ABSTRACT
Intrahepatic cholangiocarcinoma (iCCA) is a highly fatal malignancy with rapidly increasing incidence and mortality worldwide. Currently, gemcitabine-based systemic chemotherapy is the main clinical therapeutic regimen; however, its efficacy is poor, and its mechanism has not been elucidated. In this study, we use a Seahorse Extracellular Flux analyser to measure glycolysis capacity (extracellular acidification rate, ECAR) and oxygen consumption rate (OCR). The glucose uptake or lactic acid content is detected, and the effects of saikosaponin D, an active compound derived from Bupleuri Radix (a traditional Chinese medicine for soothing the liver and relieving depression), on gemcitabine cytotoxicity in norepinephrine-stimulated iCCA cells are analysed. We find that adrenergic signaling plays a fundamental role in chronic stress-induced therapeutic resistance in iCCA. Norepinephrine (NE) and epinephrine (E) enhance the proliferation of iCCA cells and interfere with the response to gemcitabine through activation of the ß2-adrenergic receptor (ADRB2). Furthermore, we find that NE upregulates the expressions of several drug efflux-related genes (such as ABCG2 and MDR1) and promotes glycolysis in iCCA cells. In addition, saikosaponin D reverses the poor response of iCCA cells to gemcitabine by downregulating ADRB2 level. Furthermore, saikosaponin D inhibits drug efflux and glycolysis in iCCA cells by regulating the expressions of MDR1, ABCG2, HK2, and GLUT1. Collectively, saikosaponin D enhances the antitumor effect of gemcitabine by controlling glucose metabolism and drug efflux by inhibiting the ADRB2 signaling. Therefore, the combination of saikosaponin D and gemcitabine may be a potential therapeutic strategy for the treatment of iCCA.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Humans , Gemcitabine , Norepinephrine/therapeutic use , Cholangiocarcinoma/genetics , Epinephrine/pharmacology , Epinephrine/therapeutic use , Bile Ducts, Intrahepatic/pathology , Bile Duct Neoplasms/genetics , Glycolysis , Receptors, Adrenergic, beta-2/geneticsABSTRACT
Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an "ingredient-gene-path" network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P < 0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P < 0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P < 0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.
Subject(s)
Action Potentials/drug effects , Anti-Arrhythmia Agents/pharmacology , Arrhythmias, Cardiac/prevention & control , Drugs, Chinese Herbal/pharmacology , Heart Rate/drug effects , Myocardial Ischemia/drug therapy , Action Potentials/genetics , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , Disease Models, Animal , Gene Expression Regulation , Gene Regulatory Networks , Heart Rate/genetics , Male , Medicine, Chinese Traditional , Myocardial Ischemia/genetics , Myocardial Ischemia/metabolism , Myocardial Ischemia/physiopathology , NAV1.5 Voltage-Gated Sodium Channel/genetics , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Protein Interaction Maps , Receptor, Muscarinic M2/genetics , Receptor, Muscarinic M2/metabolism , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Swine , Swine, Miniature , Time FactorsABSTRACT
Chronic heart failure is the terminal stage of various cardiovascular diseases. Despite the availability of several classes of drugs, there is still an unmet need for effective treatment. Based on bench work during the past two decades, we have proposed that enhancement of ß 2-adrenergic receptor signaling in combination with the presently preferred ß 1-adrenergic receptor blockade would be a promising strategy. Chinese herbal medicines have been shown to be effective in the treatment of heart failure, although the mechanisms largely remain unknown. In the present study, we screened an herbal medicine compound/extract library for ß-adrenergic receptor ligands to determine the target of certain effective botanical remedies and seek a leading compound(s) for chronic heart failure treatment. Using a high-throughput screening assay, we identified higenamine, which has a long history in chronic heart failure treatment in traditional Chinese medicine, to be a potent ß-adrenergic receptor agonist. Further experiments using specific inhibitors showed that higenamine activated both ß 1-adrenergic receptor and ß 2-adrenergic receptor. Inhibition of its action by pertussis toxin (a Gi inhibitor) indicated that it is a ß 2-adrenergic receptor Gs/Gi dual agonist. Contractility experiments demonstrated a positive inotropic effect of higenamine. In conclusion, we found an herbal compound, higenamine, to be a dual agonist for ß 1/ß 2-adrenergic receptors with no preference in stimulating the Gs and Gi pathways in ß 2-adrenergic receptor signaling. Our results elucidated not only the target of higenamine to explain its pharmacological effect in treating chronic heart failure, but also the mechanisms of its cardiac toxicity.
