells. HEK293 cells are relatively rich in AKAP5 (found mostly in association with the cell membrane); whereas A431 cells are rich in AKAP12 (found distributed both in the cytoplasm and in association with the cell membrane). In biochemical analysis of subcellular fractions and in whole-cell imaging, the membrane localization of AKAP5 was decreased in response to treating cells with the beta-adrenergic agonist isoproterenol, whereas membrane association of AKAP12 was increased initially in response to agonist treatment. These data demonstrate quantitatively a clearly different pattern of AKAP-receptor association for AKAP5 versus AKAP12. AKAP5 remains associated with its G-protein-coupled receptor, at the cell membrane, docked with the receptor during agonist-induced internalization and later receptor recycling after agonist wash-out. AKAP12-receptor docking, in contrast, is dynamic, driven by agonist stimulation (accounting for movement of AKAP12 from the cytoplasm to the cell membrane). AKAP12 then is internalized with the β2-adrenergic receptor, but segregates away from the G-protein-coupled receptor upon recycling of the internalized receptor to the cell membrane. Thus these homologous, AKAPs that dock G-protein-coupled receptors have markedly different patterns of trafficking, docking, and re-distribution. [Copyright &y& Elsevier]

Abstract: Signal transduction via the Frizzled family of seven-transmembrane receptors controls important developmental processes. Aberrant signaling caused by altered Frizzled receptor activity or by mutations in downstream signaling components has been implicated in several adult pathologies. A diverse array of intracellular signaling pathways has been suggested to transduce the signals exerted in cells when secreted ligands of the Wnt family bind to Frizzled receptors. Studies with a chimeric receptor composed of Frizzled-2 and the β2-adrenergic receptor have suggested that the binding of Wnt-5a to Frizzled-2 results in the activation of G proteins of the Gαi family, the mobilization of calcium from intracellular stores and the induction of gene transcription through nuclear factor of activated T cells. In this report, we demonstrate by using β-lactamase reporter gene technology that full-length, wild-type human Frizzled-2 does not couple to calcium-mediated signaling in HEK293 cells following stimulation with purified recombinant mouse Wnt-5a. In contrast, when stimulated with recombinant mouse Wnt-3a, Frizzled-2 activates the canonical Wnt/Frizzled signaling pathway, involving the transcriptional modulator β-catenin. Our report underlines the importance of using cell lines stably overexpressing wild-type Frizzled receptors and the use of purified ligands when studying receptor pharmacology. This approach has allowed us to measure the half-maximal concentration for activation of human Frizzled-2 (1.5±0.4 nM; avg.±SD) and human Frizzled-1 (1.3±0.5 nM) following stimulation by Wnt-3a. Our results suggest that there is receptor redundancy with regard to Wnt-3a reception. In addition, we introduce β-lactamase reporter gene technology as an alternative to luciferase-based reporters to measure Frizzled receptor modulation for the discovery of Frizzled receptor-interacting drugs. [Copyright &y& Elsevier]

Adenosine receptors (ARs) are members of the superfamily of G protein-coupled receptors. The homology models of adenosine A1 and A2A receptors were constructed. The high-resolution X-ray structure of bovine rhodopsin and crystal structure of β2-adrenergic receptor were used as templates. The binding sites of the A1 and A2A ARs were constructed by using data obtained from mutagenesis experiments as well as docking simulations of the respective AR antagonsists DPCPX and XAC. To compare rhodopsin- and β2-adrenergic-based models, the binding mode of A1 (KW-3902, LUF-5437) and A2A (KW-6002, ZM-241385) ARs antagonists were also examined. The differences in the binding ability of both models were noted during the study. The β2-adrenergic-based A2A AR model was much more capable to stabilize the ligand in the binding site cavity than the corresponding rhodopsin-based A2A AR model, however, such differences were not so clear in case of A1 AR models. It was suggested that for the A1 AR it is possible to use the crystal structure of rhodopsin as a template as well as β2-adrenergic receptor, but for A2A AR, with the now available β2-adrenergic receptor X-ray structure, docking studies should be avoided on the rhodopsin-based model. However, taking into account that the β2AR shares about 31% of the residues with the AR in comparison to 21% in case of bRho, we suggest using β2-adrenergic-based models for the A1 and A2A ARs for further in silico ligand screening also because of their generally better ability to stabilize ligands inside the binding pocket. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009 [ABSTRACT FROM AUTHOR]

For the β2-adrenergic receptor (β2AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of β2AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, β2AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which β2ARs recycled to the cell surface. In LB-treated cells, nonrecycled β2ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of β2ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on β2AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of β2ARs, a process that likely is dependent on myosin Vb. [ABSTRACT FROM AUTHOR]

