RESUMO
Neutrophil infiltration and activation in the lung are important pathophysiological features in COPD, severe asthma and bronchiectasis mostly mediated by CXCL8 and CXCL1 via CXCR1 and CXCR2. No thorough study to date has been performed to compare the anti-inflammatory effect profile of dual CXCR1/2 vs. selective CXCR2 antagonists in relevant human neutrophil assays and pulmonary inflammation models. Dual CXCR1/2 (SCH527123, diaminocyclobutandione-1) and selective CXCR2 (SB265610, thiopyrimidine-1) antagonist activity and receptor residence time were determined by [(35)S]GTPγS binding in human (h)- and guinea pig (gp)-CXCR1 and CXCR2 overexpressing membranes. h-neutrophil chemotaxis, degranulation and ROS production were established using CXCL8 or CXCL1 to evaluate dual CXCR1/2- or selective CXCR2-dependent activities. LPS-induced lung inflammation in gp was selected to assess in vivo potency. Dual CXCR1/2 antagonists blocked both CXCL8 and CXCL1-induced h-neutrophil functions and [(35)S]GTPγS binding. In contrary, selective CXCR2 antagonists displayed significantly reduced potency in CXCL8 -mediated h-neutrophil responses despite being active in CXCR2 assays. Upon LPS challenge in gp, administration of SCH527123 inhibited the increase of neutrophils in BALF, modestly reduced blood neutrophils and induced minor neutrophil accumulation in bone marrow. Differentiation of CXCR1/2 vs. CXCR2 antagonists could not be extended to in vivo due to differences in CXCR1 receptor homology between h and gp. Dual CXCR1/2 therapy may represent a promising anti-inflammatory treatment for respiratory diseases reducing more effectively neutrophil migration and activation in the lung than a CXCR2 selective treatment. However, the in vivo confirmation of this claim is still missing due to species differences in CXCR1.
Assuntos
Benzamidas/farmacologia , Ciclobutanos/farmacologia , Neutrófilos/metabolismo , Compostos de Fenilureia/farmacologia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Triazóis/farmacologia , Animais , Linhagem Celular , Cricetinae , Cobaias , Humanos , Inflamação/imunologia , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Masculino , Espécies Reativas de Oxigênio/imunologia , Transdução de SinaisRESUMO
Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15-epi-lipoxin (LX)A interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti-inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15-epi-LXA4 analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK-571 binds to FPR2/ALX, so cross-reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15-epi-LXA4 are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15-epi-LXA4-mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX-mediated signalling, enhancing guanosine triphosphate-gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15-epi-LXA4 was inactive. Furthermore, 15-epi-LXA4 showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15-epi-LXA4 showed a moderate reduction of interleukin (IL)-8-mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15-epi-LXA4 is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL-8-induced neutrophil survival and produces only moderate inhibition in IL-8-mediated neutrophil migration. Our data do not support an anti-inflammatory role of 15-epi-LXA4- FPR2/ALX interaction in IL-8-induced neutrophil inflammation.
Assuntos
Lipoxinas/farmacologia , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Receptores de Formil Peptídeo/agonistas , Receptores de Leucotrienos/metabolismo , Receptores de Lipoxinas/agonistas , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Interleucina-8/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Mucus hypersecretion and mucin MUC5AC overexpression are pathological features of chronic obstructive pulmonary disease (COPD). This study examines the inhibitory effect of aclidinium, a new long-acting muscarinic antagonist, on MUC5AC expression in human airway epithelial cells. MUC5AC mRNA (RT-PCR) and protein expression (ELISA and immunohistochemistry) were studied in human bronchial tissue and differentiated human airway epithelial cells activated with carbachol (100 µM) or cigarette smoke extract in the absence or presence of aclidinium. Carbachol increased MUC5AC mRNA and protein expression in human bronchus and cultured epithelial cells. Aclidinium inhibited the carbachol-induced MUC5AC mRNA and protein expression with potency (half maximal inhibitory concentration) ~1 nM in human bronchus and cultured airway epithelial cells. AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, inhibited carbachol-induced MUC5AC responses, indicating EGFR transactivation. Aclidinium inhibited carbachol-induced phospho-EGFR and phospho-p44/42 MAPK expression. In cultured airway epithelial cells transfected with small interfering (si)RNA against muscarinic receptor subtypes, siRNA-M3 but not siRNA-M2 blocked carbachol-induced MUC5AC expression. Cigarette smoke-induced MUC5AC upregulation in cultured airway epithelial cells was suppressed by aclidinium. In conclusion, aclidinium decreases carbachol and tobacco smoke-induced MUC5AC overexpression in human airway epithelial cells. This effect may contribute to the clinical efficacy of aclidinium in mucus hypersecretory diseases including COPD.
