RESUMO
BACKGROUND: PGD(2) is the major prostanoid released by mast cells during an allergic response. Its role in the allergic response, however, remains unclear. OBJECTIVE: Because the accumulation of eosinophils is a feature of allergic reactions, we investigated the role of PGD(2) in the modulation of eosinophil function. METHODS: Circulating human eosinophils were isolated and challenged with PGD(2). The effects of PGD(2) on various eosinophil functions were then analyzed. RESULTS: PGD(2) binds with high affinity preferentially to 2 receptors, DP and chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2). We show that both DP and CRTH2 are detectable on circulating eosinophils. We demonstrate that PGD(2) (1-10 nmol/L) induces a rapid change in human eosinophil morphology and an increase in chemokinesis and promotes eosinophil degranulation. These effects are induced by the CRTH2-selective agonist 13-14-dihydro-15-keto-PGD(2) (DK-PGD(2)) but not by the DP-selective agonist BW245C. These results suggest a role for CRTH2 in the modulation of eosinophil movement and in triggering the release of cytotoxic proteins. Finally, we demonstrate that BW245C, but not DK-PGD(2), can delay the onset of apoptosis in cultured eosinophils, presumably through interaction with DP. CONCLUSION: These data support the hypothesis that PGD(2) controls eosinophil functions through 2 pharmacologically distinct receptors with independent functions. Blockade of PGD(2)-mediated effects on human eosinophils may reduce the damage caused by these cells during an allergic response, but inhibition of both receptors may be required.
Assuntos
Apoptose , Eosinófilos/fisiologia , Receptores Imunológicos/fisiologia , Receptores de Prostaglandina/fisiologia , Degranulação Celular/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Humanos , Prostaglandina D2/farmacologia , RNA Mensageiro/análise , Receptores Imunológicos/genética , Receptores de Prostaglandina/genéticaRESUMO
Gabapentin (Neurontin, Pfizer Global R & D) is a novel anticonvulsant, antihyperalgesic, and antinociceptive agent with a poorly understood mechanism of action. In this study, we show that gabapentin (EC50 2 microM) inhibited up to 70 to 80% of the total K+-evoked Ca2+ influx via voltage-dependent calcium channels (VD-CCs) in a mouse pituitary intermediate melanotrope clonal mIL-tsA58 (mIL) cell line. mIL cells endogenously express only gamma-aminobutyric acid type B (GABA(B)) gb1a-gb2 receptors. Moreover, activity of the agonist gabapentin was dose dependently and completely blocked with the GABA(B) antagonist CGP55845 and was nearly identical to the prototypic GABA(B) agonist baclofen in both extent and potency. Antisense knockdown of gb1a also completely blocked gabapentin activity, while gb1b antisense and control oligonucleotides had no effect, indicating that gabapentin inhibition of membrane Ca2+ mobilization in mIL cells was dependent on a functional GABA(B) (gb1a-gb2) heterodimer receptor. In addition, during combined whole cell recording and multiphoton Ca2+ imaging in hippocampal neurons in situ, gabapentin significantly inhibited in a dose-dependent manner subthreshold soma depolarizations and Ca2+ responses evoked by somatic current injection. Furthermore, gabapentin almost completely blocked Ca2+ action potentials and Ca2+ responses elicited by suprathreshold current injection. However, larger current injection overcame this inhibition of Ca2+ action potentials suggesting that gabapentin did not predominantly affect L-type Ca2+ channels. The depressant effect of gabapentin on Ca2+ responses was coupled to the activation of neuronal GABA(B) receptors since they were blocked by CGP55845, and baclofen produced similar effects. Thus gabapentin activation of neuronal GABA(B) gb1a-gb2 receptors negatively coupled to VD-CCs can be a potentially important therapeutic mechanism of action of gabapentin that may be linked to inhibition of neurotransmitter release in some systems.
Assuntos
Acetatos/farmacologia , Aminas , Analgésicos/farmacologia , Anticonvulsivantes/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos , Agonistas dos Receptores de GABA-B , Células Piramidais/efeitos dos fármacos , Ácido gama-Aminobutírico , Animais , Baclofeno/farmacologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Agonistas GABAérgicos/farmacologia , Gabapentina , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/fisiologiaRESUMO
The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.
