RESUMEN
Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Transducción de Señal/fisiología , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Factor 4 Similar a Kruppel , Óxido Nítrico Sintasa de Tipo III/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Estrés MecánicoRESUMEN
AIMS/HYPOTHESES: To investigate the effects of extracellular purines on insulin secretion from mouse pancreatic islets. METHODS: Mouse islets and beta cells were isolated and examined with mRNA real-time quantification, cAMP quantification and insulin and glucagon secretion. ATP release was measured in MIN6c4 cells. Insulin and glucagon secretion were measured in vivo after glucose injection. RESULTS: Enzymatic removal of extracellular ATP at low glucose levels increased the secretion of both insulin and glucagon, while at high glucose levels insulin secretion was reduced and glucagon secretion was stimulated, indicating an autocrine effect of purines. In MIN6c4 cells it was shown that glucose does induce release of ATP into the extracellular space. Quantitative real-time PCR demonstrated the expression of the ADP receptors P2Y(1) and P2Y(13) in both intact mouse pancreatic islets and isolated beta cells. The stable ADP analogue 2-MeSADP had no effect on insulin secretion. However, co-incubation with the P2Y(1) antagonist MRS2179 inhibited insulin secretion, while co-incubation with the P2Y(13) antagonist MRS2211 stimulated insulin secretion, indicating that ADP acting via P2Y(1) stimulates insulin secretion, while signalling via P2Y(13) inhibits the secretion of insulin. P2Y(13) antagonism through MRS2211 per se increased the secretion of both insulin and glucagon at intermediate (8.3 mmol/l) and high (20 mmol/l) glucose levels, confirming an autocrine role for ADP. Administration of MRS2211 during glucose injection in vivo resulted in both increased secretion of insulin and reduced glucose levels. CONCLUSIONS/INTERPRETATION: In conclusion, ADP acting on the P2Y(13) receptors inhibits insulin release. An antagonist to P2Y(13) increases insulin release and could be evaluated for the treatment of diabetes.
Asunto(s)
Adenosina Difosfato/metabolismo , Insulina/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Animales , Apirasa/metabolismo , Línea Celular , Células Cultivadas , AMP Cíclico/metabolismo , Femenino , Glucagón/metabolismo , Técnicas In Vitro , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Reacción en Cadena de la Polimerasa , Receptores Purinérgicos P2Y1 , Tionucleótidos/farmacologíaRESUMEN
Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.
Asunto(s)
Secuencia de Bases , Encéfalo/fisiología , ADN/genética , Biblioteca de Genes , Proyecto Genoma Humano , Secuencia de Aminoácidos , Automatización , Mapeo Cromosómico , Expresión Génica , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido NucleicoRESUMEN
Continuous cardiac monitoring is usually not performed during hemodialysis treatment, although a majority of patients with kidney failure suffer from cardiovascular disease. In the present paper, a method is proposed for estimating a cardiac pressure signal by combining the arterial and the venous pressure sensor signals of the hemodialysis machine. The estimation is complicated by the periodic pressure disturbance caused by the peristaltic blood pump, with an amplitude much larger than that of the cardiac pressure signal. Using different techniques for combining the arterial and venous pressure signals, the performance is evaluated and compared to that of an earlier method which made use of the venous pressure only. The heart rate and the heartbeat occurrence times, determined from the estimated cardiac pressure signal, are compared to the corresponding quantities determined from a photoplethysmographic reference signal. Signals from 9 complete hemodialysis treatments were analyzed. For a heartbeat amplitude of 0.5 mmHg, the median absolute deviation between estimated and reference heart rate was 1.3 bpm when using the venous pressure signal only, but dropped to 0.6 bpm when combining the pressure signals. The results show that the proposed method offers superior estimation at low heartbeat amplitudes. Consequently, more patients can be successfully monitored during treatment without the need of extra sensors. The results are preliminary, and need to be verified on a separate dataset.