Abstract: The recently published X-ray structures of the β2-adrenergic receptor are the first examples of ligand-mediated GPCR crystal structures. We have previously performed computational studies that examine the potential viability of these structures for use in drug design, exploiting known ligand activities. Our previous study and a newly reported β2/Timolol X-ray complex provide validation of the computational approaches. In the present work, we use the X-ray structures to extract, via in silico high-throughput docking, compounds from proprietary and commercial databases and demonstrate the successful identification of active compounds by radioligand binding. [Copyright &y& Elsevier]

Abstract: Endurance exercise induces mitochondrial biogenesis in skeletal muscle. It has been shown that lipin-1 acts as a transcriptional coactivator in liver, and stimulates gene expression of mitochondrial enzymes. We hypothesized that lipin-1 might be involved in exercise-induced mitochondrial biogenesis in skeletal muscle. The present investigation first demonstrated that lipin-1 mRNA in rat triceps muscle was increased by approximately 2-fold after an acute bout of endurance swimming exercise. Second, ectopic expression of lipin-1 in L6 myotube increased carnitine palmitoyltransferase-1 and δ-aminolevulinate synthase gene expression. Finally, lipin-1 mRNA expression in rat triceps muscle was significantly elevated at 6h after subcutaneous injections of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or clenbuterol, which are 5′-AMP-activated protein kinase (AMPK) and β2-adrenergic receptor (β2-AR) activators, respectively. These results may suggest that enhanced expression of lipin-1 is involved in exercise-induced mitochondrial enzyme adaptations, possibly through AMPK- and β2-AR-related mechanisms. [Copyright &y& Elsevier]

Genetic variants in the β2-adrenergic receptor ( ADRB2) coding block have been associated with different parameters of asthma severity, but there is no consensus on which variants are most important. Our objective was to determine whether the genetic variants in the 5′- or 3′-flanking regions of ADRB2 impact the response to therapy. DNA was obtained initially from 72 adults hospitalized for an asthma exacerbation. We sequenced a 5,000 bp region of the ADRB2 gene that spanned the flanking regions and identified 31 single nucleotide polymorphisms (SNPs). Nonresponders to asthma therapy were defined as patients whose forced expiratory volume in 1 second (FEV1) worsened by >10% at 24 hours after admission. We then evaluated the relationship between the 19 common SNPs and response to asthma-specific therapy during acute disease exacerbations. Our results showed a significant association between nonresponders and a haplotype of five promoter SNPs in a nearly complete linkage disequilibrium. An analysis of the promoter and coding block polymorphisms in an extended cohort of 99 patients confirmed that promoter haplotype was the genetic component most strongly associated with asthmatic nonresponders, which was statistically significant among whites ( p < 0.05). An identification of this promoter haplotype may provide an alternate explanation for the variation in the asthma responses observed with ADRB2 coding block polymorphisms. [ABSTRACT FROM AUTHOR]

Abstract: The effects of 3-week exercise training on the functions of peritoneal macrophages from BALB/c mice were investigated. Lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and proinflammatory cytokine production in macrophages from trained mice was markedly higher than those from control mice. Meanwhile, exercise training decreased the steady state level of β2-adrenergic receptor (β2AR) mRNA in macrophages. Overexpression of β2AR in the macrophage cell line RAW264 by transfecting with β2AR cDNA suppressed NO synthase (NOS) II expression but dose not influenced proinflammatory cytokine expression. When expression of transfected β2AR in RAWar cells was downregulated by a tetracycline repressor-regulated mammalian expression system, NOS II mRNA expression was significantly increased; this suggested that the changes in the β2AR expression level in macrophages associated with exercise training play a role in the regulation of NO production following LPS stimulation. These findings indicate that exercise training improves macrophage innate immune function in a β2AR-dependent and -independent manner. [Copyright &y& Elsevier]