Assuntos
Leucócitos/metabolismo , Proteínas de Membrana , Receptores de Leucotrienos/biossíntese , Sangue , Humanos , Pulmão/imunologia , Receptores de Leucotrienos/análiseRESUMO
Gamma-aminobutyric acid (GABA) activates two qualitatively different inhibitory mechanisms through ionotropic GABA(A) multisubunit chloride channel receptors and metabotropic GABA(B) G protein-coupled receptors. Evidence suggests that pharmacologically distinct GABA(B) receptor subtypes mediate presynaptic inhibition of neurotransmitter release by reducing Ca2+ conductance, and postsynaptic inhibition of neuronal excitability by activating inwardly rectifying K+ (Kir) conductance. However, the cloning of GABA(B) gb1 and gb2 receptor genes and identification of the functional GABA(B) gb1-gb2 receptor heterodimer have so far failed to substantiate the existence of pharmacologically distinct receptor subtypes. The anticonvulsant, antihyperalgesic, and anxiolytic agent gabapentin (Neurontin) is a 3-alkylated GABA analog with an unknown mechanism of action. Here we report that gabapentin is an agonist at the GABA(B) gb1a-gb2 heterodimer coupled to Kir 3.1/3.2 inwardly rectifying K+ channels in Xenopus laevis oocytes. Gabapentin was practically inactive at the human gb1b-gb2 heterodimer, a novel human gb1c-gb2 heterodimer and did not block GABA agonism at these heterodimer subtypes. Gabapentin was not an agonist at recombinant GABA(A) receptors as well. In CA1 pyramidal neurons of rat hippocampal slices, gabapentin activated postsynaptic K+ currents, probably via the gb1a-gb2 heterodimer coupled to inward rectifiers, but did not presynaptically depress monosynaptic GABA(A) inhibitory postsynaptic currents. Gabapentin is the first GABA(B) receptor subtype-selective agonist identified providing proof of pharmacologically and physiologically distinct receptor subtypes. This selective agonism of postsynaptic GABA(B) receptor subtypes by gabapentin in hippocampal neurons may be its key therapeutic advantage as an anticonvulsant.
Assuntos
Acetatos/farmacologia , Aminas , Anticonvulsivantes/farmacologia , Ácidos Cicloexanocarboxílicos , Neurônios/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização , Receptores de GABA-B/metabolismo , Ácido gama-Aminobutírico , Sequência de Aminoácidos , Animais , Dimerização , Potenciais Pós-Sinápticos Excitadores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Agonistas dos Receptores de GABA-B , Gabapentina , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/fisiologia , Oócitos , Canais de Potássio/biossíntese , Canais de Potássio/genética , Isoformas de Proteínas , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.
Assuntos
Cisteína , Leucotrienos/metabolismo , Proteínas de Membrana , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Medula Suprarrenal/química , Clonagem Molecular , Humanos , Antagonistas de Leucotrienos/farmacologia , Leucotrieno C4/metabolismo , Leucotrieno D4/metabolismo , Leucotrieno E4/metabolismo , Pulmão/química , Modelos Moleculares , Miocárdio/química , Receptores de Leucotrienos/sangue , Proteínas Recombinantes/metabolismo , SRS-A/análogos & derivados , SRS-A/farmacologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Receptores de GABA-B/biossíntese , Receptores de Glutamato Metabotrópico/biossíntese , Sequência de Aminoácidos , Animais , Southern Blotting , Linhagem Celular , Membrana Celular/metabolismo , Densitometria , Citometria de Fluxo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/metabolismo , Testes de Precipitina , Receptores de GABA-B/genética , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Xenopus laevisRESUMO
A cDNA clone coding for the guinea pig leukotriene B4 (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein-Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B4 (Kd value of approximately 0.4 nM) and were expressed at high levels (Bmax values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [3H]leukotriene B4 specific binding at the recombinant guinea pig BLT receptor is leukotriene B4 > 20-OH-leukotriene B4 > 12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen -1-oic acid) > 12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen -1-oic acid) > 20-COOH-leukotriene B4 > U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexane diol) >> leukotriene C4 = leukotriene D4 = leukotriene E4. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B4 and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca2+ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of Galpha16 was required to achieve a robust Ca2+ driven response. Leukotriene B4 was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B4 analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B4 receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.
