1.Expression of adenosine receptors in human retinal pigment epithelium cells in vitro.
Wen-juan WAN ; Dong-mei CUI ; Xiao YANG ; Jian-min HU ; Chuan-xu LI ; Shou-long HU ; Klaus TRIER ; Jun-wen ZENG
Chinese Medical Journal 2011;124(8):1139-1144
BACKGROUNDAdenosine receptors (ADORs) have been reported to play a role in experimental myopia. This study aimed to determine the distribution of ADORs in human retinal pigment epithelium (RPE) cells cultured in vitro.
METHODSHuman RPE cells (cell line D407) were cultured in vitro. ADOR mRNA in RPE was detected by reverse transcription polymerase chain reaction. ADOR protein expression in RPE was confirmed by Western blotting analysis of cell lysates. Confocal fluorescence microscopy was used to study the subcellular distribution of ADORs.
RESULTSAll four subtypes of ADORs mRNA and protein were expressed in human RPE. This was confirmed by Western blotting analysis. The ADOR subtypes were differently distributed within the cells. ADORA1 was expressed in nucleus, perinucleus and cytoplasm of RPE. ADORA2A was concentrated mainly in one side of the perinucleus and cytoplasm of RPE. ADORA2B was strongly expressed in the nucleus, perinucleus and the cytoplasm, and ADORA3 was expressed weakly in the cytoplasm of RPE.
CONCLUSIONSADORs are expressed in human RPE. The different distribution at the subcellular level suggests different functions of ADOR subtypes.
Blotting, Western ; Cell Line ; Fluorescent Antibody Technique, Indirect ; Humans ; Receptors, Purinergic P1 ; genetics ; metabolism ; Retinal Pigment Epithelium ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction
2.In vitro expression and analysis of the 826 human G protein-coupled receptors.
Xuechen LV ; Junlin LIU ; Qiaoyun SHI ; Qiwen TAN ; Dong WU ; John J SKINNER ; Angela L WALKER ; Lixia ZHAO ; Xiangxiang GU ; Na CHEN ; Lu XUE ; Pei SI ; Lu ZHANG ; Zeshi WANG ; Vsevolod KATRITCH ; Zhi-Jie LIU ; Raymond C STEVENS
Protein & Cell 2016;7(5):325-337
G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.
Animals
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Computational Biology
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Crystallography, X-Ray
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Gene Expression
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Humans
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Plasmids
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genetics
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metabolism
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Protein Domains
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Receptors, Adrenergic, beta-1
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Receptors, G-Protein-Coupled
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classification
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genetics
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metabolism
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Receptors, Odorant
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metabolism
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Receptors, Purinergic P1
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genetics
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metabolism
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Sf9 Cells
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Spodoptera
3.Conditioning-induced cardioprotection: Aging as a confounding factor.
Puneet Kaur RANDHAWA ; Anjana BALI ; Jasleen Kaur VIRDI ; Amteshwar Singh JAGGI
The Korean Journal of Physiology and Pharmacology 2018;22(5):467-479
The aging process induces a plethora of changes in the body including alterations in hormonal regulation and metabolism in various organs including the heart. Aging is associated with marked increase in the vulnerability of the heart to ischemia-reperfusion injury. Furthermore, it significantly hampers the development of adaptive response to various forms of conditioning stimuli (pre/post/remote conditioning). Aging significantly impairs the activation of signaling pathways that mediate preconditioning-induced cardioprotection. It possibly impairs the uptake and release of adenosine, decreases the number of adenosine transporter sites and down-regulates the transcription of adenosine receptors in the myocardium to attenuate adenosine-mediated cardioprotection. Furthermore, aging decreases the expression of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) and subsequent transcription of catalase enzyme which subsequently increases the oxidative stress and decreases the responsiveness to preconditioning stimuli in the senescent diabetic hearts. In addition, in the aged rat hearts, the conditioning stimulus fails to phosphorylate Akt kinase that is required for mediating cardioprotective signaling in the heart. Moreover, aging increases the concentration of Na⁺ and K⁺, connexin expression and caveolin abundance in the myocardium and increases the susceptibility to ischemia-reperfusion injury. In addition, aging also reduces the responsiveness to conditioning stimuli possibly due to reduced kinase signaling and reduced STAT-3 phosphorylation. However, aging is associated with an increase in MKP-1 phosphorylation, which dephosphorylates (deactivates) mitogen activated protein kinase that is involved in cardioprotective signaling. The present review describes aging as one of the major confounding factors in attenuating remote ischemic preconditioning-induced cardioprotection along with the possible mechanisms.