Toll-like receptors (TLRs) play an important role in innate immunity while, β2-adrenergic receptors (β2AR) provide the key linkages for the sympathetic nervous system to regulate the immune system. However, their role in macrophages remains uncertain. Here, we demonstrate the cross-talk between β2AR and TLR signalling pathways. Expression of β2AR was down-regulated by TLR4 ligand lipopolysaccharide (LPS) stimulation. To investigate the physiological consequence of this down-regulation RAW264 cells, a macrophage cell line, were transfected with a β2AR expression vector (RAWar). Both LPS-stimulated inducible nitric oxide synthase (NOS II) expression and NO production were markedly suppressed in the RAWar cells. The activation of nuclear factor-κB (NF-κB) and degradation of the inhibitor of NF-κB (IκBα) in response to LPS were markedly decreased in these cells. The level of β-arrestin 2, which regulates β2AR signalling, was also reduced in RAW264 cells after stimulation with LPS, but not in RAWar cells. Overexpression of β-arrestin 2 (RAWarr2) also inhibited NO production and NOS II expression. Furthermore, we demonstrated that β-arrestin 2 interacted with cytosolic IκBα and that the level of IκBα coimmunoprecipitated by anti-β-arrestin 2 antibodies was decreased in the RAW264 cells but not in RAWar or RAWarr2 cells. These findings suggest that LPS-stimulated signals suppress β2AR expression, leading to down-regulation of β-arrestin 2 expression, which stabilizes cytosolic IκBα and inhibits the NF-κB activation essential for NOS II expression, probably to ensure rapid and sufficient production of NO in response to microbial attack. [ABSTRACT FROM AUTHOR]

Aim: To observe the effect of β2-adrenergic agonist clenbuterol on ischemia/reperfusion (I/R) injury in isolated rat hearts and hydrogen peroxide (H2O2)-induced cardiomyocyte apoptosis. Methods: Isolated rat hearts were subjected to 30 min global ischemia and 60 min reperfusion on a Langendorff apparatus. Cardiac function was evaluated by heart rate, left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure, maximal rise rate of left ventricular pressure (+d p/d tmax), and the coronary effluent (CF). Lactate dehydrogenase (LDH) in the coronary effluent, malondialdehyde (MDA), superoxide dismutase (SOD), and Ca2+-ATPase activity in the cardiac tissue were measured using commercial kits. The apoptotic cardiomyocyte was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. Bax/Bcl-2 mRNA levels and the expression of caspase-3 were detected by RT-PCR and immunoblotting, respectively. Cultured newborn rat cardiomyocytes were pre-incubated with clenbuterol, and oxidative stress injury was induced by H2O2. Cell viability and cardiomyocyte apoptosis were evaluated by flow cytometry (FCM). Results: In the isolated rat hearts after I/R injury, clenbuterol significantly improved diastolic function (LVEDP and CF) and Ca2+-ATPase activity. Treatment with clenbuterol increased SOD activity and decreased the MDA level and LDH release compared with the I/R group ( P<0.05). Moreover, clenbuterol decreased apoptosis, which was associated with a reduction in TUNEL-positive cells, Bax/Bcl-2 mRNA, and caspase-3 expression. In H2O2-induced cardiomyocyte injury, clenbuterol increased cell viability and attenuated cardiomyocyte apoptosis. Pre-treatment with ICI118551 (selective β2-adrenergic antagonist) decreased these effects compared with the clenbuterol-treated group ( P<0.05). Conclusion: Clenbuterol ameliorated ventricular diastolic function by enhaning Ca2+-ATPase activity and reduced oxidative stress and cardiac myocyte apoptosis in an experimental rat model of myocardium I/R. It decreased cardiomyocyte apoptosis induced by H2O2 in vitro. It plays a key role in the cardiac protection against myocardium I/R injury. [ABSTRACT FROM AUTHOR]

Abstract: Increasing evidence indicates that the β2-adrenergic receptor (β2-AR) may play an important role in Alzheimer''s disease (AD). We investigated the effect of two polymorphisms in the β2-AR gene: Gly16Arg and Gln27Glu for the risk of sporadic Late Onset Alzheimer''s Disease (LOAD) in 109 patients and 109 healthy controls matched for sex and age in a Han Chinese population. Results revealed that both the 16Gly allele and the 27Glu allele of the β2-AR gene were associated with an increased risk of LOAD (P =0.009, OR=1.652 and P =0.002, OR=2.846, respectively), and they also showed a highly significant interaction with the Apolipoprotein E gene (APOE) ε4 allele (OR=4.200 and 9.441, respectively). Examination of the haplotypes identified the Gly16Glu27 haplotype to increase the risk of LOAD (P =0.004). Our results suggest that variations in the β2-AR gene play an important role in the pathogenesis of sporadic LOAD, and interact with the ε4 allele to markedly increase the LOAD risk. [Copyright &y& Elsevier]

The article reports on the results of a study of beta2-adrenergic receptor genetic (ADRB2) polymorphisms and short-term bronchodilator responses in patients with chronic obstructive pulmonary disease (COPD). A description of study methods is presented. The study concluded that the genetic effects of ADRB2 gene polymorphisms may explain some of the variability in response to therapeutic doses of a short-acting beta2-agonists in patients with COPD.