Assuntos
Receptores do Leucotrieno B4/genética , Equorina/análise , Equorina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Cobaias , Humanos , Medições Luminescentes , Melanóforos/metabolismo , Dados de Sequência Molecular , Ensaio Radioligante , Receptores do Leucotrieno B4/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transfecção , XenopusRESUMO
The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.
Assuntos
Proteínas de Membrana , Receptores de Leucotrienos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células COS , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Antagonistas de Leucotrienos , Leucotrieno D4 , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Dados de Sequência Molecular , Músculo Liso/metabolismo , Receptores de Leucotrienos/química , Receptores de Leucotrienos/genética , Distribuição Tecidual , Transfecção , Cromossomo X , Xenopus laevisRESUMO
The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Isoenzimas/química , Lactonas/síntese química , Lactonas/farmacologia , Prostaglandina-Endoperóxido Sintases/química , Administração Oral , Animais , Disponibilidade Biológica , Células CHO , Cricetinae , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/química , Humanos , Indometacina/efeitos adversos , Indometacina/farmacologia , Concentração Inibidora 50 , Lactonas/administração & dosagem , Lactonas/efeitos adversos , Proteínas de Membrana , Ratos , SulfonasRESUMO
The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50) = 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50) = 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50) > 50 microM in U937 cells and IC(50) > 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50) = 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50) = 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50) = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50) = 0.24 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/metabolismo , Lactonas/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animais , Araquidonato 15-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Artrite Experimental/sangue , Artrite Experimental/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Células COS , Linhagem Celular , Cricetinae , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Sistema Digestório/efeitos dos fármacos , Cães , Edema/induzido quimicamente , Edema/prevenção & controle , Feminino , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/prevenção & controle , Técnicas In Vitro , Leucotrieno B4/biossíntese , Masculino , Proteínas de Membrana , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Ratos , Ratos Endogâmicos Lew , Saimiri , SulfonasRESUMO
We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein-coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I-galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.
Assuntos
Encéfalo/metabolismo , Receptores de Neuropeptídeos/genética , Sequência de Aminoácidos , Animais , Autorradiografia , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Ratos , Receptores Acoplados a Proteínas G , Receptores de Galanina , Receptores de Kisspeptina-1 , Receptores de Neuropeptídeos/metabolismo , Alinhamento de SequênciaRESUMO
Query of the expressed sequence tag database with the rat metabotropic GABABR1A receptor amino acid sequence using the TFASTA algorithm revealed two partial cDNA fragments whose sequence information was then used to isolate by PCR a novel full-length human cDNA encoding a putative G-protein-coupled receptor (GPCR), termed GPR 51. Sequence analysis revealed that it encoded a protein of 941 amino acids, similar in size and homology to GABAB receptors followed by metabotropic glutamate receptors but not other GPCRs. GPR 51 expressed in COS-1 cells showed no specific binding for [3H](+)baclofen and when expressed in Xenopus oocyte and Xenopus melanophore functional assays showed no activity to GABA, (-)baclofen, and glutamic acid. Northern blot analysis and in situ hybridization revealed that GPR 51 transcripts were predominantly expressed in the central nervous system with highest abundance in the cortex, thalamus, hippocampus, amygdala, cerebellum, and spinal cord. In contrast, GPR 51 receptor transcripts were almost not detected in the peripheral tissues. Gene GPR 51 was localized by radiation hybrid mapping to chromosome 9, 4.81 cR from the WI-8684 marker, and proximal to the hereditary sensory neuropathy type 1 locus.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Cromossomos Humanos Par 9/genética , Proteínas de Ligação ao GTP/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Receptores de GABA-B , Receptores de GABA/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Células COS , Mapeamento Cromossômico , Clonagem Molecular , Etiquetas de Sequências Expressas , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Saimiri/anatomia & histologia , Saimiri/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Receptores de GABA-B/metabolismo , Sequência de Aminoácidos , Animais , Azidas/metabolismo , Células COS , Dimerização , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Compostos Organofosforados/metabolismo , Canais de Potássio/metabolismo , Conformação Proteica , RNA Mensageiro/metabolismo , Ratos , Receptores de GABA-B/genética , Relação Estrutura-Atividade , Xenopus laevisRESUMO
Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.