Assuntos
Mucina-5AC/antagonistas & inibidores , Antagonistas Muscarínicos/farmacologia , Sistema Respiratório/efeitos dos fármacos , Fumar/tratamento farmacológico , Tropanos/farmacologia , Carbacol/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucina-5AC/análise , Mucina-5AC/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Fumar/efeitos adversosRESUMO
Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.
Assuntos
Resistência à Insulina , Fígado/metabolismo , Músculos/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas/metabolismo , Receptor de Insulina/metabolismo , Animais , Células CHO , Cricetinae , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Jejum , Insulina/farmacologia , Masculino , Camundongos , Camundongos Obesos , Fosforilação , RatosRESUMO
IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.
Assuntos
Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO/metabolismo , Cricetinae , Humanos , Proteínas Substratos do Receptor de Insulina , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tirosina/metabolismoRESUMO
Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Desenvolvimento Embrionário e Fetal , Biblioteca Gênica , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina , Camundongos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/genética , RNA Mensageiro/análise , Receptor de Insulina/metabolismo , Seleção Genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição TecidualRESUMO
In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
Assuntos
Isoenzimas/genética , Fígado/enzimologia , Piruvato Quinase/genética , RNA Mensageiro/biossíntese , Vanadatos/farmacologia , Animais , Northern Blotting , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Glucoquinase/genética , Glucose/farmacologia , Insulina/farmacologia , Fígado/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Endogâmicos , Tionucleotídeos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
A common pharmacophore for compounds structurally related to nitraquazone has been derived. Using this pharmacophore, new structures have been designed, synthesized, and evaluated for their inhibitory potencies against cyclic adenosine 5'-monophosphate (cAMP) specific phosphodiesterase (PDE 4). From these compounds, 4-benzylamino-2-butylthieno[3,2-d]pyrimidine (4) was selected for optimization. The effects of changes to the lipophilic groups and the amino linkage on the PDE 4 activity have been investigated. As a result, some potent PDE 4 inhibitors, selective with respect to PDE 3, have been identified. A selected group of compounds have been further evaluated for their ability to displace [3H]rolipram from its binding site and also to potentiate isoprenaline-induced cAMP accumulation in isolated guinea pig eosinophils. Of these, 2-butyl-4-cyclohexylaminothieno[3,2-d]pyrimidine (33) has an interesting profile, with an important improvement in PDE 4/[3H]rolipram ratio with respect to reference drugs, and good activity in cAMP potentiation, consistent with efficient cell penetration.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Antiasmáticos/síntese química , Desenho de Fármacos , Inibidores de Fosfodiesterase/síntese química , Pirimidinas/síntese química , Tiofenos/síntese química , Animais , Antiasmáticos/química , Antiasmáticos/metabolismo , Antiasmáticos/farmacologia , Ligação Competitiva , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Avaliação Pré-Clínica de Medicamentos , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Modelos Moleculares , Miocárdio/enzimologia , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirrolidinonas/metabolismo , Rolipram , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/metabolismo , Tiofenos/farmacologiaRESUMO
The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders. The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Benzotiadiazinas/síntese química , Inibidores Enzimáticos/síntese química , Isoenzimas/antagonistas & inibidores , Tiadiazinas/síntese química , Benzotiadiazinas/química , Benzotiadiazinas/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7 , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Recombinantes/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Tiadiazinas/química , Tiadiazinas/farmacologiaRESUMO
A series of 3,4-diaryloxazolones were prepared and evaluated for their ability to inhibit cyclooxygenase-2 (COX-2). Extensive structure-activity relationship work was carried out within this series, and a number of potent and selective COX-2 inhibitors were identified. The replacement of the methyl sulfone group on the 4-phenyl ring by a sulfonamide moiety resulted in compounds with superior in vivo antiinflammatory properties. In the sulfonamide series, the introduction of a methyl group at the 5-position of the oxazolone ring gave rise to very COX-2-selective compounds but with decreased in vivo activity. Selected 3,4-diaryloxazolones exhibited excellent activities in experimental models of arthritis and hyperalgesia. The in vivo activity of these compounds was confirmed with the evaluation of their antipyretic effectiveness and their ability to inhibit migration of proinflammatory cells. As expected from their COX-2 selectivity, most of the active compounds lacked gastrointestinal toxicity in vivo in rats after a 4-day treatment of 100 mg/kg/day. Within this novel series, sulfonamides 9-11 have been selected for further preclinical evaluation.