Asunto(s)
Presión Arterial , Corazón/fisiología , Monitoreo Fisiológico/métodos , Diálisis Renal , Procesamiento de Señales Asistido por Computador , Presión Venosa , Frecuencia Cardíaca , HumanosRESUMEN
The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at -10 degrees C compared with 0 degrees C. The influences of glycerol, ethylene glycol, N,N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed.
Asunto(s)
Membranas Sinápticas/efectos de los fármacos , Animales , Bovinos , Glicerol/farmacología , L-Lactato Deshidrogenasa/análisis , Polímeros/farmacología , Solubilidad , Solventes/farmacología , Temperatura , ViscosidadRESUMEN
Crude synaptic membranes isolated from calf brain cortex were subjected to an aqueous two-phase system and the partition of the various membrane constituents and activities between the phases were studied. These constituents were phosphate, cholesterol and protein. The activities measured were acetyl-cholinesterase, succinate dehydrogenase, 2',3'-cyclicnucleotide-3'-phosphohydrolase and stereospecific opiate-binding. The successful fractionation of the membranes was achieved by the use of an aqueous two-phase system in a counter-current distribution process. A ligand bound to poly(ethylene glycol) with an affinity for opiate receptors was synthesized by reacting 6-aminonaloxone with tresylpoly(ethylene glycol). The ligand-polymer was used to extract membrane-bound opiate receptors into the upper, poly(ethylene glycol)-rich phase. This use of affinity partitioning resulted in membrane fractions with a 3-4 fold higher ability to bind stereospecifically etorphine than the original preparations of synaptic membranes.
Asunto(s)
Corteza Cerebral/metabolismo , Naloxona/metabolismo , Receptores Opioides/análisis , Membranas Sinápticas/análisis , Animales , Bovinos , Cromatografía de Afinidad/métodos , Distribución en Contracorriente , Polietilenglicoles , Cloruro de Potasio , Extractos de Tejidos/análisis , UltracentrifugaciónRESUMEN
SK-N-MC human neuroblastoma cells express functional D1, but not D5, dopaminergic receptors. Stimulating cells with dopamine or the D1-selective agonist, SKF R-38393, rapidly (t(1/2) = 1 h) resulted in > 95% attenuation of dopamine-mediated accumulation of cyclic AMP, without any change in D1 dopamine receptor levels. Prolonged (> 4 h) exposure of cells to dopamine attenuated D1 receptor levels to 45-50% of control (t(1/2) = 8 h) and was accompanied by a loss of high-affinity binding sites. At the molecular level, the expression of D1 receptor messenger RNA was bimodal: an initial increase (by approximately 60%) of receptor messenger RNA within 2 h of treatment of cells with dopamine was followed by a decline to 50% below control messenger RNA levels. Low concentrations (1-10 nM) of dopamine also potentiated D1 messenger RNA levels (up to 48%), resulting in a twofold increase in receptor levels. Transfection studies with the cloned human D1 promoter construct, pGL-D1P, indicated that the up-regulation of D1 messenger RNA was due to activation of promoter by dopamine. The dopamine-mediated up-regulation of both D1 receptor messenger RNA and promoter was prevented by the D1-selective antagonist, SCH 23390. The results suggest that dopamine regulates D1 receptor gene and protein expression in a bimodal manner, partly through activation of the receptor promoter. Moreover, the effects of dopamine are independent of the second messenger, cyclic AMP.