Assuntos
Clonagem Molecular , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/metabolismo , Linhagem Celular , Humanos , Isomerismo , Ligantes , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Ratos , Receptores de Galanina , Receptores de Neuropeptídeos/fisiologia , Ribonucleases , Transdução de Sinais/fisiologia , Suínos , Xenopus laevisRESUMO
In a search of the Caenorhabditis elegans DNA data base, an expressed sequence tag of 327 base pairs (termed cm01c7) with strong homology to the human leukotriene A4 (LTA4) hydrolase was found. The use of cm01c7 as a probe, together with conventional hybridization screening and anchored polymerase chain reaction techniques resulted in the cloning of the full-length 2.1 kilobase pair C. elegans LTA4 hydrolase-like homologue, termed aminopeptidase-1 (AP-1). The AP-1 cDNA was expressed transiently as an epitope-tagged recombinant protein in COS-7 mammalian cells, purified using an anti-epitope antibody affinity resin, and tested for LTA4 hydrolase and aminopeptidase activities. Despite the strong homology between the human LTA4 hydrolase and C. elegans AP-1(63% similarity and 45% identity at the amino acid level), reverse-phase high pressure liquid chromatography and radioimmunoassay for LTB4 production revealed the inability of the C. elegans AP-1 to use LTA4 as a substrate. In contrast, the C. elegans AP-1 was an efficient aminopeptidase, as demonstrated by its ability to hydrolyze a variety of amino acid p-nitroanilide derivatives. The aminopeptidase activity of C. elegans AP-1 resembled that of the human LTA4 hydrolase/aminopeptidase enzyme with a preference for arginyl-p-nitroanilide as a substrate. Hydrolysis of the amide bond of arginyl-p-nitroanilide was inhibited by bestatin with an IC50 of 2.6 +/- 1.2 microM. The bifunctionality of the mammalian LTA4 hydrolase is still poorly understood, as the physiological substrate for its aminopeptidase activity is yet to be discovered. Our results support the idea that the enzyme originally functioned as an aminopeptidase in lower metazoa and then developed LTA4 hydrolase activity in more evolved organisms.
Assuntos
Aminopeptidases/genética , Caenorhabditis elegans/genética , Sequência de Aminoácidos , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/biossíntese , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Caenorhabditis elegans/enzimologia , Clonagem Molecular , DNA Complementar/genética , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Evolução Molecular , Etiquetas de Sequências Expressas , Expressão Gênica , Genes de Helmintos , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Leucina/análogos & derivados , Leucina/farmacologia , Leucotrieno B4/biossíntese , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Inibidores de Proteases/farmacologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Animais , Anti-Inflamatórios não Esteroides/química , Arginina/química , Células CHO/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cricetinae , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Relação Dose-Resposta a Droga , Humanos , Immunoblotting , Isoenzimas/efeitos dos fármacos , Isoenzimas/metabolismo , Proteínas de Membrana , Mutagênese Sítio-Dirigida , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , TransfecçãoRESUMO
Human cyclooxygenase-2 (hCox-2) is a key enzyme in the biosynthesis of prostaglandins and the target of nonsteroidal anti-inflammatory drugs. Recombinant hCox-2 overexpressed in a vaccinia virus (VV)-COS-7 system comprises two glycoforms. Removal of the N-glycosylation consensus sequence at Asn580 (N580Q and S582A mutants) resulted in the expression of protein comprising a single glycoform, consistent with the partial N-glycosylation at this site in the wild-type (WT) enzyme. The specific cyclooxygenase activities of the purified WT and N580Q mutant were equivalent (40 +/- 3 mumol O2/min/mg) and titrations with diclofenac showed no difference in inhibitor sensitivities of WT and both mutants. Results of the expression of WT and N580Q hCox-2 in a Drosophila S2 cell system were also consistent with the N-glycosylation at this site, but low levels of activity were obtained. High levels of N-glycosylation heterogeneity are observed in hCox-2 expressed using recombinant baculovirus (BV) in Sf9 cells. Expression of a double N-glycosylation site mutant in Sf9 cells, N580Q/N592Q, resulted in a decrease in glycosylation but no clear decrease in heterogeneity, indicating that the high degree of N-glycosylation heterogeneity observed with the BV-Sf9 system is not due to partial glycosylation of both Asn580 and Asn592. N-linked oligosaccharide profiling of purified VV and BV WT and S582A mutant hCox-2 showed the presence of high mannose structures, (Man)n (GlcNAc)2, n = 9, 8, 7, 6. The S582A mutant was the most homogeneous with (Man)9(GlcNAc)2 comprising greater than 50% of oligosaccharides present. Analysis of purified VV WT and S582A mutant hCox-2 by liquid chromatography-electrospray ionization-mass spectrometry showed an envelope of peaks separated by approximately 160 Da, corresponding to differences of a single monosaccharide. The difference between the highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide.
