Structural and Functional Analysis of a ß2-Adrenergic Receptor Complex with GRK5.

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

S-nitrosylation is required for ß2AR desensitization and experimental asthma.

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.

Diverse pathways in GPCR-mediated activation of Ca2+ mobilization in HEK293 cells.

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signaling. Ca2+ is a well-known second messenger that can be induced by GPCR activation through the primary canonical pathways involving Gαq- and Gßγ-mediated activation of phospholipase C-ß (PLCß). While some Gs-coupled receptors are shown to trigger Ca2+ mobilization, underlying mechanisms remain elusive. Here we evaluated whether Gs-coupled receptors including the ß2-adrenergic receptor (ß2AR) and the prostaglandin EP2 and EP4 receptors (EP2R and EP4R) that are endogenously expressed in HEK293 cells utilize common pathways for mediating Ca2+ mobilization. For the ß2AR, we found an essential role for Gq in agonist-promoted Ca2+ mobilization while genetic or pharmacological inhibition of Gs or Gi had minimal effect. ß-agonist-promoted Ca2+ mobilization was effectively blocked by the Gq-selective inhibitor YM-254890 and was not observed in ΔGαq/11 or ΔPLCß cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the ß2AR with Gq. For the EP2R, which couples to Gs, agonist treatment induced Ca2+ mobilization in a pertussis toxin (PTX)-sensitive but YM-254890-insensitive manner. In contrast, EP4R, which couples to Gs and Gi, exhibited Ca2+ mobilization that was sensitive to both PTX and YM-254890. Interestingly, both EP2R and EP4R were largely unable to induce Ca2+ mobilization in ΔGαs or ΔPLCß cells, supporting a strong dependency on Gs signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are utilized to mediate Ca2+ mobilization in HEK293 cells where the ß2AR primarily utilizes Gq, EP2R uses Gs and Gi, and EP4R utilizes Gs, Gi and Gq.

Structure optimization of Cmpd-15 as negative allosteric modulators for the ß2-adrenergic receptor.

19 derivatives of 1-benzyl-3-arylpyrazole-5-carboxamides (H1-H19) and 5 derivatives of 1-benzyl-5-arylpyrazole-3-carboxamides (J1-J5) have been designed and synthesized as potential negative allosteric modulators (NAMs) for the ß2-adrenergic receptor (ß2AR). The new pyrazole derivatives were screened on the classic G-protein dependent signaling pathway at ß2AR. The majority of 1-benzyl-3-aryl-pyrazole-5-carboxamide derivatives show more potent allosteric antagonistic activity against ß2AR than Cmpd-15, the first reported ß2AR NAM. However, the 1-benzyl-5-arylpyrazole-3-carboxamide derivatives exhibit very poor or even no allosteric antagonistic activity for ß2AR. Furthermore, the active pyrazole derivatives have relative better drug-like profiles than Cmpd-15. Taken together, we discovered a series of derivatives of 1-benzyl-3-arylpyrazole-5-carboxamides as a novel scaffold of ß2AR NAM.

Sympathetic ß2-adrenergic receptor blockade overcomes docetaxel resistance in prostate cancer.

PURPOSE: Due to the limited effective therapies, resistance to docetaxel is ordinarily fatal and remains a critical clinical challenge.ß2-adrenergic receptor(ß2-AR)can promote the metastasis and invasion of prostate cancer, but the role in chemotherapy-resistant prostate cancer remains unclear. METHODS: By downloading the GEO database in NCBI, the expression of ß2-AR in different prostate tissues was analyzed. We constructed docetaxel-resistant prostate cancer cell lines by the method of dose-escalation. LC3B-labeled stable cells and shAtg5 knockdown stable cells were constructed by lentivirus infection. The molecular mechanism of ß2-AR affecting docetaxel sensitivity through apoptosis and autophage were investigated by flow cytometry, mitochondrial membrane potential and western blot. Then we detected the interaction between autophagy and apoptotic by performing immunoprecipitation assay. RESULTS: We show that restraining the activity of ß2-AR sensitized the cell response and reduced the resistance to docetaxel. The mechanism involves the regulation of ß2-AR in the cellular response to docetaxel through apoptosis and autophagy via caspase signaling and Atg5/AMPK/mTOR pathway as well as the effect of ß2-AR on the crosstalk between apoptosis and autophagy via p38 MAPK and JNK/c-Jun/FOXO3a signaling pathways. CONCLUSION: Our data demonstrate that ß2-AR inhibitor-induced autophagy and apoptosis contribute to the effectiveness responses to docetaxel in castration-resistant prostate cancer, and in combination with pharmacological agents of ß2-AR and autophagy inhibitors may provide a potential therapeutic strategy to enhance the limited capacity of docetaxel to control castration-resistant prostate cancer.