Asunto(s)
2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , AMP Cíclico/metabolismo , Dopamina/fisiología , Regulación Neoplásica de la Expresión Génica , Receptores de Dopamina D1/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Benzazepinas/análogos & derivados , Benzazepinas/metabolismo , Benzazepinas/farmacología , Bucladesina/farmacología , División Celular/efectos de los fármacos , Colforsina/farmacología , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Neuroblastoma , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/genética , Receptores de Dopamina D1/genética , Proteínas Recombinantes/metabolismo , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
A cDNA encoding a serotonin receptor has been isolated from a Caenorhabditis elegans mixed stage cDNA library. The nematode serotonin receptor, designated 5HT-Ce, was permanently expressed in murine Ltk-cells, where it mediates adenylate cyclase attenuation. Sequence analysis and the pharmacological profiles demonstrate its relatedness not only to Drosophila and Lymnae 5HT receptors but also to mammalian 5HT1a receptors. The 5HT-Ce-gene does not map close to the position of any known serotonergic mutations.
Asunto(s)
Caenorhabditis elegans/genética , Receptores de Serotonina/genética , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Secuencia de Bases , Células COS/enzimología , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica/fisiología , Datos de Secuencia Molecular , Mutación/fisiología , Receptores de Serotonina/fisiología , Homología de Secuencia de Aminoácido , Serotonina/farmacologíaRESUMEN
Receptors for the neurotransmitter and neuroendocrine peptides, vasoactive intesinal peptide (VIP) and secretin, both belong to the Type B subfamily of G-protein-coupled receptors. This group is evolutionally as well as structurally distinct from the much larger Type A, or rhodopsin-type, subfamily. We have mapped the ligand-activating epitopes of the human VIP1 receptor by the use of hybrid receptor constructs with the human secretin receptor. Twelve chimeras were synthesized the successively replacing portions of the former receptor with corresponding portions of the latter receptor, or by interchanging the first extracellular loops. Each of the different chimeric receptor DNAs were then expressed in murine reporter cells, and their ability to activate cAMP production was investigated on stimulation with the respective natural peptide ligands. We stimulated the reporter cells with secretion or VIP following transient expression of the receptor chimeras. The experiments indicated that there are two molecular domains of importance for the recognition and activation of these peptides, namely, the inner portion of the extracellular tail and the first extracellular loop of the two receptors.
Asunto(s)
Epítopos/genética , Activación del Canal Iónico/fisiología , Receptores de la Hormona Gastrointestinal/genética , Receptores de Péptido Intestinal Vasoactivo/genética , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , Humanos , Ligandos , Ratones , Mutagénesis/fisiología , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Péptido Intestinal Vasoactivo/química , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Proteínas Recombinantes de Fusión/genética , Sistemas de Mensajero Secundario/genética , TransfecciónRESUMEN
Aqueous two-phase systems composed of water, dextran and poly(ethylene glycol) can be used for the separation of biological particles. The adjustment of the partition of such particles between the two phases and the interface between them has been studied by using a preparation of synaptosomes (from calf brain cortex) also containing free mitochondria. The partition has been affected by variation of polymer concentrations and addition of salts, e.g. phosphates and chloride. The time for separation of the phases showed a bimodal behaviour with an initially rapid formation of bulk phases followed by a slow phase separation. The relative amount of mixed phases at the time of the transition was proportional to the amount of particles included. Counter-current distribution with moderate time for the phase separation was carried out in such way that the interface material travelled with approximately half the speed of the moving upper phase. In this way the distribution of the particles between the upper phase and the interface as well as between the interface and the lower phase could be studied in the same experiment. The heterogeneity of the synaptosome preparation was clearly demonstrated by counter-current distribution at low polymer concentrations while no separation was obtained when the system contained larger amounts of polymers. Possible reasons for this behaviour are discussed.
Asunto(s)
Corteza Cerebral/ultraestructura , Sinaptosomas/ultraestructura , Animales , Tampones (Química) , Bovinos , Fraccionamiento Celular/métodos , Distribución en Contracorriente , Dextranos , Indicadores y Reactivos , PolietilenglicolesRESUMEN
BACKGROUND: The citric cycle intermediate succinate has recently been identified as a ligand for the G-protein-coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. OBJECTIVE: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. METHODS AND RESULTS: Using real-time-PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y(1) receptor. Light transmission aggregation experiments showed dose-dependent aggregation induced by succinate, reaching a maximum response at 0.5 mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb-IIIa and P-selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3-kinase-ß activation, and receptor desensitization. Furthermore, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A(2), and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y(12) receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. CONCLUSIONS: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate-induced platelet aggregation depends on thromboxane A(2) generation, ATP release, and P2Y(12) activation.