Subject(s)
Adrenergic beta-1 Receptor Agonists/pharmacology , Adrenergic beta-2 Receptor Agonists/pharmacology , Alkaloids/pharmacology , Drugs, Chinese Herbal/chemistry , High-Throughput Screening Assays/methods , Tetrahydroisoquinolines/pharmacology , Alkaloids/chemistry , Animals , Cell Line , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/pharmacology , Humans , Molecular Docking Simulation , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Rats, Sprague-Dawley , Receptors, Adrenergic, beta-1/genetics , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Tetrahydroisoquinolines/chemistryABSTRACT
Preclinical studies have demonstrated that anti-galectin-3 (Gal-3) interventions are effective in attenuating cardiac remodeling, fibrosis, and dysfunction. We determined, in a transgenic (TG) mouse model of fibrotic cardiomyopathy, whether Gal-3 expression was elevated and whether Gal-3 played a critical role in disease development. We studied mice with fibrotic cardiomyopathy attributable to cardiac overexpression of human ß2-adrenoceptors (ß2-TG). Cardiac expression levels of Gal-3 and fibrotic or inflammatory genes were determined. The effect of Gal-3 inhibition in ß2-TG mice was studied by treatment with Gal-3 inhibitors ( N-acetyllactosamine and modified citrus pectin) or by deletion of Gal-3 through crossing ß2-TG and Gal-3 knockout mice. Changes in cardiomyopathy phenotypes were assessed by echocardiography and biochemical assays. In ß2-TG mice at 3, 6, and 9 mo of age, upregulation of Gal-3 expression was observed at mRNA (~6- to 15-fold) and protein (~4- to 8-fold) levels. Treatment of ß2-TG mice with N-acetyllactosamine (3 wk) or modified citrus pectin (3 mo) did not reverse cardiac fibrosis, inflammation, and cardiomyopathy. Similarly, Gal-3 gene deletion in ß2-TG mice aged 3 and 9 mo did not rescue the cardiomyopathy phenotype. In conclusion, the ß2-TG model of cardiomyopathy showed a robust upregulation of Gal-3 that correlated with disease severity, but Gal-3 inhibitors or Gal-3 gene deletion had no effect in halting myocardial fibrosis, remodeling, and dysfunction. Gal-3 may not be critical for cardiac fibrogenesis and remodeling in this cardiomyopathy model. NEW & NOTEWORTHY We showed a robust upregulation of cardiac galectin-3 (Gal-3) expression in a mouse model of cardiomyopathy attributable to cardiomyocyte-restricted transgenic activation of ß2-adrenoceptors. However, pharmacological and genetic inhibition of Gal-3 did not confer benefit in this model, implying that Gal-3 may not be a critical disease mediator of cardiac remodeling in this model.
Subject(s)
Cardiomyopathies/metabolism , Galectin 3/metabolism , Myocytes, Cardiac/metabolism , Receptors, Adrenergic, beta-2/metabolism , Ventricular Remodeling , Amino Sugars/pharmacology , Animals , Cardiomyopathies/etiology , Cardiomyopathies/genetics , Cardiomyopathies/physiopathology , Disease Models, Animal , Fibrosis , Galectin 3/antagonists & inhibitors , Galectin 3/deficiency , Galectin 3/genetics , Genetic Predisposition to Disease , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Pectins/pharmacology , Phenotype , Receptors, Adrenergic, beta-2/genetics , Severity of Illness Index , Up-Regulation , Ventricular Remodeling/drug effectsABSTRACT
Beige adipocytes emerge postnatally within the white adipose tissue in response to certain environmental cues, such as chronic cold exposure. Because of its highly recruitable nature and relevance to adult humans, beige adipocytes have gained much attention as an attractive cellular target for antiobesity therapy. However, molecular circuits that preferentially promote beige adipocyte biogenesis remain poorly understood. We report that a combination of mild cold exposure at 17°C and capsinoids, a nonpungent analog of capsaicin, synergistically and preferentially promotes beige adipocyte biogenesis and ameliorates diet-induced obesity. Gain- and loss-of-function studies show that the combination of capsinoids and cold exposure synergistically promotes beige adipocyte development through the ß2-adrenoceptor signaling pathway. This synergistic effect on beige adipocyte biogenesis occurs through an increased half-life of PRDM16, a dominant transcriptional regulator of brown/beige adipocyte development. We document a previously unappreciated molecular circuit that controls beige adipocyte biogenesis and suggest a plausible approach to increase whole-body energy expenditure by combining dietary components and environmental cues.
