A large number of β-adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of cardiovascular diseases and other diseases. Nonetheless, it remains unclear whether these commonly used β-AR drugs can activate downstream β- arrestin-biased signaling pathways. The objective of this study was to investigate β-arrestin2 recruitment effects of β-AR agonists and antagonists that were commonly used in clinical practice. We used TANGO (transcriptional activation following arrestin translocation) assay to detect the β-arrestin2 recruitment by β-AR ligands in HEK293 cell line (HTLA cells) stably transfected with tetracycline transactivator protein (tTA) dependent luciferase reporter and β-arrestin2-TEV fusion gene. Upon activation of β-AR by a β-AR ligand, β-arrestin2 was recruited to the C terminus of the receptor, followed by cleavage of the G protein-coupled receptors (GPCRs) fusion protein at the TEV protease-cleavage site. The cleavage resulted in the release of tTA, which, after being transported to the nucleus, activated transcription of the luciferase reporter gene. The results showed that β-AR non-selective agonists epinephrine, noradrenaline and isoprenaline all promoted β-arrestin2 recruitment at β1-AR and β2-AR. β1-AR selective agonists dobutamine and denopamine both promoted β-arrestin2 recruitment at β1-AR. β2-AR selective agonists procaterol and salbutamol promoted β-arrestin2 recruitment at β2-AR. β-AR non-selective antagonists alprenolol and pindolol promoted β-arrestin2 recruitment at β1-AR. β1-AR selective antagonists celiprolol and bevantolol showed β-arrestin2 recruitment at β1-AR. β2-AR selective antagonists butoxamine showed β-arrestin2 recruitment at β1-AR. These results provide some clues for the potential action of β-AR drugs, and lay a foundation for the screening of β-arrestin-biased β-AR ligands.
Humans ; beta-Arrestin 2/metabolism* ; HEK293 Cells ; Adrenergic beta-Agonists/pharmacology* ; Isoproterenol/pharmacology* ; Receptors, Adrenergic, beta-2/metabolism* ; Norepinephrine/pharmacology*

OBJECTIVES: To study the association of β2-drenergic receptor ( METHODS: A total of 143 children with asthma who attended the hospital from October 2016 to October 2020 were enrolled as the asthma group, among whom 61 children had mild symptoms (mild group) and 82 children had moderate-to-severe symptoms (moderate-to-severe group). A total of 137 healthy children were enrolled as the control group. Peripheral venous blood samples were collected from the two groups. The SNaPshot SNP technique was used to analyze the SNP and haplotypes of the RESULTS: Polymorphisms were observed in the CONCLUSIONS SNP/haplotype of the
Asthma/genetics* ; Case-Control Studies ; Child ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Humans ; Polymorphism, Single Nucleotide ; Receptors, Adrenergic, beta-2/genetics* ; Regulatory Sequences, Nucleic Acid

Alleles ; General Surgery ; methods ; Genetic Variation ; Humans ; Hypotension ; drug therapy ; Receptors, Adrenergic, beta-2 ; genetics ; metabolism ; Vasoconstrictor Agents ; administration & dosage

OBJECTIVE: To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy. METHODS: Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of β2 adrenergic receptor (β2-AR). RESULTS: The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were β2-AR positive, but the proportion of β2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%. CONCLUSION A method, which is simple and operable, for knocking out β2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of β2-AR in human T cells.
CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; RNA, Guide ; Receptors, Adrenergic, beta-2 ; genetics ; T-Lymphocytes

OBJECTIVETo investigate the role of long non-coding RNA (lncRNA) BC088414 in hypoxic-ischemic injury of neural cells.

METHODSRat adrenal pheochromocytoma (PC12) cells were divided into four groups: normoxic, oxygen glucose deprivation (OGD), siRNA-normoxic (siRNA group) and siRNA-OGD (n=3 each). Cells were incubated in glucose-free and serum-free DMEM medium under the conditions of 37℃ and 1% O2+99% N2/CO2 for 6 hours to establish an in vitro hypoxic-ischemic model. Quantitative real-time PCR was used to measure mRNA expression of lncRNA BC088414, β2-adrenoceptor (Adrb2), and caspase-6 (CASP6). siRNAs were used to inhibit BC088414 expression in PC12 cells. The TUNEL method was used to measure cell apoptosis.