Loss of bronchoprotection with ICS plus LABA treatment, ß-receptor dynamics, and the effect of alendronate.

BACKGROUND: Loss of bronchoprotection (LOBP) with a regularly used long-acting ß2-adrenergic receptor agonist (LABA) is well documented. LOBP has been attributed to ß2-adrenergic receptor (B2AR) downregulation, a process requiring farnesylation, which is inhibited by alendronate. OBJECTIVE: We sought to determine whether alendronate can reduce LABA-associated LOBP in inhaled corticosteroid (ICS)-treated patients. METHODS: We conducted a randomized, double-blind, placebo-controlled, parallel-design, proof-of-concept trial. Seventy-eight participants with persistent asthma receiving 250 µg of fluticasone twice daily for 2 weeks were randomized to receive alendronate or placebo while initiating salmeterol for 8 weeks. Salmeterol-protected methacholine challenges (SPMChs) and PBMC B2AR numbers (radioligand binding assay) and signaling (cyclic AMP ELISA) were assessed before randomization and after 8 weeks of ICS plus LABA treatment. LOBP was defined as a more than 1 doubling dose reduction in SPMCh PC20 value. RESULTS: The mean doubling dose reduction in SPMCh PC20 value was 0.50 and 0.27 with alendronate and placebo, respectively (P = .62). Thirty-eight percent of participants receiving alendronate and 33% receiving placebo had LOBP (P = .81). The after/before ICS plus LABA treatment ratio of B2AR number was 1.0 for alendronate (P = .86) and 0.8 for placebo (P = .15; P = .31 for difference between treatments). The B2AR signaling ratio was 0.89 for alendronate (P = .43) and 1.02 for placebo (P = .84; P = .44 for difference). Changes in lung function and B2AR number and signaling were similar between those who did and did not experience LOBP. CONCLUSION: This study did not find evidence that alendronate reduces LABA-associated LOBP, which relates to the occurrence of LOBP in only one third of participants. LOBP appears to be less common than presumed in concomitant ICS plus LABA-treated asthmatic patients. B2AR downregulation measured in PBMCs does not appear to reflect LOBP.

Design, synthesis, and functional assessment of Cmpd-15 derivatives as negative allosteric modulators for the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2AR), a G protein-coupled receptor, is an important therapeutic target. We recently described Cmpd-15, the first small molecule negative allosteric modulator (NAM) for the ß2AR. Herein we report in details the design, synthesis and structure-activity relationships (SAR) of seven Cmpd-15 derivatives. Furthermore, we provide in a dose-response paradigm, the details of the effects of these derivatives in modulating agonist-induced ß2AR activities (G-protein-mediated cAMP production and ß-arrestin recruitment to the receptor) as well as the binding affinity of an orthosteric agonist in radio-ligand competition binding assay. Our results show that some modifications, including removal of the formamide group in the para-formamido phenylalanine region and bromine in the meta-bromobenzyl methylbenzamide region caused dramatic reduction in the functional activity of Cmpd-15. These SAR results provide valuable insights into the mechanism of action of the NAM Cmpd-15 as well as the basis for future development of more potent and selective modulators for the ß2AR based on the chemical scaffold of Cmpd-15.

The ß2-adrenergic receptor is involved in differences in the protein degradation level of the pectoral muscle between fast- and slow-growing chicks during the neonatal period.

