RESUMO
BACKGROUND: Mast cells are specialized secretory cells releasing multiple inflammatory mediators when activated. Activation requires antigen/IgE cross-linking of FcÉRI receptors, initiating a complex intracellular signalling cascade. Ceramide kinase (CERK) is a novel lipid kinase implicated in several inflammatory cellular signalling processes. OBJECTIVE: We sought to investigate a role for CERK in FCÉRI/IgE-mediated mast-cell activation. METHODS: The rat and human mast cell-lines RBL-2H3 and LAD-2, respectively, were stimulated via FcÉRI or with the active product of CERK [ceramide-1-phosphate (C-1P)]. Multiple end-points were measured by enzyme-linked immunosorbent assay; histamine (pre-formed early-phase mediator), prostaglandin D2 (PGD2 - rapidly metabolized early-phase mediator) and interleukin (IL) -13 (de novo transcribed late-phase mediator). RESULTS: We demonstrated that C-1P alone induced release of histamine and PGD2 and was additive to antigen-mediated activation. C-1P did not stimulate IL-13 by a statistically significant amount. Using a specific inhibitor of CERK, antigen-mediated release of histamine and PGD2 was significantly inhibited. Finally, we identified that, for histamine, CERK was downstream of spleen tyrosine kinase, phosphoinositol-3 kinases and phospholipase C, but upstream of c-jun N-terminal kinase (JNK); while for PGD2 CERK was positioned upstream of JNK, mitogen-activated protein kinase kinase and cyclooxygense. CONCLUSIONS AND CLINICAL RELEVANCE: We have identified a differential role for CERK in mast-cell activation and begun to elucidate its position in the mast cell-signalling cascade, thereby suggesting a model by which CERK may be mediating its effects. This type of study is essential for complete understanding of activation pathways that may eventually be used to identify new targets for drug discovery in inflammatory diseases.
Assuntos
Mastócitos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Animais , Western Blotting , Degranulação Celular/imunologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Liberação de Histamina/imunologia , Humanos , Mastócitos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prostaglandina D2/metabolismo , RatosRESUMO
BACKGROUND AND PURPOSE: The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart. EXPERIMENTAL APPROACH: Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP. KEY RESULTS: Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.
Assuntos
Venenos Elapídicos/metabolismo , Guanilato Ciclase/metabolismo , Artéria Torácica Interna/metabolismo , Músculo Liso Vascular/metabolismo , Peptídeos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Vasodilatação , Vasodilatadores/metabolismo , Adrenomedulina/farmacologia , Sequência de Aminoácidos , Fator Natriurético Atrial/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Relação Dose-Resposta a Droga , Venenos Elapídicos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Guanilato Ciclase/análise , Guanilato Ciclase/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Artéria Torácica Interna/química , Artéria Torácica Interna/efeitos dos fármacos , Microscopia Confocal , Modelos Biológicos , Dados de Sequência Molecular , Músculo Liso Vascular/química , Músculo Liso Vascular/efeitos dos fármacos , Peptídeos/farmacologia , Ligação Proteica , Receptores do Fator Natriurético Atrial/análise , Receptores do Fator Natriurético Atrial/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Hepatitis C virus (HCV) infection is an issue of global concern, and studies are ongoing to identify new therapies that are both effective and safe. PF-4878691 is a Toll-like receptor 7 (TLR7) agonist modeled so as to dissociate its antiviral activities from its inflammatory activities. In a proof-of-mechanism study in healthy volunteers who received doses of 3, 6, and 9 mg of PF-4878691 twice a week for 2 weeks, PF-4878691 induced biomarkers of the immune and interferon (IFN) responses in a dose-dependent and dose-frequency-related manner. A novel finding was induction of TLR7 expression in vivo in response to PF-4878691, leading to an amplified biomarker response. A nonresponder at the 9-mg dose had a polymorphism in the IFN-α receptor 1 subunit (Val168Leu). Two subjects who had received 9-mg doses experienced serious adverse events (SAEs), characterized by flu-like symptoms, hypotension, and lymphopenia, leading to early termination of the study. TLR7 stimulation results in a pharmacologic response at levels commensurate with predicted antiviral efficacy, but these doses are associated with SAEs, raising concerns about the therapeutic window of this class of compounds for the treatment of HCV infection.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/imunologia , Imunidade Inata/efeitos dos fármacos , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/imunologia , Adulto , Estudos de Coortes , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor 7 Toll-Like/biossíntese , Resultado do Tratamento , Adulto JovemRESUMO
We have identified and characterised a novel member of the PDE7 family of cyclic nucleotide phosphodiesterases (PDE), which we have designated PDE7B. Mouse and human full-length cDNAs were isolated encoding a protein of 446 and 450 amino acids, respectively. The predicted protein sequence of PDE7B showed highest homology (70% identity) to that of PDE7A. Northern blot analysis identified a single 5.5-kb transcript with highest levels detected in brain, heart, and liver. Kinetic analysis of the mouse and human purified recombinant enzymes show them to specifically hydrolyse cAMP with a Km of 0.1 and 0.2 microM respectively. Inhibitor studies show sensitivity to dipyridamole, IC50 of 0.51 and 1.94 microM, and IBMX, IC50 of 3.81 and 7.37 microM, for the mouse and human enzymes, respectively. This shows that dipyridamole is not selective for cGMP over cAMP PDEs as previously believed. Other standard PDE inhibitors including zaprinast, rolipram, and milrinone do not significantly inhibit PDE7B.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Primers do DNA/genética , Feminino , Humanos , Técnicas In Vitro , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição TecidualRESUMO
Three distinct genes encode alpha(1)-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the alpha(1)-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of alpha(1)-adrenoceptor dimerization are discussed.