Subject(s)
Acclimatization , Adipocytes, Beige/physiology , Adipogenesis , Anti-Obesity Agents/therapeutic use , Capsaicin/analogs & derivatives , Dietary Supplements , Obesity/prevention & control , Adipocytes, Beige/cytology , Adipocytes, Beige/drug effects , Adipocytes, Beige/pathology , Adipogenesis/drug effects , Adrenergic beta-2 Receptor Agonists/pharmacology , Adrenergic beta-2 Receptor Agonists/therapeutic use , Adrenergic beta-2 Receptor Antagonists/pharmacology , Adrenergic beta-2 Receptor Antagonists/toxicity , Animals , Anti-Obesity Agents/agonists , Anti-Obesity Agents/antagonists & inhibitors , Capsaicin/agonists , Capsaicin/antagonists & inhibitors , Capsaicin/chemistry , Capsaicin/therapeutic use , Cells, Cultured , Cold Temperature , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Energy Metabolism/drug effects , Gene Expression Regulation/drug effects , Hydrogenation , Male , Mice, Inbred C57BL , Mice, Transgenic , Obesity/chemically induced , Obesity/metabolism , Obesity/pathology , Oxygen Consumption/drug effects , Protein Stability/drug effects , Random Allocation , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/drug effects , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Epilepsy or seizure disorder is among the least understood chronic medical conditions affecting over 65 million people worldwide. Here, we show that disruption of the polycystic kidney disease 2-like 1 (Pkd2l1 or Pkdl), encoding polycystin-L (PCL), a non-selective cation channel, increases neuronal excitability and the susceptibility to pentylenetetrazol-induced seizure in mice. PCL interacts with ß2-adrenergic receptor (ß2AR) and co-localizes with ß2AR on the primary cilia of neurons in the brain. Pkdl deficiency leads to the loss of ß2AR on neuronal cilia, which is accompanied with a remarkable reduction in cAMP levels in the central nervous system (CNS). The reduction of cAMP levels is associated with a reduction in the activation of cAMP response element-binding protein, but not the activation of Ca(2+)/calmodulin-dependent protein kinase II, Akt or mitogen-activated protein kinases. Our data, thus, indicate for the first time that a ciliary protein complex is required for the control of neuronal excitability in the CNS.
Subject(s)
Calcium Channels/genetics , Cerebral Cortex/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Epilepsy/genetics , Hippocampus/metabolism , Receptors, Adrenergic, beta-2/genetics , Receptors, Cell Surface/genetics , Thalamus/metabolism , Animals , Calcium Channels/deficiency , Cerebral Cortex/pathology , Cilia/metabolism , Cilia/pathology , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Disease Models, Animal , Disease Susceptibility , Epilepsy/chemically induced , Epilepsy/metabolism , Epilepsy/pathology , Excitatory Postsynaptic Potentials , Gene Deletion , Gene Expression Regulation, Developmental , Hippocampus/pathology , Humans , Ion Transport , Mice , Neurons/metabolism , Neurons/pathology , Pentylenetetrazole , Receptors, Adrenergic, beta-2/metabolism , Receptors, Cell Surface/deficiency , Signal Transduction , Thalamus/pathologyABSTRACT
Hypoglycemic effect is an efficient means to modulate elevated blood glucose levels in patients with diabetes. We found that the extract of lotus plumule (the germ of Nelumbo nucifera Gaertn. seed) showed potent glucose uptake enhancement activity against L6 myotubes, which results in a hypoglycemic effect. This activity was further investigated, and an active constituent was identified as a single bioactive compound, higenamine 4'-O-ß-d-glucoside. Mechanistic studies employing phosphatidylinositol 3-kinase (PI3K) inhibitor, AMP-activated protein kinase (AMPK) inhibitor, or adrenergic receptor antagonist showed that the compound induced its activity through ß2-adrenergic receptor. Patients with type II diabetes mellitus frequently develop insulin resistance. Owing to the differences between the mechanism of action of insulin and of the isolated compound, the compound or lotus plumule itself may have the possibility of modulating blood glucose levels in insulin-resistant patients effectively.