The aim of this study was to investigate whether ß2-AR mRNA expression is involved in either atrogin-1/MAFbx mRNA expression or protein degradation in chicken skeletal muscle by comparing fast- and slow-growing chicks during the neonatal period. Based on their body weight gain from 1 to 5 days of age, 5-day-old chicks (Gallus gallus domestics) were divided into a slow-growing and a fast-growing group, the mean weight gains of which were 6.3 ±â€¯1.3 g/day and 11.3 ±â€¯0.9 g/day, respectively. The ratio of pectoral muscle weight to total body weight was higher in the fast-growing group of chicks than in the slow-growing group. In addition, the plasma 3-methylhistidine concentration, an index of protein degradation in skeletal muscle, was significantly lower in the fast-growing than in the slow-growing chicks. The mRNA expression of ß2-AR, which we previously found is involved in decreasing muscle protein degradation by suppression atrogin-1/MAFbx mRNA expression, was significantly higher in the pectoral muscle of the fast-growing group compared with that of the slow-growing group. Concordantly, lower mRNA expression of atrogin-1/MAFbx was observed in the pectoral muscle of the fast-growing chicks. However, in the sartorius muscle, which is a muscle in the thigh, the ratio of the muscle weight to total body weight was not significantly different between the two groups of chicks at 5 days of age. In addition, there was no significant difference in the mRNA expressions of ß2-AR and atrogin-1/MAFbx in the sartorius muscle between these two groups. These results suggest that ß2-AR expression levels might be physiologically significant in the control of protein degradation in the pectoral muscle of neonatal chicks.

ß2-receptor agonists salbutamol and terbutaline attenuated cytokine production by suppressing ERK pathway through cAMP in macrophages.

ß2-receptor agonists are used in the treatment of inflammatory obstructive lung diseases asthma and COPD as a symptomatic remedy, but they have been suggested to possess anti-inflammatory properties, also. ß2-receptor activation is considered to lead to the activation of ERK pathway through G-protein- and cAMP-independent mechanisms. In this study, we investigated the effects of ß2-receptor agonists salbutamol and terbutaline on the production of inflammatory factors in macrophages. We found that ß2-receptor agonists inhibited LPS-induced ERK phosphorylation and the production of MCP-1. A chemical cAMP analog 8-Br-cAMP also inhibited ERK phosphorylation and TNF and MCP-1 release. As expected, MAPK/ERK kinase (MEK)1/2 inhibitor PD0325901 inhibited ERK phosphorylation and suppressed both TNF and MCP-1 production. In conclusion, we suggest that ß2-receptor agonists salbutamol and terbutaline inhibit inflammatory gene expression partly by a mechanism dependent on cAMP leading to the inhibition of ERK signaling in macrophages. Observed anti-inflammatory effects of ß2-receptor agonists may contribute to the clinical effects of these drugs.

IL-13 desensitizes ß2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism.

BACKGROUND: ß2-Adrenergic receptor (ß2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of ß2-agonists is well studied, type 2 inflammation could also affect ß2AR function. OBJECTIVE: We sought to evaluate the effect of the type 2 cytokine IL-13 on ß2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2). METHODS: HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and ß2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2. RESULTS: Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in ß2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients. CONCLUSION: IL-13 treatment of HAECs leads to ß2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) ß2ARs, suggesting that the beneficial effects of ß2-agonists could be blunted in patients with type 2 associated asthma.

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through ß-arrestin-2 in THP-1 cell line.

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a ß2-adrenergic receptor (ß2-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of ß2-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and ß-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1ß (IL-1ß) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of ß-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1ß (IL-1ß) release, thus ß-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1ß release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the ß2-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1ß release via ß-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases.

ß2-Adrenergic receptors in immunity and inflammation: stressing NF-κB.

ß2-Adrenergic receptors (ß2-ARs) transduce the effects of (nor)epinephrine on a variety of cell types and act as key mediators of the body's reaction to stress. ß2-ARs are also expressed on immune cells and there is ample evidence for their role in immunomodulation. A key regulator of the immune response and a target for regulation by stress-induced signals is the transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB shapes the course of both innate and adaptive immune responses and plays an important role in susceptibility to disease. In this review, we summarise the literature that has been accumulated in the past 20years on adrenergic modulation of NF-κB function. We here focus on the molecular basis of the reported interactions and show that both physiological and pharmacological triggers of ß2-ARs intersect with the NF-κB signalling cascade at different levels. Importantly, the action of ß2-AR-derived signals on NF-κB activity appears to be highly cell type specific and gene selective, providing opportunities for the development of selective NF-κB modulators.

A systematic study on the influence of the main ingredients of an ivy leaves dry extract on the ß2-adrenergic responsiveness of human airway smooth muscle cells.