Assuntos
Isoenzimas/química , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/genética , Animais , Baculoviridae/genética , Sequência de Bases , Células COS , Sequência de Carboidratos , Linhagem Celular , Sequência Consenso , Ciclo-Oxigenase 2 , Drosophila , Expressão Gênica , Glicosilação , Humanos , Espectrometria de Massas , Proteínas de Membrana , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/genética , Oligossacarídeos/química , Oligossacarídeos/genética , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Spodoptera , Vaccinia virus/genéticaRESUMO
The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1 alpha, IL-1 beta and TNF alpha in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were preincubated with inhibitors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1-30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2-0.4 microM). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released more PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.
Assuntos
Isoenzimas/biossíntese , Isoenzimas/metabolismo , Osteossarcoma/enzimologia , Osteossarcoma/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismo , Ácido Araquidônico/farmacologia , Ácido Araquidônico/fisiologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/química , Citocinas/metabolismo , Citocinas/farmacologia , Dexametasona/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/efeitos dos fármacos , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Aspirin (ASA) acetylates Ser516 of prostaglandin G/H synthase-2 (PGHS-2) resulting in a modified enzyme that converts arachidonic acid to 15(R)-hydroxy-eicosatetraeroic acid [15(R)-HETE]. ASA has pharmacological benefits that may not all be limited to inhibition of prostaglandin synthesis, and this study was initiated to further investigate the properties of ASA-acetylated PGHS-2 and of the mutation of Ser516 to methionine, which mimics ASA acetylation. Both the S516M mutant and ASA-acetylated form of PGHS-2 (ASA-PGHS-2) synthesize 15(R)-HETE and have apparent K(m) values for arachidonic acid within 10-fold of the apparent K(m) value for untreated PGHS-2. The time courses of turnover-dependent inactivation were similar for reactions catalyzed by PGHS-2 and ASA-PGHS-2, whereas the PGHS-2(S516M) showed a decrease in both the initial rate of 15-HETE production and rate of enzyme inactivation. The production of 15-HETE by modified PGHS-2 was sensitive to inhibition by most nonsteroidal anti-inflammatory drugs (NSAIDs), including selective PGHS-2 inhibitors. As observed for the cyclooxygenase activity of PGHS-2, the inhibition of 15-HETE production by indomethacin was time-dependent for both ASA-PGHS-2 and PGHS-2(S516M). However, two potent, structurally related NSAIDs, diclofenac and meclofenamic acid, do not inhibit either ASA-PGHS-2 or the PGHS-2(S516M) mutant. These results demonstrate that the sensitivity to inhibition by NSAIDs of the 15-HETE production by ASA-treated PGHS-2 is different than that of prostaglandin production by PGHS-2 and that Ser516 plays an important role in the interaction with fenamate inhibitors. The results also indicate that the conversion of arachidonic acid to 15-HETE by ASA-PGHS-2 is an efficient process providing a unique mechanism among NSAIDs that will not lead to arachidonic acid accumulation or shunting to other biosynthetic pathways.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Acetilação , Animais , Ácido Araquidônico/metabolismo , Células COS , Dinoprostona/biossíntese , Ácidos Hidroxieicosatetraenoicos/biossínteseRESUMO
Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.