Subject(s)
Adrenergic beta-Agonists/pharmacology , Alkaloids/chemistry , Glucose/metabolism , Glucosides/pharmacology , Hypoglycemic Agents/pharmacology , Nelumbo/chemistry , Receptors, Adrenergic, beta-2/metabolism , Tetrahydroisoquinolines/chemistry , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Adrenergic Antagonists/pharmacology , Adrenergic beta-Agonists/chemistry , Adrenergic beta-Agonists/isolation & purification , Animals , Cell Line , Chromones/pharmacology , Gene Expression Regulation , Glucosides/chemistry , Glucosides/isolation & purification , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/isolation & purification , Mice , Morpholines/pharmacology , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Plant Extracts/chemistry , Propranolol/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Adrenergic, beta-2/genetics , Seeds/chemistry , Signal TransductionABSTRACT
The family of G protein-coupled receptors (GPCRs) remains a central focus of basic pharmacology and drug discovery efforts. Convenient methods to assess the efficacy of potentially therapeutic reagents for GPCRs are strongly required for high-throughput screening (HTS) assay. We recently developed a rapid, sensitive, and quantitative method for detecting potential chemicals that act on GPCRs using split luciferase complementation. In principle, this is based on the detection of interactions of GPCR with ß-arrestin, which translocates to the activated GPCRs. This method can facilitate the construction of HTS systems in a multi-well plate format. Particularly, the method is compatible with single-cell imaging and animal models and even deeper tissues such as organs, because of its high sensitivity, suggesting that promising candidates from HTS assay can be moved easily to the next phase for additional analysis. This system can contribute to the effective evaluation of potentially therapeutic reagents and expedite the development of new drugs for GPCRs.
Subject(s)
Arrestins/metabolism , High-Throughput Screening Assays , Receptors, Adrenergic, beta-2/metabolism , Receptors, Somatostatin/metabolism , Recombinant Fusion Proteins/metabolism , Single-Cell Analysis/methods , Adrenergic beta-2 Receptor Agonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Animals , Arrestins/genetics , Drug Discovery , Drug Evaluation, Preclinical , Female , Gene Expression , Genes, Reporter , HEK293 Cells , Humans , Isoproterenol/pharmacology , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements , Mice, Inbred BALB C , Plasmids/chemistry , Plasmids/metabolism , Propranolol/pharmacology , Receptors, Adrenergic, beta-2/genetics , Receptors, Somatostatin/agonists , Receptors, Somatostatin/genetics , Recombinant Fusion Proteins/genetics , Somatostatin/pharmacology , Transfection , beta-ArrestinsABSTRACT
ß-Adrenergic agonists (ß-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of ß-agonists, a ß2-adrenergic receptor (ß2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant ß2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of ß-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized ß2-AR proteins by ß-agonists. The IC50 and limit of detection values for ractopamine were 30.38µgL(-1) and 5.20µgL(-1), respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of ß-agonists in animal feeds.
Subject(s)
Adrenergic beta-Agonists/analysis , Biosensing Techniques/methods , DNA Restriction Enzymes/metabolism , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Baculoviridae/genetics , Cloning, Molecular , Drug Evaluation, Preclinical , Mesocricetus , Receptors, Adrenergic, beta-2/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sf9 Cells , SpodopteraABSTRACT
OBJECTIVE: To observe the cardioprotective effect of electroacupuncture (EA) stimulation at "Neiguan" (PC 6) in mice with myocardial ischemia (MI) and to explore the involvement of cardiac beta-adrenergic receptors (beta-AR) in the cardioprotective effect of EA intervention. METHODS: Adult male C 57 BL/6 mice of wild type and beta1/beta2-AR double-knockout (beta1/beta2-AR(-/-)) mice were randomly and respectively divided into control group and EA group, with 8 mice in each group. These mice were subject to procedures of basic control, fatigue swimming and fatigue swimming + EA. The model was established by fatigue swimming for inducing acute MI. EA (2 Hz, 0.5 mA) was applied to bilateral "Neiguan" (PC 6) for 30 min, once daily for 7 days in both EA groups. The ST-segment of electrocardiogram (ECG) of standard limb lead II, heart rate were recorded by using a biophysical amplifier and the arrhythmia score was assessed according to Curtis and Walker's methods (1988). RESULTS: Self-comparison showed that, compared with the baseline, the amplitude of ECG-ST II segment was obviously increased in swimming fatigue C 57 BL/6 mice (P < 0.01) and further obviously increased in beta1/beta2-AR(-/-) mice (P < 0.01), suggesting an acute MI in C 57 BL/6 mice and a worsened MI in beta1/beta2-AR(-/-) mice. Simultaneously, the heart rate was markedly decreased (P < 0.05), and the score of arrhythmia obviously increased in both C 57 BL/6 and beta1/beta2-AR(-/-) mice (P < 0.05). Compared with the modeling procedures, ECG-ST II amplitude and arrhythmia score were significantly decreased in C 57 BL/6 mice of the EA group (P < 0.01, P < 0.05), rather than in the beta1/beta2-AR(-/-) mice of the EA group (P > 0.05). In addition, heart rate levels of both C 57 BL/6 mice and beta1/beta2-AR(-/-) mice had no significant differences between control and EA groups (P > 0.05). CONCLUSION: EA at "Neiguan" (PC 6) can protect the heart from swimming fatigue-induced MI in C 57 BL/6 mice not in beta1/beta2-AR(-/-) mice, suggesting an involvement of beta1/beta2-AR in the protective effect of EA.