Abstract: We have previously shown that the ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein, is important for the internalization of several G protein-coupled receptors. Here, we propose to elucidate the molecular steps controlled by ARF6 in the endocytic process of the angiotensin II type 1 receptor (ATR), a model receptor being internalized via the clathrin-coated vesicle pathway. In HEK 293 cells, angiotensin II stimulation leads to the formation of a complex including ARF6, the β-subunit of AP-2 and the heavy chain of clathrin. In vitro experiments indicate that the interactions between ARF6 and the β-subunit of AP-2 as well as with the heavy chain of clathrin are direct, and dependent upon the nature of the nucleotide bound to ARF6. β2-adaptin binds to ARF6-GDP while clathrin preferentially interacts with ARF6 when loaded with GTP. These interactions have an important physiological consequence. Indeed, depletion of ARF6 prevents the agonist-dependent recruitment of β2-adaptin and clathrin to the activated ATR. Interestingly, in these cells, the plasma membrane redistribution of either β2-adaptin-GFP or βarrestin 2-GFP, following Ang II stimulation, is altered. Both proteins are defective in clustering into large punctated structure at the plasma membrane compared to control conditions. Taken together, these results suggest that the cycling of ARF6 between its GDP-and GTP-bound states coordinates the recruitment of AP-2 and clathrin to activated receptors during the endocytic process. [Copyright &y& Elsevier]

Rising asthma morbidity rates and often insufficient control of the disease achievable by means of currently available methods make it necessary to find new, safe and efficient therapeutic options. Long-acting beta-agonists have well-documented effect on the bronchoconstriction component of the asthma, but they lack the anti-inflammatory effect. Thus, they should not be used as the first line therapy in asthma treatment. The available data suggest that in patients whose asthma is not sufficiently controlled by means of anti-inflammatory drugs alone, the addition of LABA may bring good results. At the same time it has been stressed that the antiasthmatic therapy should be individualized and take into consideration all coexisting factors and individual patients' response to the therapy. The aim of this review article is presentation of current results of the research on the mechanism of action of long-acting beta-agonists and the objective evaluation of their safety and efficacy. [ABSTRACT FROM AUTHOR]

Abstract: Generation of reactive oxygen species (ROS) can occur upon agonist stimulation of surface receptors to modulate downstream signaling processes. Here, we show that activation of the β2 adrenergic receptor (β2AR) by stimulation with the agonist isoproterenol leads to generation of ROS that is required for β2AR signal transduction. Specifically, we show that inhibition of NADPH oxidase with diphenyliodonium chloride, inhibition of the small GTPase Rac1 with NSC23766, and inhibition of formed ROS with the antioxidant N-acetyl-l-cysteine decreases β2AR-mediated cAMP formation, protein kinase A activation, and receptor phosphorylation and internalization, but does not impact ligand binding. The results also show that inhibition of ROS attenuates active β2AR-mediated binding of GTP to α subunits of heterotrimeric G proteins. Based on these results, we propose that agonist-dependent ROS formation is needed for β2AR signal transduction, perhaps through stabilization of active receptor conformers by redox-mediated modification of receptor and/or Gα proteins cysteine residues. [Copyright &y& Elsevier]

Cystic fibrosis (CF), an autosomal recessive disease affecting the lung, pancreas, gut, liver, and reproductive tract, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cyclic adenosine 3′, 5′ monophosphate-regulated chloride channel. The variability of disease progression among patients with CF suggests effects of genetic modifiers of disease. Beta-2 adrenergic receptors (β2AR), which are abundant in airway epithelial cells, accelerate the formation of cyclic adenosine 3′, 5′ monophosphate, which can modulate CFTR activity and affect smooth muscle contractility. We tested the hypothesis that genetic variants of the β2AR gene, which have been shown to influence receptor desensitization, are more frequent in patients than in controls.We genotyped 130 adult CF patients and 1 : 1 age-matched, sex-matched, and ethnicity-matched normal volunteers for Gly16Arg and Gln27Glu β2AR.We found that CF patients were more likely than controls to be Gly16 homozygotes (48 and 32%, respectively) (P<0.01) and Glu27 homozygotes (29 and 10%, respectively) (P<0.01).Our results, showing a higher frequency of Gly16 and Glu27 β2AR alleles in adult CF patients than in the control population, contrast with data from children with CF, who are reported to have lower frequency of Gly16 and similar frequency of G1u27, and with data from young adults with CF, who showed no differences in frequencies of β2AR variants. The Gly16Glu27 variant of β2AR may have properties that lead to enhanced β2AR function, resulting in the upregulation of CFTR activity and the improvement of CF disease. [ABSTRACT FROM AUTHOR]