Asunto(s)
AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Transducción de Señal , Ácido Succínico/farmacología , Plaquetas/metabolismo , Western Blotting , Citometría de Flujo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Microscopía Fluorescente , Reacción en Cadena de la PolimerasaRESUMEN
In vitro studies suggest that the membrane G protein-coupled receptor GPR30 is a functional estrogen receptor (ER). The aim of the present study was to determine the possible in vivo role of GPR30 as a functional ER primarily for the regulation of skeletal parameters, including bone mass and longitudinal bone growth, but also for some other well-known estrogen-regulated parameters, including uterine weight, thymus weight, and fat mass. Three-month-old ovariectomized (OVX) GPR30-deficient mice (GPR30(-/-)) and wild-type (WT) mice were treated with either vehicle or increasing doses of estradiol (E(2); 0, 30, 70, 160, or 830 ng.mouse(-1).day(-1)). Body composition [bone mineral density (BMD), fat mass, and lean mass] was analyzed by dual-energy-X ray absorptiometry, while the cortical and trabecular bone compartments were analyzed by peripheral quantitative computerized tomography. Quantitative histological analyses were performed in the distal femur growth plate. Bone marrow cellularity and distribution were analyzed using a fluorescence-activated cell sorter. The estrogenic responses on most of the investigated parameters, including increase in bone mass (total body BMD, spine BMD, trabecular BMD, and cortical bone thickness), increase in uterine weight, thymic atrophy, fat mass reduction, and increase in bone marrow cellularity, were similar for all of the investigated E(2) doses in WT and GPR30(-/-) mice. On the other hand, E(2) treatment reduced longitudinal bone growth, reflected by decreased femur length and distal femur growth plate height, in the WT mice but not in the GPR30(-/-) mice compared with vehicle-treated mice. These in vivo findings demonstrate that GPR30 is not required for normal estrogenic responses on several major well-known estrogen-regulated parameters. In contrast, GPR30 is required for a normal estrogenic response in the growth plate.
Asunto(s)
Desarrollo Óseo/fisiología , Estrógenos/metabolismo , Ovariectomía , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tejido Adiposo/anatomía & histología , Tejido Adiposo/crecimiento & desarrollo , Animales , Densidad Ósea , Femenino , Fémur/citología , Fémur/crecimiento & desarrollo , Placa de Crecimiento/citología , Placa de Crecimiento/crecimiento & desarrollo , Ratones , Ratones Mutantes , Tamaño de los Órganos , Receptores de Estrógenos/metabolismo , Timo/anatomía & histología , Timo/crecimiento & desarrollo , Útero/anatomía & histología , Útero/crecimiento & desarrolloRESUMEN
Free fatty acids (FFA) have generally been proposed to regulate pancreatic insulin release by an intracellular mechanism involving inhibition of CPT-1. The recently de-orphanized G-protein coupled receptor, FFA(1)R/GPR40, has been shown to be essential for fatty-acid-stimulated insulin release in MIN6 mouse insulinoma cells. The CPT-1 inhibitor, 2-bromo palmitate (2BrP), was investigated for its ability to interact with mouse FFA(1)R/GPR40. It was found to inhibit phosphatidyl inositol hydrolysis induced by linoleic acid (LA) (100 muM in all experiments) in HEK293 cells transfected with FFA(1)R/GPR40 and in the MIN6 subclone, MIN6c4. 2BrP also inhibited LA-stimulated insulin release from mouse pancreatic islets. Mouse islets were subjected to antisense intervention by treatment with a FFA(1)R/GPR40-specific morpholino oligonucleotide for 48 h. Antisense treatment of islets suppressed LA-stimulated insulin release by 50% and by almost 100% when islets were pretreated with LA for 30 min before applying the antisense. Antisense treatment had no effect on tolbutamide-stimulated insulin release. Confocal microscopy using an FFA(1)R/GPR40-specific antibody revealed receptor expression largely localized to the plasma membrane of insulin-producing cells. Pretreating the islets with LA for 30 min followed by antisense oligonucleotide treatment for 48 h reduced the FFA(1)R/GPR40 immunoreactivity to background levels. The results demonstrate that FFA(1)R/GPR40 is inhibited by the CPT-1 inhibitor, 2BrP, and confirm that FFA(1)R/GPR40 is indeed necessary, at least in part, for fatty-acid-stimulated insulin release.