Subject(s)
Electroacupuncture , Fatigue/complications , Myocardial Ischemia/therapy , Receptors, Adrenergic, beta-1/metabolism , Acupuncture Points , Animals , Fatigue/genetics , Fatigue/metabolism , Heart Rate , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardial Ischemia/etiology , Myocardial Ischemia/metabolism , Myocardial Ischemia/physiopathology , Receptors, Adrenergic, beta-1/genetics , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , SwimmingABSTRACT
The present review discusses and summarizes the up-to-date body of knowledge concerning human nutrigenomic studies with Mediterranean diet (MedDiet) and olive oil (OO) interventions, at real-life doses and conditions. A literature review was carried out until March 2012. Original articles assessing the nutrigenomic effect of the MedDiet and its main source of fat, OO, on gene expression were selected. State-of-the-art data in this field, although scarce, are promising. Despite a great diversity among studies, the attributed health benefits of the MedDiet and its components, such as OO, could be explained by a transcriptomic effect on atherosclerosis, inflammation, and oxidative stress-related genes (i.e. ADRB2, IL7R, IFNγ, MCP1, TNFα). Gene expression changes toward a protective mode were often associated with an improvement in systemic markers for oxidation and inflammation. The suggested underlying molecular pathways responsible for these changes, and the extent to which evidence exists of a MedDiet and OO nutrigenomic effect, are also discussed.
Subject(s)
Diet, Mediterranean , Health Promotion , Plant Oils/administration & dosage , Transcriptome , Atherosclerosis/genetics , Atherosclerosis/prevention & control , Biomarkers/blood , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Humans , Inflammation/genetics , Inflammation/prevention & control , Interferon-gamma/genetics , Nutrigenomics , Olive Oil , Oxidation-Reduction , Oxidative Stress , Polyphenols/administration & dosage , Polyphenols/analysis , Randomized Controlled Trials as Topic , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Receptors, Interleukin-7/genetics , Receptors, Interleukin-7/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Allergy and asthma are complex diseases influenced by many genes and molecular mechanisms. Recently a number of genome-wide association studies (GWAS) have investigated asthma- and allergy-related phenotypes. Results suggest the existence of sub phenotypes of asthma and document a need to better define the disease. Genetics may also help to identify groups of patients susceptible for specific forms of treatment and those at risk for adverse effects of therapy. Thus, genetics may represent a key tool to achieve individualised medicine in asthma and allergy in the future.
Subject(s)
Asthma/genetics , Dermatitis, Atopic/genetics , Rhinitis, Allergic, Perennial/genetics , Adrenal Cortex Hormones/genetics , Asthma/epidemiology , Asthma/therapy , Biological Therapy , Dermatitis, Atopic/epidemiology , Genome-Wide Association Study , Humans , Molecular Epidemiology , Pharmacogenetics , Phenotype , Receptors, Adrenergic, beta-2/genetics , Rhinitis, Allergic, Perennial/epidemiologyABSTRACT
Diabetes mellitus is a heterogeneous disease and nutritional therapy forms a necessary dimension for its long-term management. Traditional medicinal plants and vitamins are the potentially useful natural products for diabetes control. Diabetes causes atrophy and wasting of skeletal muscles resulting in major peripheral damage. The current study was designed to investigate the therapeutic effect of vitamin D3 and curcumin treatment on ß2-adrenoceptors, transcription factor CREB, insulin receptors, protein kinase B (Akt) and malate dehydrogenase activity in the skeletal muscle of diabetic rats. Radioreceptor binding assay was done for ß2-adrenoceptors using specific ligand, [³H] propranolol and gene expression studies of ß2-adrenoceptors, transcription factor CREB, insulin receptors and Akt were also done using specific probes. The results of the ß2-adrenoceptor assay showed significant increase in binding parameters, receptor number (B(max)) and equilibrium dissociation constant (K(d)) in the diabetic group in comparison to control. Similarly, an up regulation of ß2-adrenoceptor and CREB gene expression was observed in the diabetic group whereas the insulin receptor expression was down regulated which signifies the increased glycogenolysis, gluconeogenesis and decreased glycogenesis in the muscles. Expression of Akt was found to be up regulated in the diabetic group. Malate dehydrogenase activity was significantly decreased in both cytosolic and mitochondrial fractions of the diabetic group. All these molecular aspects were reversed to near control with vitamin D3 and curcumin treatment. Our results suggest the rising potential of both vitamin D3 and curcumin in the management of peripheral complications associated with diabetes.