The bronchospasmolytic and secretolytic effects of ivy leaves dry extracts can be explained by an increased ß2-adrenergic responsiveness of the bronchi. Recently, it was shown that α-hederin inhibits the internalization of ß2-adrenergic receptors (ß2AR) under stimulating conditions. α-Hederin pretreated alveolar type II cells and human airway smooth muscle cells revealed an increased ß2AR binding and an elevated intracellular cAMP level, respectively. In order to identify whether additional compounds also mediate an increased ß2-adrenergic responsiveness, we examined the ingredients of an ivy leaves dry extract (EA 575) protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, 3,4-, 3,5- and 4,5-dicaffeoylquinic acid, hederacoside B, and ß-hederin. Within all the tested substances, only ß-hederin inhibited the internalization of GFP-tagged ß2AR in stably transfected HEK293 cells. Using fluorescence correlation spectroscopy ß-hederin (1 µM, 24 h) pretreated HASM cells showed a statistically significant increase in the ß2AR binding from 33.0 ± 8.9% to 44.1 ± 11.5% which was distributed with 36.0 ± 9.5% for τbound1 and 8.1 ± 2.6% for τbound2, respectively (n = 8, p < 0.05). The increased binding was selectively found for the receptor-ligand complex with unrestricted lateral mobility (τbound1 of 0.9 ± 0.1 ms, D1 = 9.1 ± 0.2 µm(2)/s, n = 8), whereas the binding of ß2AR with hindered lateral mobility (τbound2 of 64.2 ± 47.6 ms, D2 = 0.15 ± 0.02 µm(2)/s, n = 8) was not affected. Compared to control cells, a statistically significant increase of 17.5 ± 6.4% (n = 4, p < 0.05) and 24.2 ± 5.8% (n = 4, p < 0.001) in the cAMP formation was found for ß-hederin pretreated HASM cells after stimulation with 10 µM of terbutaline and simultaneous stimulation with 10 µM terbutaline and 10 µM forskolin, respectively. Within this systematic study focusing on the influence of the ingredients of an ivy leaves dry extract on HASM cells it was possible to identify ß-hederin as further component presumably responsible for the ß2-mimetic effects.

Comparison of airway dimensions with once daily tiotropium plus indacaterol versus twice daily Advair(®) in chronic obstructive pulmonary disease.

BACKGROUND: Current guidelines recommend combining long-acting bronchodilators with different modes of action in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD). We evaluated the effects of airway dimensions and pulmonary function with tiotropium plus indacaterol versus Advair(®). METHODS: Subjects (n = 46) were randomized to receive tiotropium (18 µg once daily) plus indacaterol (150 µg once daily) or Advair(®) (50/250 µg twice daily) for 16 weeks. Airway geometry was determined by quantitative computed tomography (luminal area, Ai; total area of the airway, Ao; wall area, WA; and percentage wall area, WA/Ao and wall thickness, T). Spirometry (forced expiratory volume in 1 s, FEV1; forced vital capacity, FVC and inspiratory capacity, IC) and St. George's Respiratory Questionnaire (SGRQ) were evaluated. RESULTS: Tiotropium plus indacaterol significantly increased CT-indices including Ai corrected for body surface area (Ai/BSA), and decreased WA/BSA, WA/Ao and T/√BSA compared with Advair(®) (p < 0.05, respectively). In physiological parameters, mean difference in IC was significantly higher under treatment with tiotropium plus indacaterol than Advair(®) (p < 0.05). The changes in Ai/BSA, WA/BSA, WA/Ao and T/√BSA were significantly correlated with changes in IC (r = 0.535, p = 0.011; r = -0.688, p < 0.001; r = -0.555, p = 0.002 and r = -0.542, p = 0.007; respectively). There were more significant improvements in SGRQ scores after treatment with tiotropium plus indacaterol than Advair(®). CONCLUSIONS: These findings suggest that dual bronchodilation with tiotropium plus indacaterol is superior in airway geometry and lung function compared with Advair(®) in COPD.

A genome-wide association study of bronchodilator response in Latinos implicates rare variants.