RESULTSThe OGD group had a significantly higher cell apoptotic index than the normoxic group (P<0.01). After inhibition of BC088414 expression, the OGD group had a significantly reduced apoptotic index (P<0.05). The OGD group had significantly higher mRNA expression levels of lncRNA BC088414, Adrb2, and CASP6 compared with the normoxic group (P<0.05). The siRNA -normoxic group had significantly lower mRNA expression levels of Adrb2 and CASP6 than the normoxic group (P<0.05), and the siRNA-OGD group also had significantly lower mRNA expression levels of Adrb2 and CASP6 than the OGD group (P<0.05).

CONCLUSIONSLncRNA BC088414 may promote apoptosis through Adrb2 and CASP6 and aggravate neural cell injury induced by hypoxia-ischemia.

Animals ; Apoptosis ; Caspase 6 ; genetics ; physiology ; Cell Hypoxia ; Neurons ; pathology ; PC12 Cells ; RNA, Long Noncoding ; physiology ; RNA, Messenger ; analysis ; Rats ; Receptors, Adrenergic, beta-2 ; genetics ; physiology

BACKGROUND AND PURPOSE: The purpose of this meta-analysis was to determine the precise association between beta-2 adrenergic receptor (beta2AR) polymorphism and Ischemic stroke. METHODS: Published case control studies on association between beta2AR and ischemic stroke were searched from electronic databases. Pooled Odds ratio and 95% Confidence interval were calculated by using software RevMan version 5.2. RESULTS: A total of three studies involving 1,642 cases and 1,673 controls, which were published from 2007 to 2014, were subjected to meta-analysis for allelic association and 518 cases and 510 controls for genotypic association. Pooled analysis of two studies for genotypic association suggested that subjects carrying Gln27Glu polymorphism of beta2AR had an increased risk for Ischemic stroke under recessive model (OR 2.09; 95% CI; 1.20 to 3.64) and under dominant model (OR 1.47; 95% CI 1.14 to 1.90). Pooled analysis of three studies for allelic association showed a significantly higher Glu27 allele of beta2AR in the patients with ischemic stroke (OR 1.58; 95% CI; 1.38 to 1.81). CONCLUSIONS: The present meta-analysis suggests that Gln27Glu polymorphism of beta2AR gene is associated with increased risk for ischemic stroke.
Alleles ; Case-Control Studies ; Cerebral Infarction ; Humans ; Odds Ratio ; Receptors, Adrenergic* ; Receptors, Adrenergic, beta-2 ; Stroke*

OBJECTIVETo investigate the significance of beta-adrenergic receptor 2 (beta2-AR) and vascular endothelial growth factor-2 (VEGFR-2) in the occurrence and development of infantile hemangioma through detecting the expression of beta2-AR and VEGFR-2 in the different stages of infantile hemangiomas.

METHODSAccording to the Mulliken's classification standard, we classified the specimens as proliferating group (32 cases), involuting group (17 cases) and involuted group (11 cases). Normal skin tissue surrounding the hemangioma from 7 cases were chosen as control group. The expression of beta2-AR and VEGFR-2 was detected by immunohistochemical technique in proliferating hemangioma, involuting hemangioma, involuted hemangioma. The mean optical density was measured by image analysis system (Image Pro Plus 6.0) and SPSS 16.0 software was applied for statistical analysis.

RESULTSThe expression of beta2-AR and VEGFR-2 was strongly positive in proliferating hemangioma, while positive in involuting hemangioma and weakly positive in the involuted stage. The mean optical density of each phase was 0.064 751 2 +/- 0.012 747, 0.031 6017 +/- 0.006 848,0.011 869 8 +/- 0.039 349 for beta2-AR, and 0.068 940 9 +/- 0.029 274, 0.028 445 5 +/- 0.006 396, 0.011 184 1 +/- 0.004 198 for VEGFR-2. The differences between different stages had a statistically significance (P < 0.05). Correlation analysis on the mean optical density between beta2-AR and VEGFR-2 had a statistically significance (P < 0. 05).

CONCLUSIONSBeta2-AR and VEGFR-2 may be involved in the occurrence and development of infantile hemangioma.

Child ; Child, Preschool ; Female ; Hemangioma ; metabolism ; Humans ; Infant ; Male ; Receptors, Adrenergic, beta-2 ; metabolism ; Receptors, Vascular Endothelial Growth Factor ; metabolism

BACKGROUNDSequence variants in the &beta;-adrenergic receptor (ADRB) genes have a close relationship with the development of coronary artery disease (CAD) and the patient's prognosis. However, there is a lack of data on the role of the variants in ADRBs genes in Han Chinese patients with CAD. We aimed to investigate the association of genetic variants in the ADRB1 and ADRB2 genes with the incidence of major adverse cardiac event (MACE) in Han Chinese patients with CAD.