Subject(s)
Cholecalciferol/pharmacology , Curcumin/pharmacology , Diabetes Mellitus, Experimental/metabolism , Muscle, Skeletal/metabolism , Receptor, Insulin/genetics , Receptors, Adrenergic, beta-2/genetics , Animals , Blood Glucose/analysis , Cyclic AMP Response Element-Binding Protein/genetics , Diabetes Mellitus, Experimental/blood , Gene Expression Regulation/drug effects , Insulin/blood , Malate Dehydrogenase/metabolism , Male , Muscle, Skeletal/drug effects , Proto-Oncogene Proteins c-akt/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , StreptozocinABSTRACT
Regulation of CaV1.2 channels in cardiac myocytes by the ß-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the ß-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.
Subject(s)
A Kinase Anchor Proteins/metabolism , Brain/cytology , Calcium Channels, L-Type/metabolism , Neurons/metabolism , A Kinase Anchor Proteins/genetics , Animals , Calcium Channels, L-Type/genetics , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Electrophoresis, Polyacrylamide Gel , Electrophysiology , Hippocampus/cytology , Immunoblotting , Immunohistochemistry , Immunoprecipitation , Mice , Mice, Mutant Strains , Phosphorylation , Protein Binding , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
AIM: To investigate the efficacy of glycyrrhizin (GL) combined with salbutamol (SA) as an anti-asthma therapy. METHODS: Rat lung beta2-adrenergic receptor (beta(2)-AR) mRNA level was measured by real-time RT PCR. Intracellular cAMP accumulation was evaluated with a reporter gene assay. An in vitro acetylcholine-induced guinea pig tracheal strip contraction model was used to test the relaxing effects of GL and SA. The anti-inflammatory effects of GL and SA were tested using tumor necrosis factor-alpha-induced NF-kappaB transcriptional activation reporter assay, I-kappaB Western blotting and interleukin-8 ELISA. An in vivo guinea pig asthma model was used to prove further the synergistic effect of GL and SA. RESULTS: GL (0.3 micromol/L) increased mRNA levels of beta(2)-AR in vivo and the accumulation of cAMP in vitro. The combination of GL and SA also resulted in significant complementary anti-inflammatory effects via inhibition of NF-kappaB activation, degradation of I-kappaB and production of interleukin-8. A significant synergistic effect of the combination was detected both in vitro and in vivo in a guinea pig mode. CONCLUSION: The results demonstrate that GL and SA have synergistic anti-asthmatic effects and offer the possibility of a therapeutic application of GL in combination with beta(2)-AR agonists in the treatment of asthma.