Asunto(s)
Ácidos Grasos/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Femenino , Humanos , Hipoglucemiantes/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ácido Linoleico/metabolismo , Ratones , Oligonucleótidos Antisentido/metabolismo , Palmitatos/metabolismo , Fosfatidilinositoles/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The heterogeneity of a synaptosomal preparation was studied by the use of affinity partitioning in combination with centrifugal counter-current distribution. Hexaethonium-poly(ethyleneglycol) was used as the extracting agent. The fractions were analyzed for: light scattering, protein, choline acetyltransferase, L-glutamate decarboxylase, glutamine synthetase, 2',3'-cyclicnucleotide-3'-phosphohydrolase, acetylcholinesterase and succinate dehydrogenase. The material was fractionated into three main fractions which differed in their content of marker-enzymes.
Asunto(s)
Sinaptosomas , Animales , Química Encefálica , Bovinos , Fraccionamiento Celular/métodos , Centrifugación , Distribución en Contracorriente , Ensayo de Inmunoadsorción Enzimática , Fotometría , Polietilenglicoles , Compuestos de Amonio CuaternarioRESUMEN
Ligands with an apparent affinity for various structural elements on the surface of synaptic membrane fragments have been bound to the polymers poly(ethylene glycol) and dextran. The ligand-polymer derivatives have been included in aqueous two-phase systems composed of water, poly(ethylene glycol) and dextran. The uneven distribution of the polymers resulted in the concentration of the polymer-bound ligand in one of the two phases. The effect of the ligand-polymer on the partition of membranes was studied by using synaptic membranes from calf brain, obtained by standard centrifugation methods. By using ligand-containing two-phase systems for nine-step counter-current distribution of membranes, it was shown that the distribution behaviour of various parts of the membrane preparation could be affected. The distribution was followed by determination of opiate binding, acetylcholinesterase, and total membrane (using protein and light-scattering measurements).
Asunto(s)
Encéfalo/ultraestructura , Fraccionamiento Celular/métodos , Dextranos , Polietilenglicoles , Membranas Sinápticas/ultraestructura , Acetilcolinesterasa/análisis , Animales , Bovinos , Distribución en Contracorriente , Cloruro de Potasio , Receptores Opioides/metabolismo , AguaRESUMEN
Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively.