BACKGROUND: The primary rescue medication to treat acute asthma exacerbation is the short-acting ß2-adrenergic receptor agonist; however, there is variation in how well a patient responds to treatment. Although these differences might be due to environmental factors, there is mounting evidence for a genetic contribution to variability in bronchodilator response (BDR). OBJECTIVE: To identify genetic variation associated with bronchodilator drug response in Latino children with asthma. METHODS: We performed a genome-wide association study (GWAS) for BDR in 1782 Latino children with asthma using standard linear regression, adjusting for genetic ancestry and ethnicity, and performed replication studies in an additional 531 Latinos. We also performed admixture mapping across the genome by testing for an association between local European, African, and Native American ancestry and BDR, adjusting for genomic ancestry and ethnicity. RESULTS: We identified 7 genetic variants associated with BDR at a genome-wide significant threshold (P < 5 × 10(-8)), all of which had frequencies of less than 5%. Furthermore, we observed an excess of small P values driven by rare variants (frequency, <5%) and by variants in the proximity of solute carrier (SLC) genes. Admixture mapping identified 5 significant peaks; fine mapping within these peaks identified 2 rare variants in SLC22A15 as being associated with increased BDR in Mexicans. Quantitative PCR and immunohistochemistry identified SLC22A15 as being expressed in the lung and bronchial epithelial cells. CONCLUSION: Our results suggest that rare variation contributes to individual differences in response to albuterol in Latinos, notably in SLC genes that include membrane transport proteins involved in the transport of endogenous metabolites and xenobiotics. Resequencing in larger, multiethnic population samples and additional functional studies are required to further understand the role of rare variation in BDR.

Pharmacogenetics: implications of race and ethnicity on defining genetic profiles for personalized medicine.

Pharmacogenetics is being used to develop personalized therapies specific to subjects from different ethnic or racial groups. To date, pharmacogenetic studies have been primarily performed in trial cohorts consisting of non-Hispanic white subjects of European descent. A "bottleneck" or collapse of genetic diversity associated with the first human colonization of Europe during the Upper Paleolithic period, followed by the recent mixing of African, European, and Native American ancestries, has resulted in different ethnic groups with varying degrees of genetic diversity. Differences in genetic ancestry might introduce genetic variation, which has the potential to alter the therapeutic efficacy of commonly used asthma therapies, such as ß2-adrenergic receptor agonists (ß-agonists). Pharmacogenetic studies of admixed ethnic groups have been limited to small candidate gene association studies, of which the best example is the gene coding for the receptor target of ß-agonist therapy, the ß2-adrenergic receptor (ADRB2). Large consortium-based sequencing studies are using next-generation whole-genome sequencing to provide a diverse genome map of different admixed populations, which can be used for future pharmacogenetic studies. These studies will include candidate gene studies, genome-wide association studies, and whole-genome admixture-based approaches that account for ancestral genetic structure, complex haplotypes, gene-gene interactions, and rare variants to detect and replicate novel pharmacogenetic loci.

The effects of intraperitoneal clenbuterol injection on protein degradation and myostatin expression differ between the sartorius and pectoral muscles of neonatal chicks.

The purpose of this study was to investigate the effects of injection of the ß2-adrenergic receptor agonist clenbuterol on the skeletal muscles of neonatal chicks (Gallus gallus domesticus). One-day-old chicks were randomly divided into four groups and given a single intraperitoneal injection of clenbuterol (0.01, 0.1, or 1mg/kg) or phosphate-buffered saline. Twenty-four hours after the injection, the sartorius muscles (which consist of both slow- and fast-twitch fibers) of chicks that received 0.01 or 0.1mg/kg clenbuterol were significantly heavier than those of controls, while there were no between-group differences in the weight of the pectoral muscles, which consist of only fast-twitch fibers. Muscle free N(t)-methylhistidine, regarded as an index of myofibrillar proteolysis, was decreased in the sartorius muscle of the clenbuterol-injected chicks, while it was not affected in the pectoral muscles. In the sartorius muscle of the clenbuterol-injected chicks, myostatin and atrogin-1/MAFbx mRNA expressions were decreased, while insulin-like growth factor-I was unaffected. These observations suggested, in 1-day-old chicks, clenbuterol might increase mass of the sartorius muscle by decreasing myostatin gene expression and protein degradation.

The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease.