Subject(s)
Adrenergic beta-Agonists/therapeutic use , Albuterol/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Glycyrrhizic Acid/therapeutic use , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-2 Receptor Agonists , Adrenergic beta-Agonists/pharmacology , Albuterol/pharmacology , Animals , Anti-Asthmatic Agents/pharmacology , Cell Line , Cyclic AMP/metabolism , Drug Synergism , Glycyrrhizic Acid/pharmacology , Guinea Pigs , Humans , Lung/drug effects , Lung/metabolism , Male , NF-kappa B/immunology , RNA, Messenger/genetics , Rats , Receptors, Adrenergic, beta-2/genetics , Trachea/drug effects , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Hederacoside C, alpha-hederin, and hederagenin are saponins of dry extracts obtained from the leaves of ivy (Hedera helix L.). Internalization of beta(2)-adrenergic receptor-GFP fusion proteins after stimulation with 1 microM terbutaline was inhibited by preincubation of stably transfected HEK293 cells with 1 microM alpha-hederin for 24 h, whereas neither hederacoside C nor hederagenin (1 microM each) influenced this receptor regulation. After incubation of A549 cells with 5 nM Alexa532-NA, two different diffusion time constants were found for beta(2)AR-Alexa532-NA complexes by fluorescence correlation spectroscopy. Evaluation of the autocorrelation curve revealed diffusion time constants: tau(bound1) = 1.4 +/- 1.1 ms (n = 6) found for receptor-ligand complexes with unrestricted lateral mobility, and tau(bound2) = 34.7 +/- 14.1 ms (n = 6) for receptor-ligand complexes with hindered mobility. The distribution of diffusion time constants was 24.3 +/- 2.5% for tau(bound1) and 8.7 +/- 4.3% for tau(bound2) (n = 6). A549 cells pretreated with 1 microM alpha-hederin for 24 h showed dose-dependent alterations in this distribution with 37.1 +/- 5.5% for tau(bound1) and 4.1 +/- 1.1% for tau(bound2). Simultaneously, the level of Alexa532-NA binding was significantly increased from 33.0 +/- 6.8 to 41.2 +/- 4.6%. In saturation experiments, alpha-hederin did not influence the beta(2)-adrenergic receptor density (B(max)), whereas the K(D) value for Alexa532-NA binding decreased from 36.1 +/- 9.2 to 24.3 +/- 11.1 nM. Pretreatment of HASM cells with alpha-hederin (1 microM, 24 h) revealed an increased intracellular cAMP level of 13.5 +/- 7.0% under stimulating conditions. Remarkably, structure-related saponins like hederacoside C and hederagenin did not influence either the binding behavior of beta(2)AR or the intracellular cAMP level.
Subject(s)
Hedera , Oleanolic Acid/analogs & derivatives , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/metabolism , Saponins/chemistry , Thermodynamics , Cell Line , Cell Line, Tumor , Endocytosis , Humans , Oleanolic Acid/chemistry , Oleanolic Acid/physiology , Protein Binding , Receptors, Adrenergic, beta-2/geneticsABSTRACT
Pharmacological modulators of beta-adrenoceptors can influence bone mineral density and fracture risk in humans. Studies reported that beta-adrenoceptor ligands stimulate bone resorption by enhancing the expression of RANK-L, whereas the mechanisms by which beta-adrenoreceptors regulate bone formation are poorly understood. Here we show that beta2-adrenoceptor is predominantly expressed by bone cells, although low levels of beta1- and beta3-adrenoceptors were detectable. Noradrenaline and the selective beta2-adrenoceptor agonists isoprenaline and salmeterol stimulated osteoclast formation and bone resorption in BM osteoblast co-cultures and increased expression of RANK-L by osteoblasts. All three ligands enhanced RANK-L induced osteoclast formation and increased osteoclast multinuclearity. There was no significant effect of noradrenaline or isoprenaline on osteoblast growth, differentiation or function. These findings confirm the importance of the sympathetic nervous system in the regulation of bone mass, and demonstrate that pharmacological agonists of beta2-adrenoceptors directly and indirectly stimulate osteoclast formation, but have no direct effect on osteoblast growth, differentiation or function.
Subject(s)
Osteoclasts/cytology , Receptors, Adrenergic, beta-2/physiology , Animals , Animals, Newborn , Bone Development/physiology , Bone Marrow Cells/cytology , Calcitriol/pharmacology , Cell Culture Techniques , Cell Differentiation/drug effects , Coculture Techniques , DNA, Complementary/genetics , Gene Expression , Isoproterenol/pharmacology , Mice , Norepinephrine/pharmacology , Osteoclasts/drug effects , Polymerase Chain Reaction , RANK Ligand/genetics , RANK Ligand/physiology , RNA/genetics , Receptors, Adrenergic, beta-2/drug effects , Receptors, Adrenergic, beta-2/geneticsABSTRACT
Bronchial asthma is a chronic inflammatory disorder of the airways, in which inflammation causes bronchial hyper-responsiveness and flow limitation in the presence of various stimuli. Pulmonary function in asthmatic patients frequently deteriorates between midnight and early morning, which has suggested a role for chronotherapy. Although relationships between bronchial asthma and the function of clock genes remain unclear, some medications given for asthma such as glucocorticoids or beta(2)-adrenoceptor agonists may influence clock genes in vivo. In our studies of clock gene mRNA expressions in human bronchial epithelial cells in vitro and peripheral blood cells in vivo, we demonstrated that glucocorticoid or beta(2)-adrenoceptor agonist treatment strongly induced human Per1 mRNA expression both in vitro and in vivo. Human peripheral blood cells provide a useful indication of peripheral clock gene mRNA expression in vivo.