Background: β2-adrenergic receptors (β2-AR) mediate vasorelaxation in response to adrenergic agents. Genetic polymorphisms of β2-AR were implicated in various cardiovascular and noncardiovascular traits. Methods: We tested the role of the β2AR-16 and β2AR-27 gene variants in the susceptibility to the development of ischemic stroke in a genetically homogenous and clinically well-characterized case-control sample that included 294 cases and 286 controls from Sardinia, Italy. This population was shown to be an optimal study sample for carrying out genetic analyses. Results: Age, hypertension, dyslipidemia, and atrial fibrillation were independent risk factors for stroke in this cohort. We found that the presence of the Glu27 allelic variant was associated with a significantly increased risk of stroke when assuming a recessive mode of inheritance (odds ratio [OR], 1.68; 95% confidence interval [CI], 1.17–2.41; P = .005). The same results were obtained for the subgroup of ischemic strokes of arterial origin (n = 215): OR, 1.71; 95% CI, 1.14–2.57; P = .009. Furthermore, haplotype analysis confirmed that the presence of the Glu27 allele increased the risk of cerebrovascular accidents. Conclusions: Our data suggest that the Glu27 allelic variant of the β2-AR gene may be a determinant of ischemic stroke. [Copyright &y& Elsevier]

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the β2-adrenergic receptor (β2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single β2AR is incorporated per rHDL particle. The monomeric β2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization. [ABSTRACT FROM AUTHOR]

Abstract: In wild-type FVB mice, leukocyte recruitment to lipopolysaccharide was sexually dimorphic, with a greater number of leukocytes recruited in females. In male β2-adrenergic receptor knock out mice (bred on a congenic FVB background) the number of leukocytes recruited was increased ∼ 4-fold, while in females there was no change, eliminating sexual dimorphism in leukocyte migration. While there were significantly fewer recruited CD62L+ and CD11a+ leukocytes in wild-type males, only in male β-adrenergic receptor knock out mice was there an increase in the number of recruited CD11a+ leukocytes, again eliminating sexual dimorphism. Thus, leukocyte migration and CD11a+ adhesion molecule expression in male, but not in female, leukocytes is β-adrenergic receptor-dependent. Our findings provide support for a role of β2-adrenergic receptor mechanisms in the inflammatory response, and suggest that β2-adrenergic receptor on male leukocytes contributes to sexual dimorphism in the effect of stress on inflammatory diseases. [Copyright &y& Elsevier]

Background: Genetic polymorphisms of β3-adrenergic receptor (AR) Trp64Arg and β2-AR Gln27Glu may result in significant change in the functions of these receptors. The aims of the present study were to investigate the association between Trp64Arg, Arg16Gly and Gln27Glu polymorphisms and the susceptibility to obesity and hypertension in a Chinese population. Methods: A total of 437 Chinese subjects including 149 obese hypertensive patients, 139 non-obese essential hypertensive patients, and 149 non-obese normotensive healthy controls were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific (AS)-PCR assays were used to identify Trp64Arg, Arg16Gly, and Gln27Glu genotypes. Results: The allele frequencies of 64Arg and 27Glu in the obese hypertensive group were 0.178 and 0.128, respectively. Both were significantly higher than in the non-obese hypertensive and the control groups (p<0.05). Further analysis showed that this association existed only in male hypertensive patients. Conclusions: These data reveal that frequencies of β3-AR 64Arg and β2-AR 27Glu were significantly higher in our obese hypertensive patients than in the non-obese hypertensive population and healthy controls. β3-AR Trp64Arg and β2-AR Gln27Glu genetic polymorphisms are associated with obesity in Chinese male hypertensive patients. Clin Chem Lab Med 2007;45:493–8. [ABSTRACT FROM AUTHOR]

Abstract: Cadmium (Cd) is a widely spread toxicant with endocrine disrupting properties. Under experimental conditions it suppresses sex steroid synthesis in the male as well as the female. Testicular steroidogenesis is primarily regulated by gonadotropins, but is also influenced by catecholamines. We have previously shown that Cd exposure affects rat testosterone synthesis by down-regulating luteinizing hormone (LH) receptor mRNA expression. In this study, rats were given 10μmol/kg Cd subcutaneously and sacrificed 0.48–144h later. We investigated the effects of Cd on testicular gene expression of two adrenergic receptors. In addition, mRNA levels of the androgen-regulated house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured. In contrast to the suppressive influence on LH receptor expression Cd lacked effect on the expression of α1A- and β2-adrenergic receptors. GAPDH gene expression, on the other hand, was up-regulated 1.6-fold after exposure to 10μmol/kg Cd. These data suggest that the influence of Cd on testicular gene expression involves a specific effect on the LH receptor and not a general effect on seven-transmembrane-spanning receptors. Also, data indicate that the increased expression of GAPDH may be secondary to Cd-induced testosterone deprivation, suggesting future studies of androgen-regulated genes in the toxicity of Cd. [Copyright &y& Elsevier]