Glucocorticoids are primary stress hormones necessary for life that regulate numerous physiologic processes in an effort to maintain homeostasis. Synthetic derivatives of these hormones have been mainstays in the clinic for treating inflammatory diseases, autoimmune disorders, and hematologic cancers. The physiologic and pharmacologic actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors. Ligand-occupied GR induces or represses the transcription of thousands of genes through direct binding to DNA response elements, physically associating with other transcription factors, or both. The traditional view that glucocorticoids act through a single GR protein has changed dramatically with the discovery of a large cohort of receptor isoforms with unique expression, gene-regulatory, and functional profiles. These GR subtypes are derived from a single gene by means of alternative splicing and alternative translation initiation mechanisms. Posttranslational modification of these GR isoforms further expands the diversity of glucocorticoid responses. Here we discuss the origin and molecular properties of the GR isoforms and their contribution to the specificity and sensitivity of glucocorticoid signaling in healthy and diseased tissues.

16α-OHE1 alleviates hypoxia-induced inflammation and myocardial damage via the activation of ß2-Adrenergic receptor.

OBJECTIVE: Myocardial injuries resulting from hypoxia are a significant concern, and this study aimed to explore potential protective strategies against such damage. Specifically, we sought to investigate the cardioprotective effects of 16α-hydroxyestrone (16α-OHE1). METHODS: Male Sprague‒Dawley (SD) rats were subjected to hypoxic conditions simulating high-altitude exposure at 6000 m in a low-pressure chamber for 7 days. Before and during hypoxic exposure, estradiol (E2) and various doses of 16α-OHE1 were administered for 14 days. Heart weight/body weight (HW/BW), myocardial structure, Myocardial injury indicators and inflammatory infiltration in rats were measured. H9C2 cells cultured under 5% O2 conditions received E2 and varying doses of 16α-OHE1; Cell viability, apoptosis, inflammatory infiltration, and Myocardial injury indicators were determined. Expression levels of ß2AR were determined in rat hearts and H9C2 cells. The ß2AR inhibitor, ICI 118,551, was employed to investigate ß2AR's role in 16α-OHE1's cardioprotective effects. RESULTS: Hypoxia led to substantial myocardial damage, evident in increased heart HW, CK-MB, cTnT, ANP, BNP, structural myocardial changes, inflammatory infiltration, and apoptosis. Pre-treatment with E2 and 16α-OHE1 significantly mitigated these adverse changes. Importantly, the protective effects of E2 and 16α-OHE1 were associated with the upregulation of ß2AR expression in both rat hearts and H9C2 cells. However, inhibition of ß2AR by ICI 118,551 in H9C2 cells nullified the protective effect of 16α-OHE1 on myocardium. CONCLUSION: Our findings suggest that 16α-OHE1 can effectively reduce hypoxia-induced myocardial injury in rats through ß2ARs, indicating a promising avenue for cardioprotection.

Transdermal nitroglycerin for the treatment of preterm labor: a systematic review and metaanalysis.

OBJECTIVE: The purpose of this study was to evaluate the efficacy and safety of transdermal nitroglycerin as a tocolytic agent in women with preterm labor. STUDY DESIGN: We conducted a systematic review and metaanalysis of randomized controlled trials. RESULTS: Thirteen studies were included (1302 women) comparing transdermal nitroglycerin vs placebo (2 studies; n = 186); ß2-adrenergic receptor agonists (9 studies; n = 1024); nifedipine (1 study; n = 50); and magnesium sulfate (1 study; n = 42). There were no significant differences between transdermal nitroglycerin and placebo for delivery within 48 hours of the initiation of treatment or at <28, <34, or <37 weeks of gestation, adverse neonatal outcomes, and neurodevelopmental status at 24 months of life. Nevertheless, 1 study found a marginally significant reduction in the risk of a composite outcome of major neonatal morbidity and perinatal death (3/74 [4.1%] vs 11/79 [13.9%]; relative risk, 0.29; 95% confidence interval, 0.08-1.00). When compared with ß2-adrenergic receptor agonists, transdermal nitroglycerin was associated with a significant reduction in the risk of preterm birth at <34 and <37 weeks of gestation, admission to the neonatal intensive care unit, use of mechanical ventilation, and maternal side effects. There were no significant differences between transdermal nitroglycerin and nifedipine and magnesium sulfate in delivery within 48 hours of treatment and pregnancy prolongation, respectively. Overall, women who received transdermal nitroglycerin had a higher risk of headache. CONCLUSION: Although transdermal nitroglycerin appears to be more effective than ß2-adrenergic receptor agonists, the current evidence does not support its routine use as a tocolytic agent for the treatment of preterm labor. Further double-blind placebo-controlled trials are needed.