METHODSA total of 545 Han Chinese patients with CAD undergoing percutaneous coronary intervention (PCI) were recruited to the study and followed for one year. Three variant sites in ADRB1 (rs1801253) and ADRB2 (rs1042713 and rs1042714) were genotyped. The effect of the ADRB1 and ADRB2 genotypes on MACE within one year was assessed.

RESULTSThere were 47 cases of MACE during follow-up. There was no significant difference in the incidence of MACE among patients carrying different genotypes of the three variants in ADRB1 and ADRB2 (Log-rank, all P > 0.05). Cox regression analysis showed no association between three variants in ADRB1 and ADRB2 genes and the incidence of MACE during one-year follow-up, the adjusted hazard ratios (95% confidence interval) for rs1801253, rs1042713 and rs1042714 were 1.05 (0.54-2.02), 1.24 (0.58-2.64) and 1.66 (0.81-3.42), respectively.

CONCLUSIONOur data did not support a relationship between the three polymorphisms of ADRB1 (rs1801253) and ADRB2 (rs1042713 and rs1042714) genes and risk of subsequent cardiovascular events after PCI in Han Chinese patients with CAD.

Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; Coronary Artery Disease ; genetics ; Female ; Genotype ; Humans ; Incidence ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Polymorphism, Single Nucleotide ; genetics ; Receptors, Adrenergic, beta ; genetics ; Receptors, Adrenergic, beta-1 ; genetics ; Receptors, Adrenergic, beta-2 ; genetics

Sustained activation of &beta; adrenergic receptor (&beta;AR) leads to pathologic cardiac hypertrophy. However, the related mechanisms still remain unclear. In this study, we observe how N-acetylcysteine (NAC) affects the oxidative stress and calcium/calmodulin-dependent protein kinase II (CaMKII) expression in heart of isoproterenol (ISO)-stimulated rats, and investigate whether oxidative stress and CaMKII contribute to the development of sustained &beta;AR-stimulated cardiac hypertrophy. Healthy male Wistar rats were randomly separated into 4 groups: control (CTRL), ISO-treated (ISO), control with NAC supplement (CTRL+NAC) and ISO-treated with NAC supplement (ISO+NAC) groups (6 rats in each group). Systolic blood pressure (SBP) was measured in awake rats with the tail-cuff method every week for two weeks. Heart weight/body weight ratio (HW/BW) and HE staining were used for the detection of myocardial hypertrophy. Myocardial mitochondrial reactive oxygen species (ROS) levels were measured by DCF fluorometry. The expressions of activated-CaMKII (p-CaMKII/CaMKII) and NADPH oxidase 4 (NOX(4)) were determined by Western blot analysis. The results showed that ISO-treated (i.p., daily 3 mg/kg, 2 weeks) rats developed an obvious cardiac hypertrophy as expressed by increases of HW/BW and myocyte cross-section area. Cardiac mitochondrial ROS level was significantly enhanced in ISO group as compared to CTRL group (P < 0.05). The expressions of NOX(4) and p-CaMKII in ISO group were also up-regulated as compared to CTRL group (1.4 and 1.6 times of CTRL, respectively, P < 0.05). NAC supplement significantly suppressed the hypertrophic development of heart in ISO-stimulated rats. The cardiac mitochondrial ROS level showed a significant decrease in rats of ISO+NAC group (P < 0.05 vs ISO). In accordance with this, ISO+NAC group rats also showed marked reductions in the expressions of NOX(4) and p-CaMKII/CaMKII compared to ISO group rats (P < 0.05). There were no significant differences of the detected indices between the rats from CTRL+NAC and CTRL groups. SBP showed no differences among four groups. These results suggest that both oxidative stress and CaMKII play important roles in sustained &beta;AR-stimulated cardiac hypertrophy. NAC may suppress ISO-induced cardiac hypertrophy by down-regulating the expression of activated-CaMKII, and by reducing the level of oxidative stress originated from mitochondria and NADPH oxidase pathways.
Acetylcysteine ; pharmacology ; Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cardiomegaly ; physiopathology ; Isoproterenol ; pharmacology ; Male ; Mitochondria, Heart ; metabolism ; Myocardium ; pathology ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Receptors, Adrenergic, beta ; metabolism

Aged ; Base Sequence ; Coronary Angiography ; Coronary Vasospasm ; genetics ; physiopathology ; DNA Primers ; Female ; Gene Frequency ; Heterotrimeric GTP-Binding Proteins ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Receptors, Adrenergic, beta-2 ; genetics