Human β2-adrenergic receptor is a G-protein–coupled receptor with seven transmembrane helices, and is important in pharmaceutical targeting on pulmonary and cardiovascular diseases. N-terminal histidine-tagged gene constructs with optimized codon usage were designed so as to obtain Pichia pastoris transformants with a high expression level. The constructs were inserted into the pPIC9 vector, and then electroporated into the SMD1168 strains. The highest expression level obtained was about 4 mg/liter-culture broth. The dissociation constant of the receptor in the membrane fraction was 1.2 nM toward CGP-12177 antagonist. The receptor was solubilized with sucrose monolaurate and purified with a series of chromatography steps including anion-exchange, Ni-Sepharose, alprenolol-Agarose, and hydroxyapatite columns. The receptor was heterogeneously glycosylated, showing broad SDS-PAGE bands around 70–90 kDa. After endoglycosidase treatment, the receptor appeared as a single band around 45 kDa, and was further purified with hydroxyapatite and gel-filtration columns. The receptor was eluted as a sharp peak at the gel-filtration elution volume corresponding to a molecular mass of 117 kDa. The saccharide-trimmed receptor thus purified is homogeneous as analyzed with SDS-PAGE, shows the dissociation constant of 4.7 nM toward CGP-12177 antagonist, and is suitable for crystallization experiments. [ABSTRACT FROM PUBLISHER]

Abstract: Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) is currently understood to be mediated by matrix metalloproteases (MMPs) and the release of EGF-like ligands. This ligand-mediated process also suggests that downstream of EGFR the signalling in response to GPCR ligands or EGF appears to be indistinguishable. Here we provide evidence that transactivation of EGFR by the β2-adrenergic receptor (β2-AR) is independent of MMPs and results in an incomplete downstream signalling involving extracellular signal-activated kinase (ERK) but not PLCγ1 and Akt. In contrast, β2-AR has the ability to activate PLCγ1 when the EGFR is primed either by co-stimulation with EGF or by increased basal activity due to over-expression. In that way but not via the β2-AR-mediated transactivation the EGFR docking sites pY992 and pY1173 may be generated which are critical for PLCγ1. This EGFR-supported transactivation is strongly dependent on EGFR tyrosine kinase, c-Src, and the c-Src-specific EGFR tyrosine residue 845 and represents a novel paradigm of EGFR transactivation. [Copyright &y& Elsevier]

Pharmacogenetics promises to individualize therapeutics. Concerns, however, exist about the lack of replication of discoveries. Selective use of different endpoints, times of assessment, types of interventions and genetic groups across studies may lead to spurious results. Here, we examined the variability of definitions of endpoints and analyses reported across studies addressing the association of the Arg16Gly and/or Gln27Glu polymorphisms of the β2-adrenergic receptor gene with clinical response to β2-agonist therapy in asthma.We systematically calculated the number and type of endpoints and analyses reported across studies and recorded the appraisal of their statistical significance.Across 21 studies, the total number of probed and reported associations was 487 when the multiple endpoints and types of comparisons presented by multiple comparisons were considered (337 for Arg16Gly, 98 for Gln27Glu and 52 for their haplotypes): 465 (95%) were probed only once; only six associations were probed twice and two associations were probed five times, for the same endpoint, time of assessment, type of interventions and genetic group. Most studies (17/21) claimed at least one significant association. Overall, however, 243/487 (49.9%) probed and reported associations were not statistically significant, 120 (24.6%) were of unspecified statistical significance, 86 (17.7%) were statistically significant only for specific selected genetic contrasts and only 38 (7.8%) were genuinely statistically significant for the comparison between all available genetic groups.The multifarious outcomes in this literature are inconsistent across studies and susceptible to selective reporting. The lack of standardization hinders the evaluation of replication validity for reported discoveries. [ABSTRACT FROM AUTHOR]

The article cites a study on the relationship between nonspecific airway hyperresponsiveness (AHR) and Arg16Gly β2-adrenergic receptor (AR) gene polymorphism in asymptomatic healthy Japanese individuals. The study showed that a particular β2-AR gene polymorphism at the 16th sequence could be a genetic determinant of AHR, a hallmark of asthma, based on the bronchoconstriction caused by metacholine in asymptomatic healthy subjects.