Assuntos
Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/farmacologia , Oxazóis/síntese química , Oxazóis/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/uso terapêutico , Febre/tratamento farmacológico , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Oxazóis/uso terapêutico , Prostaglandina-Endoperóxido Sintases/sangue , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 microM to 100 microM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 microM-100 microM) produced a consistent positive inotropic response in the presence of a concentration (1 microM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not gamma-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 microM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and beta-carboline-3-carboxylic acid methyl ester (betaCCMe), or by the inverse agonists, beta-carboline-3-carboxylic acid N-methylamide (betaCCMa) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0.1 microM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 microM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [3H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Diazepam/farmacologia , Coração/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Animais , Canais de Cloreto/fisiologia , AMP Cíclico/metabolismo , Interações Medicamentosas , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Cobaias , Coração/fisiologia , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , Ratos , Receptores de GABA-A/metabolismo , Rolipram , Trítio , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/fisiologiaRESUMO
1. Cyclo-oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX-1 and COX-2 which are encoded by different genes. COX-1 is expressed constitutively and is known to be the site of action of aspirin and other nonsteroidal anti-inflammatory drugs. COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the development of inflammation has been claimed. 2. Endothelial cells are an important physiological source of prostanoids and the selective induction of COX-2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines. 3. The HUV-EC-C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12-myristate 13-acetate (PMA) and interleukin-1 beta (IL-1 beta). 4. Both PMA and IL-1 beta produced dose- and time-dependent increases of the synthesis of the COX-derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2. 5. The increase of COX activity by PMA or IL-1 beta correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected. 6. Treatment of the cells with PMA induced a time-dependent increase in the expression of both COX-1 and COX-2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX-2 isoenzyme at the protein level. 7. The enzymatic activity of the PMA-induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX-1 selective assay. Classical non-steroidal anti-inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX-2 inhibitors (SC-58125, NS-398 and nimesulide) showed higher potency towards the COX of PMA-treated HUV-EC-C. 8. Overall, it appears that the stimulation of the HUV-EC-C line with PMA selectively induces the COX-2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin.
Assuntos
Endotélio/efeitos dos fármacos , Interleucina-1/farmacologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Indometacina/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Isolated vegetative tumour cells from mice bearing the Lewis lung carcinoma showed low rates of basal respiration with both low oxygen uptake rates and cytochrome-c oxidase activity. The cells were affected by a marked Crabtree effect and a high rate of lactate production in the presence of 10 mM glucose. The glycolytic capacity of the tumour was also assessed through the measurement of the maximum activities for hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase. These activities were similar to the ones found in other fast-growing, undifferentiated tumours. The concentration of fructose-2,6-bisphosphate in the tumour was 2,3 nmoles/g fresh tissue wt., a value which is of the same order of magnitude as that found in other types of highly glycolytic cells. It is concluded that the Lewis lung carcinoma follows the same pattern as other undifferentiated tumours with a high capacity for both glucose and amino acid utilization.