Subject(s)
Asthma/genetics , Asthma/physiopathology , Biological Clocks/genetics , Biological Clocks/physiology , Central Nervous System/physiology , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Animals , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/physiologyABSTRACT
Association and linkage studies of beta2-adrenergic receptor (beta2-ADR) polymorphisms in relation to the expression of asthmatic phenotypes and immune regulatory mechanisms have shown inconsistent results. In order to analyse the relevance of particular combinations of single nucleotide polymorphisms (SNPs) or haplotypes of beta2-ADR gene to bronchial asthma, bronchodilator response and total immunoglobulin E (IgE) we determined by direct DNA sequencing five SNPs (in positions: -47, -20, 46, 79, 252) in a group of 180 Caucasian subjects (110 patients with grass allergy and 70 nonatopic controls). The eight different beta2-ADR haplotypes were identified, with three the most common of them representing 92% of the studied cohort. Significantly higher (pcor = 0.0045) bronchodilator response was observed in patients with homozygotic genotype 46A/A in comparison with respective homo- and hetero-zygotes. There was no significant difference in bronchodilator response when beta2-ADR haplotypes were analysed. Significantly higher (pcor = 0.0005) total IgE levels were found in patients with beta2-ADR haplotype -47T/-20T/46A/79C/252G and homozygotic carriers of 46A (pcor = 0.0015) and 79C (pcor = 0.003) genotypes. No significant associations were found in regards to asthmatic phenotype and atopy. These results indicate that depending on phenotype studied, either an individual beta2-ADR SNP or beta2-ADR haplotype might affect disease manifestation.
Subject(s)
Hypersensitivity/etiology , Hypersensitivity/genetics , Poaceae/adverse effects , Receptors, Adrenergic, beta-2/genetics , Adult , Bronchospirometry , Female , Haplotypes , Humans , Hypersensitivity/blood , Hypersensitivity/diagnosis , Immunoglobulin E/blood , Male , Poland , Pollen/adverse effects , Polymorphism, Single NucleotideABSTRACT
OBJECTIVE: Chronic mechanical unloading induces left ventricular (LV) atrophy, which may impair functional recovery during support with an LV-assist device. Clenbuterol, a beta2-adrenergic receptor (AR) agonist, is known to induce myocardial hypertrophy and might prevent LV atrophy during LV unloading. Furthermore, beta2-AR stimulation is reported to improve Ca2+ handling and contribute to antiapoptosis. However, there is little information on the effects of clenbuterol during LV unloading. METHODS AND RESULTS: We investigated LV atrophy and function after LV unloading produced by heterotopic heart transplantation in isogenic rats. After transplantation, rats were randomized to 1 of 2 groups (n=10 each). The clenbuterol group received 2 mg.kg(-1).d(-1) of the drug for 2 weeks; the control group received normal saline. The weight of unloaded control hearts was 48% less than that of host hearts after 2 weeks of unloading. Clenbuterol significantly increased the weight of the host hearts but did not prevent unloading-induced LV atrophy. Papillary muscles were isolated and stimulated, and there was no difference in developed tension between the 2 groups. However, the inotropic response to the beta-AR agonist isoproterenol significantly improved in the clenbuterol group. The mRNA expression of myocardial sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) and fetal gene shift (myosin heavy chain [MHC] mRNA isozyme) was also significantly improved by clenbuterol treatment. There was no difference in beta1-AR mRNA expression between the 2 groups. In contrast, beta2-AR mRNA was significantly decreased in the clenbuterol-treated, unloaded heart. This indicates that clenbuterol may downregulate beta2-ARs. In the evaluation of apoptosis, mRNA expression of caspase-3, which is the central pathway for apoptosis, tended to be better in the clenbuterol group. CONCLUSIONS: During complete LV unloading, clenbuterol did not prevent myocardial atrophy but improved gene expression (SERCA2a, beta-MHC) and beta-adrenergic responsiveness and potentially prevented myocardial apoptosis. However, chronic administration of clenbuterol may be associated with downregulation of beta2-ARs.