Adenosine
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Aging*
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Animals
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Catalase
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Heart
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Metabolism
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Myocardium
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Negotiating
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Oxidative Stress
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Phosphorylation
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Phosphotransferases
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PPAR gamma
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Protein Kinases
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Rats
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Receptors, Purinergic P1
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Reperfusion Injury
4.Regulation of epithelial sodium channel α-subunit expression by adenosine receptor A₂a in alveolar epithelial cells.
Wang DENG ; Dao-Xin WANG ; Wei ZHANG ; Chang-Yi LI
Chinese Medical Journal 2011;124(10):1551-1555
BACKGROUNDThe amiloride-sensitive epithelial sodium channel α-subunit (α-ENaC) is an important factor for alveolar fluid clearance during acute lung injury. The relationship between adenosine receptor A(2a) (A(2a)AR) expressed in alveolar epithelial cells and α-ENaC is poorly understood. We targeted the A(2a)AR in this study to investigate its role in the expression of α-ENaC and in acute lung injury.
METHODSA549 cells were incubated with different concentrations of A(2a)AR agonist CGS-21680 and with 100 µmol/L CGS-21680 for various times. Rats were treated with lipopolysaccharide (LPS) after CGS-21680 was injected. Animals were sacrificed and tissue was harvested for evaluation of lung injury by analysis of the lung wet-to-dry weight ratio, lung permeability and myeloperoxidase activity. RT-PCR and Western blotting were used to determine the mRNA and protein expression levels of α-ENaC in A549 cells and alveolar type II epithelial cells.
RESULTSBoth mRNA and protein levels of α-ENaC were markedly higher from 4 hours to 24 hours after exposure to 100 µmol/L CGS-21680. There were significant changes from 0.1 µmol/L to 100 µmol/L CGS-21680, with a positive correlation between increased concentrations of CGS-21680 and expression of α-ENaC. Treatment with CGS-21680 during LPS induced lung injury protected the lung and promoted α-ENaC expression in the alveolar epithelial cells.
CONCLUSIONActivation of A(2a)AR has a protective effect during the lung injury, which may be beneficial to the prognosis of acute lung injury.
Acute Lung Injury ; metabolism ; Adenosine ; analogs & derivatives ; pharmacology ; Animals ; Blotting, Western ; Cell Line ; Epithelial Sodium Channels ; genetics ; metabolism ; Humans ; Male ; Phenethylamines ; pharmacology ; Pulmonary Alveoli ; cytology ; metabolism ; Purinergic P1 Receptor Agonists ; pharmacology ; Rats ; Receptors, Purinergic P1 ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction
5.Roles of Adenosine and Serotonin Receptors on the Antinociception of Sildenafil in the Spinal Cord of Rats.
Hyung Gon LEE ; Woong Mo KIM ; Cheon Hee PARK ; Myung Ha YOON
Yonsei Medical Journal 2010;51(6):960-964
PURPOSE: The phosphodiesterase 5 inhibitor sildenafil has antinociceptive effects, mediated by an increase in cGMP. This study examined the role of spinal adenosine and serotonin receptors played in the antinociceptive effects of intrathecal sildenafil. MATERIALS AND METHODS: Intrathecal catheters were inserted into the subarachnoid space of Sprague-Dawley male rats as a drug delivery device. Pain was induced by injecting formalin into the plantar surface of rats and observing nociceptive behavior (flinching response) for 60 mininutes. Then, the effects of intrathecal adenosine and serotonin receptor antagonists on the antinociceptive activity of intrathecal sildenafil were examined. RESULTS: Intrathecal sildenafil suppressed the flinching response in a dose-dependent manner during phases 1 and 2 in the formalin test. Both CGS 15943 and dihydroergocristine decreased the antinociceptive effects of sildenafil during phases 1 and 2 in the formalin test. CONCLUSION: Intrathecal sildenafil effectively attenuated the pain evoked by formalin injection. Both adenosine and serotonin receptors may be involved in the antinociceptive action of sildenafil at the spinal level.
Adenosine/metabolism
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Analgesics/*therapeutic use
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Animals
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Cyclic GMP/metabolism
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Dihydroergocristine/pharmacology
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Injections, Spinal
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Male
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Pain/*drug therapy
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Piperazines/*pharmacology
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Purines/pharmacology
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Rats
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Rats, Sprague-Dawley
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Receptors, Purinergic P1/*metabolism
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Receptors, Serotonin/*metabolism
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Spinal Cord/*metabolism
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Sulfones/*pharmacology
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Vasodilator Agents/therapeutic use