Assuntos
Receptores Adrenérgicos alfa 1/química , Receptores Acoplados a Proteínas G/química , Animais , DNA/química , DNA Complementar/metabolismo , Dimerização , Humanos , Ligantes , Ligação Proteica , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Phosphodiesterase 11A (PDE11A) is a recently identified family of cAMP and cGMP hydrolyzing enzymes. Thus far, a single splice variant designated as PDE11A1 has been reported. In this study, we identify and characterize two additional splice variants of PDE11A, PDE11A2 and PDE11A3. The full-length cDNAs are 2,141 bp for PDE11A2 and 2205 bp for PDE11A3. The ORF of PDE11A2 predicts a protein of 576 aa with a molecular mass of 65.8 kDa. The ORF of PDE11A3 predicts a protein of 684 aa with a molecular mass of 78.1 kDa. Comparison of the PDE11A2 sequence with that of PDE11A1 indicates an additional 86 aa at the N terminus of PDE11A2. Part of this sequence extends the potential cGMP binding region (GAF domain) present in PDE11A1. Compared with PDE11A2, PDE11A3 has an additional 108 N-terminal amino acids. Sequence analysis of PDE11A3 indicates the presence of another GAF domain in this region. This diversification of regulatory sequences in the N-terminal region of PDE11A splice variants suggests the interesting possibility of differential regulation of these enzymes. Recombinant PDE11A2 and -A3 proteins expressed in the Baculovirus expression system have the ability to hydrolyze both cAMP and cGMP. The K(m) values for cAMP hydrolysis are 3.3 microM and 5.7 microM for PDE11A2 and PDE11A3, respectively. The K(m) values for cGMP hydrolysis are 3.7 microM and 4.2 microM for PDE11A2 and PDE11A3, respectively. Both PDEs showed a V(max) ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an IC(50) of 1.8 microM, and to zaprinast, with an IC(50) of 28 microM. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with IC(50) values of 0.82 and 5 microM for dipyridamole and zaprinast, respectively.
Assuntos
Processamento Alternativo , Diester Fosfórico Hidrolases/genética , 3',5'-GMP Cíclico Fosfodiesterases , Sequência de Aminoácidos , Animais , Catálise , Linhagem Celular , Clonagem Molecular , Humanos , Masculino , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Spodoptera/citologiaRESUMO
Single cell microfluorimetry was used to study intracellular calcium ion signals ([Ca(2+)](i)) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCI-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, [Ca(2+)](i) was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in [Ca(2+)](i) was dependent upon both Ca(2+) influx and Ca(2+) mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate or KCI-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, [Ca(2+)](i) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentration. As in undifferentiated EC cells, the ACh-mediated increases in [Ca(2+)](i) were governed by both Ca(2+) influx and Ca(2+) mobilization. In contrast, the effects of NMDA, kainate and KCI did not involve intracellular Ca(2+) mobilization. The appearance of glutamate and KCI responsiveness was not detected in non-neuronal differentiated derivatives of NTERA2 cells. Using a number of pharmacologically defined muscarinic receptor antagonists we found that NTERA2 EC cells express M(1), M(3), M(4) and possibly M(5) receptor subtypes linked to changes in [Ca(2+)](i), whilst only M(3) and M(5) are present in NTERA2-derived neurons. The results were supported by PCR analysis of the muscarinic mRNA species expressed in the cells. The data demonstrate that differentiation of NTERA2 EC cells into neurons involves the induction of functional glutamate receptors coupled to rises in [Ca(2+)](i), and changes in the expression of muscarinic ACh receptor subtypes.
Assuntos
Cálcio/metabolismo , Carcinoma Embrionário/metabolismo , Carcinoma Embrionário/patologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Membranas Intracelulares/metabolismo , Agonistas Muscarínicos/farmacologia , Neurônios/patologia , Diferenciação Celular , Citosol/metabolismo , Humanos , Neurônios/metabolismo , Concentração Osmolar , Receptores Muscarínicos/classificação , Receptores Muscarínicos/metabolismo , Células Tumorais CultivadasRESUMO
We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in this study compared to previous reports for both human and bovine alpha-1cAR membrane preparations. All six alpha-1AR subtypes couple to phosphoinositide hydrolysis in a pertussis toxin-insensitive manner, including the cloned human alpha-1a/dAR which had not been expressed previously. In spite of significant sequence differences between human alpha-1ARs and their other species counterparts, previously established ligand selectivity remains fairly comparable. In summary, these data represent the first side-by-side comparison of pharmacological properties between species homologs of alpha-1AR subtypes and should facilitate the development of alpha-1AR subtype selective drugs for clinical use.