Asunto(s)
Quimera , Células Clonales , Proteínas de Unión al GTP/metabolismo , Genes Reporteros , Receptores de Superficie Celular/metabolismo , Animales , Secuencia de Bases , Línea Celular , Separación Celular , Cartilla de ADN , Citometría de Flujo , Humanos , Plásmidos , RatasRESUMEN
We recently described a novel chemoattractant receptor, provisionally named CMKRL1, which has turned out to be the first cloned leukotriene (LT) receptor. Present binding assays using tritiated LTB4 and isolated membranes from COS-7 cells, transiently transfected with cDNA encoding this receptor, yielded a linear Scatchard plot showing expression of only a single, high-affinity receptor population with a mean Kd of 2.1 nM and Bmax of 17.0 pmoles/mg protein. Sham-transfected cells exhibited no specific binding. LTB4 elicited concentration-dependent increases in intracellular calcium measured with Fura-2 in individual CHO cells stably expressing CMKRL1. No response was seen with sham-transfected control cells, or in calcium-free medium which suggests that calcium mainly originates from extracellular sources. The LTB4-induced cellular calcium increment was blocked in the presence of a monoclonal antibody, raised against a synthetic peptide corresponding to the extracellular tail of CMKRL1 and capable of visualizing the receptor by fluorescence immunocytochemistry. Taken together the analyses show that LTB4 is the endogenous ligand for CMKRL1 which is, thus, identical to the LTB4 receptor, designated BLTR according to the NC-IUPHAR nomenclature.
Asunto(s)
Leucotrieno B4/metabolismo , Leucotrieno B4/fisiología , Receptores de Leucotrienos/metabolismo , Animales , Anticuerpos Monoclonales/química , Células CHO , Células COS , Clonación Molecular , Cricetinae , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/inmunología , Receptores de Leucotrieno B4 , Receptores Purinérgicos P2 , TransfecciónRESUMEN
With the application of a homology screening strategy, including PCR amplification and southern blot hybridization, a novel cDNA was cloned from rat liver and anterior pituitary libraries. It was found to encode a 371-amino acid protein which has the characteristics of a heptahelix receptor and shows structural identity to members of the chemoattractant receptor family. A primary receptor message of 3.5 kb size was identified by northern blot hybridization. This RNA species showed high expression in heart and lung, while expression was lower in small intestines, colon, kidney, liver, uterus, and in brain. Another larger RNA species of 6.3 kb appeared in heart and lung. In situ hybridization histochemistry performed on tissue from liver and kidney revealed a mainly vascular distribution of the receptor message.
Asunto(s)
Sistema Cardiovascular/química , Proteínas de la Membrana , Receptores de Quimiocina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Hibridación in Situ , Hígado , Datos de Secuencia Molecular , Adenohipófisis , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Neural membranes isolated from calf brain have been partitioned in aqueous two-phase systems containing dextran and polyethyleneglycol. When the partition was repeated several times, using counter-current distribution technique, the distribution of the membranes between the upper phase and the interface changed in a non-ideal manner and in favour of the interface. By using a centrifugal counter-current distribution device the time for the experiment could be reduced by a factor of 7-8 and the distribution was similar to what could be expected for ideally behaving membranes. The time-dependent change of the membranes is discussed in terms of aggregation and lateral membrane perturbations. Despite this effect a certain fractionation has been achieved as deduced from analysis of cholesterol content, opiate receptor activity and acetylcholinesterase activity along the counter-current distribution row of fractions. Compared to the starting material these activities were enriched some two-fold in certain fractions.
Asunto(s)
Química Encefálica , Animales , Bovinos , Centrifugación , Distribución en Contracorriente/instrumentación , Membranas/análisis , Neuronas/análisis , Cloruro de Potasio/análisis , Membranas Sinápticas/análisisRESUMEN
Synaptosomes isolated from calf brain cortex were lysed and fragmented by Yeda press treatment. The obtained membranes have previously been fractionated in a counter-current distribution process using a liquid-liquid two-phase system consisting of water, dextran, Ficoll and poly(ethylene glycol) [J. Chromatogr., 358 (1986) 147]. Using the fact that there are discrete membrane populations, a rapid preparative method for isolation of the two main fractions is presented in the present work, as well as a subfractionation of one of them using liquid-liquid extraction with dextran-bound Procion yellow HE-3G. The content of several membrane constituents, i.e. protein, acetylcholinesterase, succinate dehydrogenase and ATPase, as well as opiate binding, were determined for the three fractions. Counter-current distribution of the fractions elucidates their heterogeneity and the effectiveness of the purification.