Abstract: In the COS-7 cell signalling network high levels of cAMP produced, for example, by co-stimulation of β2-adrenergic receptor (β2-AR) and bradykinin B2 receptor (BKR) may affect epidermal growth factor receptor (EGFR)-mediated activation of extracellular signal-stimulated kinase (ERK). In contrast, co-stimulation of either β2-AR or B2R with EGFR leads to synergistic activation of ERK. Due to triple stimulation of these receptors the synergistic effects on ERK activation as well as cAMP accumulation are diminished. Here we demonstrate that EGF is capable of inducing Src-mediated phosphorylation of the tyrosine residues 177 and 347 of BKR. Their replacement by phenylalanine led to BKR mutants which are unable to activate the cAMP pathway. Using these mutants we can show that EGF attenuates but does not completely inhibit the BKR/cAMP pathway which is counteracting the EGFR signalling to ERK. Our findings suggest that the EGFR may control the cellular network rather by balancing mechanisms then by switch on/off reactions. [Copyright &y& Elsevier]

Background: As a result of the finding that the mutation of Arg into Gly at β2-adrenergic receptor (β2-AR)16 loci could promote the downregulation effect triggered by the β2-agonist, it was supposed that Gly16 might be associated with the downregulation of β2-AR in patients with nocturnal asthma. Objective: It was the aim of this study to analyze the association between β2-AR genetic polymorphisms and nocturnal asthmatic patients of Chinese Han nationality. Methods: A polymerase chain reaction allele-specific oligonucleotide hybridization assay was used to determine 16 and 27 loci alleles of β2-AR genetic polymorphisms in 25 nocturnal asthmatic patients (nocturnal asthma group), 22 non-nocturnal asthmatic patients (non-nocturnal asthma group), and 72 healthy people (control group). All people investigated were of Chinese Han nationality. Results: The distribution frequency of genotype Arg/Arg, Arg/Gly, and Gly/Gly at β2-AR 16 loci was 12, 16 and 72% in the nocturnal asthma group; and 27, 41 and 32% in the non-nocturnal asthma group. There was a significant increase in the frequency of genotype Gly/Gly and allele Gly in the nocturnal asthma group compared with the non-nocturnal asthma group (p < 0.01). However, there was no significant difference in the frequency of genotype Gly/Gly and allele Gly in the non-nocturnal asthma group, compared with the control group. There was no significance in the frequency of the genotypes and alleles of β2-AR 27 loci among the three groups (p > 0.05). Conclusion: The Gly16 polymorphism of β2-AR was overrepresented in nocturnal asthmatic patients, correlated with nocturnal asthma, and therefore appeared to be an important genetic factor in the expression of this asthmatic phenotype. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The β2-adrenergic receptor gene (ADRB2) is a target molecule of β2-agonists. Single nucleotide polymorphisms (SNPs) in the ADRB2 are related to the effectiveness of β2-agonists. However, there are some discrepancies in the results of pharmacogenetic studies of ADRB2 among different ethnic groups. The aims of this study were to determine the ADRB2 haplotypes and diplotypes in Japanese asthmatic and nonasthmatic subjects and to examine their relation to asthma and to compare these results with previous studies done in other ethnic groups. Methods: Complete sequences for 3 kb promoter and 1.2 kb structural regions of ADRB2 were analyzed in 48 Japanese asthmatics and 100 controls, and haplotypes and diplotypes of SNPs were analyzed. Results: Fifteen SNPs including a novel one in -839 were observed. Allele frequencies for all SNPs were similar between asthmatics and controls. We also identified 42 haplotypes and 54 diplotypes of ADRB2 in a Japanese population. The frequencies were similar between the two groups. They were classified into 17 and 23 types, respectively, according to Drysdale's haplotype-organization system, and a significant ethnic difference was observed between the Japanese and Caucasian populations. Conclusions: The frequencies of SNPs and ADBR2 haplotypes in Japanese are different from those in Caucasians and African Americans. These divergences might imply the need for independent pharmacogenetic studies for ADBR2 in each ethnic group. [ABSTRACT FROM AUTHOR]

Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin-dependent, but caveolin-independent. Functional assays demonstrate the cargo-dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated β2-adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin-mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM–Ub interactions to overcome the low affinity binding of mono-Ub to UIM. [ABSTRACT FROM AUTHOR]

Abstract: Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsα, or the Gs-coupled β2-adrenergic receptor (β2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsα-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the β2AR. Purified RGS2 selectively bound to the third intracellular loop of the β2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and β2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production. [Copyright &y& Elsevier]

Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although β-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that β-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the β-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing β2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways. [ABSTRACT FROM AUTHOR]