Assuntos
Glicólise/fisiologia , Neoplasias Pulmonares/metabolismo , Consumo de Oxigênio/fisiologia , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Frutosedifosfatos/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Lactatos/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Fosfofrutoquinase-1/metabolismo , Piruvato Quinase/metabolismoRESUMO
The incubation of isolated rat hepatocytes with vanadate increased the concentration of fructose 2,6-bisphosphate without modifying 6-phosphofructo-2-kinase activity. Vanadate also reverted and prevented the decrease of fructose 2,6-bisphosphate levels, of the "active" form of the 6-phosphofructo 2-kinase and of the pyruvate kinase activity ratio produced by glucagon, by probably counteracting the increase in cyclic AMP concentration.
Assuntos
Glucagon/farmacologia , Fígado/metabolismo , Vanadatos/farmacologia , Animais , AMP Cíclico/metabolismo , Jejum , Alimentos , Frutosedifosfatos/metabolismo , Cinética , Fígado/efeitos dos fármacos , Masculino , Fosfofrutoquinase-1/metabolismo , Fosforilação , Piruvato Quinase/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Streptozotocin diabetic rats showed more than a 4-fold increase in blood glucose levels, whereas hepatic glycogen, fructose 2,6-bisphosphate concentration, and 6-phosphofructo-2-kinase activity were decreased. The "total" 6-phosphofructo-2-kinase and the "active" (nonphosphorylated) form of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Vanadate administration for a 2-week period restored the altered values in the diabetic rats without modifying significantly in the control animals any of the parameters studied. Glucokinase activity was essentially lacking in the diabetic animals, and vanadate treatment restored the activity to about 65% of its control value, a good correlation between the recovery of the enzyme and the blood glucose level being observed. These results show an insulin-like effect of vanadate in the whole animal and suggest that insulin and vanadate possess similar actions on hepatic intracellular events.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Frutosedifosfatos/metabolismo , Glucoquinase/metabolismo , Hexosedifosfatos/metabolismo , Insulina/farmacologia , Fígado/enzimologia , Vanadatos/farmacologia , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fígado/efeitos dos fármacos , Glicogênio Hepático/metabolismo , Fosfofrutoquinase-2 , Fosfotransferases/metabolismoRESUMO
The presence of vanadate in primary cultures of rat hepatocytes produced a significant increase in the concentration of fructose 2,6-bisphosphate and in the activity of 6-phosphofructo 2-kinase. Compared with insulin, vanadate had a more potent action on the metabolite increase, but a similar effect on the 6-phosphofructo 2-kinase activity. Both the insulin- and the vanadate-dependent enhancements of 6-phosphofructo 2-kinase were inhibited by cycloheximide which specifically blocks protein synthesis on the translational level, suggesting that the increase of the enzyme activity was due to induction rather than to a change in the catalytic activity.