Background: β2-Adrenergic receptor (β2-AR) polymorphisms occurring at amino acid position 16 (Arg-Gly) and 27 (Gln-Glu) are known to be functionally relevant and also disease-modifying in subjects with asthma. It has been found in Caucasoid asthmatic patients that the Gln27 genotype β2-AR was associated with an increase in total serum IgE levels. The association between β2-AR genetic polymorphisms and total serum IgE in asthmatic patients of Chinese Han nationality remains to be established.Objectives: It was the aim of this study to investigate the association between β2-AR genetic polymorphisms and total serum IgE in asthmatic patients of Chinese Han nationality. Methods: All 59 asthmatic patients investigated (27 males and 32 females, aged between 16 and 60 years) were people of Chinese Han nationality. They were tested for their total serum IgE levels with the enzyme-linked immunosorbent assay test, and β2-AR genetic polymorphisms were tested with the polymerase chain reaction allele-specific oligonucleotide hybridization assay. Results: There was a significant difference of serum IgE levels among three β2-AR 27 loci groups (p < 0.0001), with the highest IgE level [(1.24 ± 0.25) × 106 IU/l] in the Gln/Gln group and the lowest IgE level [(0.48 ± 0.06) × 106 IU/l] in the Glu/Glu group. No polymorphism of β2-AR 16 loci was found to be associated with total serum IgE (p > 0.05). Conclusions: Our research suggested that in asthmatic patients of Chinese Han nationality, the β2-AR genetic polymorphism at 27 loci could be associated with serum IgE levels and it might therefore play an important role in the determination of phenotypes of bronchial asthma. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, β2-adrenergic receptor (β2AR) in S49 lymphoma cells, α-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced β-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of β2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys− cells that lack the α subunit of Gs were used. Stimulation of β2AR in these cells failed to increase intracellular cAMP, but β-arrestin translocation still occurred, suggesting that feedback from β2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous α subunit of Go (Goα) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced β-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Gαo and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (β2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents that induce intracellular cAMP (prostaglandin E2, forskolin, and butyryl cAMP). LPS in combination with β2-agonists and cAMP elevating agents had an additional effect on the release of VEGF, OSM, and CXCL6. These proteins are up-regulated after 16–24 h of exposure and this is mediated by the β2-AR, as determined by time course experiments and the use of a specific β2-AR antagonist (ICI 118551). Beta2-AR agonists are used as bronchodilators in the treatment of asthma, but appear to have no effect on the chronic inflammation of the disease. However, the up-regulation of VEGF, OSM, and CXCL6 may have adverse effects on the inflammatory process of asthma. These mediators are involved in the recruitment of neutrophils, airway remodelling and angiogenesis, known features of chronic inflammatory diseases. We propose that the up-regulation of these proteins could play a role in the adverse effects of prolonged excessive usage of β2-AR agonists on the airways besides the desensitization of the β2-AR. [Copyright &y& Elsevier]

Abstract: To understand the complexity of mechanisms involved in the regulation of adaptive immunity by the sympathetic neurotransmitter norepinephrine and adrenergic receptor stimulation, there must be a rich history of basic science and clinical findings upon which to form hypotheses for testing, as well as a rich supply of individuals trained in two or more disciplines. This review is intended to offer a tour of the past, present, and future discoveries that have been made in the area of adrenergic regulation of adaptive immunity, as well as share a vision of how our field of study will progress years from now, given that every individual who contributes to the interdisciplinary nature of our research is valued. And finally, this review will discuss how the lessons from the past can help us to attain a vision of interdisciplinary research for the future. [Copyright &y& Elsevier]

The human β2-adrenoceptor is a member of the 7-transmembrane family of receptors, encoded by a gene on chromosome 5, and widely distributed in the respiratory tract. Intracellular signaling after β2-adrenoceptor activation is largely affected through cyclic adenosine monophosphate and protein kinase A. Differences in the mechanism of interaction of short- and long-acting β2-agonists and the β2-receptor are reflected in the kinetics of airway smooth muscle relaxation and the onset and duration of bronchodilation in asthmatic patients. β-Adrenoceptor desensitization associated with prolonged β2-agonist activation differs depending on the cell type and is reflected in different profiles of clinical tolerance to chronic β2-agonist therapy. A number of genetic polymorphisms of the β2-receptor have been described that appear to alter the behavior of the receptor, including the response to β2-agonists. The synergy between the β2-receptor and the glucocorticoid receptor functions has implications for the combined use of β2-agonists and corticosteroids in the treatment of respiratory disease. [Copyright &y& Elsevier]