Assuntos
Frutosedifosfatos/metabolismo , Fígado/efeitos dos fármacos , Fosfofrutoquinase-1/efeitos dos fármacos , Vanadatos/farmacologia , Animais , Células Cultivadas , Feminino , Insulina/farmacologia , Fígado/enzimologia , Fígado/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Vanadate and insulin were administered to diabetic (streptozotocin) rats to compare their effects on the activity and mRNA content of 6-phosphofructo-2-kinase and L-type pyruvate kinase in the liver. The activity of 6-phosphofructo-2-kinase in livers of diabetic rats was about 40% of that found in normal rats. A similar decrease was found for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase content, measured by immunoprecipitation, and for mRNA, measured by hybridization of Northern blots. Administration of vanadate to the diabetic rats led to a progressive recovery of 6-phosphofructo-2-kinase activity, and 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase content and mRNA. This recovery, which was complete after 15 days of oral treatment, was also obtained after 60 h of insulin administration. L-type pyruvate kinase activity and mRNA were also decreased by about 70% in livers of diabetic rats. Both parameters normalized after 15 days of vanadate treatment, whereas insulin administration (60 h) raised L-pyruvate kinase mRNA three-fold above control values. Oral treatment for 15 days with vanadate can thus mimic the effect of insulin on both pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase in livers of diabetic rats.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Fígado/enzimologia , Fosfotransferases/metabolismo , RNA Mensageiro/metabolismo , Vanadatos/farmacologia , Administração Oral , Animais , Glicemia/metabolismo , Citosol/enzimologia , Diabetes Mellitus Experimental/genética , Imunoensaio , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Fosfofrutoquinase-2 , Fosfotransferases/genética , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Valores de Referência , Vanadatos/administração & dosagemRESUMO
Mice bearing the Lewis lung carcinoma showed a high tumour glutaminase activity and significantly higher concentrations of most amino acids than in both the liver and the skeletal muscle of the host. Tumour tissue slices showed a marked preference for glutamine, especially for oxidation of its skeleton to CO2. It is proposed that the metabolism of this particular carcinoma is focused on amino acid degradation, glutamine being its preferred substrate.
Assuntos
Aminoácidos/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Carcinoma de Células Escamosas/enzimologia , Glucose/metabolismo , Glutaminase/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Neoplasias Pulmonares/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Transplante de Neoplasias , Proteínas/metabolismoRESUMO
The respiratory O2 consumption of vegetative, non-necrotic, Lewis lung carcinoma cells was found to be very low compared with that of non-tumor tissues and was highly resistant to cyanide. However, the resistant rate was inhibited by salicylhydroxamic acid (SHAM) in either isolated cells or tissue fragments. In addition, this compound did not affect their cytochrome-c oxidase activity. The results support the existence of an alternative oxidase system that significantly contributes to oxygen uptake in Lewis carcinoma cells. To our knowledge, this is the first report showing significant SHAM-sensitivity of tumor respiration and perhaps of higher animal cell respiration.
Assuntos
Cianetos/farmacologia , Neoplasias Pulmonares/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Cianeto de Potássio/farmacologia , Animais , Antiprotozoários/farmacologia , Resistência a Medicamentos , Camundongos , Camundongos Endogâmicos , Salicilamidas/farmacologia , Células Tumorais Cultivadas/metabolismoRESUMO
The CD150 (SLAM) family consists of nine leukocyte cell-surface proteins involved in lymphocyte activation that belong to the immunoglobulin (Ig) superfamily. Six members of this family--CD84, CD150 (SLAM), CD229 (Ly9), CD244 (2B4), NTB-A, and CS1--associate with adapter proteins--SLAM-associated protein (SAP) and EAT-2. SAP is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative disease. Flow cytometric analysis of the expression of CD84, CD150, CD229, and CD244 cell-surface receptors on several leukocyte and lymphocyte subsets was performed. CD84 and CD150 were present on thymocytes, mature T cells and antigen-presenting cells. The expression of CD84 and CD150 was high on memory T cells. CD150 expression was strongly up-regulated after cell activation. In contrast to CD84, CD150 was absent on resting monocytes and immature dendritic cells (DCs). CD229 presented a pattern of expression restricted to lymphocytes. CD244 was preferentially expressed on natural killer cells, CD8(+) effector cells, resting monocytes, basophils, and eosinophils. We describe a broader distribution of CD84, CD150, CD229, and CD244 than previously reported and show that they are differentially expressed on hematopoietic cells. The heterogeneous expression of these receptors indicates that these molecules may play non-redundant functions in the regulation of both innate and adaptive immune responses.