RESUMO
BACKGROUND: Extracellular ATP contributes to the pathogenesis of asthma via signalling at purinergic receptors. However, the precise purinergic receptors subtypes mediating the pro-asthmatic effects of ATP have not been identified, yet. METHODS: In vivo studies were performed using the OVA-alum model. Functional expression of the P2Y(2) purinergic receptor subtype on human monocyte-derived dendritic cells and eosinophils was investigated using real-time PCR, migration assays, and production of reactive oxygen species. RESULTS: Compared to wild-type animals P2Y(2) -/- mice showed reduced allergic airway inflammation which can be explained by defective migration of blood myeloid DCs towards ATP in vitro and in vivo, whereas the influence of ATP on maturation and cytokine production was not changed. Additionally, ATP failed to induce migration of bone marrow-derived eosinophils from P2Y(2) R-deficient animals. The relevance of our findings for humans was confirmed in functional studies with human monocyte-derived DCs and eosinophils. Interestingly, stimulation of human DCs derived from allergic individuals with house dust mite allergen induced functional up-regulation of the P2Y(2) R subtype. Furthermore, eosinophils isolated from asthmatic individuals expressed higher levels of P2Y(2) R compared to healthy controls. This was of functional relevance as these eosinophils were more sensitive to ATP-induced migration and production of reactive oxygen metabolites. CONCLUSIONS: In summary, P2Y(2) R appears to be involved in asthmatic airway inflammation by mediating ATP-triggered migration of mDCs and eosinophils, as well as reactive oxygen species production. Together our data suggest that targeting P2Y(2) R might be a therapeutic option for the treatment of asthma.
Assuntos
Quimiotaxia de Leucócito/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Hipersensibilidade/imunologia , Pneumonia/imunologia , Receptores Purinérgicos P2Y2/imunologia , Trifosfato de Adenosina/imunologia , Animais , Linhagem Celular , Células Dendríticas/metabolismo , Eosinófilos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio , Receptores Purinérgicos P2Y2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Feixe Acessório Atrioventricular , Ablação por Cateter , Humanos , Imageamento Tridimensional , Fascículo Atrioventricular , Sistema de Condução Cardíaco/cirurgia , Feixe Acessório Atrioventricular/cirurgia , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/cirurgia , EletrocardiografiaRESUMO
Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.
Assuntos
Apoptose/fisiologia , Polirribossomos/metabolismo , RNA Ribossômico 28S/metabolismo , Ribonucleases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Bovinos , Permeabilidade da Membrana Celular , Células Cultivadas , AMP Cíclico/farmacologia , Dano ao DNA/fisiologia , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Ribossômico 28S/genética , Ratos , Ribossomos/químicaRESUMO
Nucleotides are ubiquitous intercellular messengers whose actions are mediated by specific receptors. Since the first clonings in 1993, it is known that nucleotide receptors belong to two families: the ionotropic P2X receptors and the metabotropic P2Y receptors. Five human P2Y receptor subtypes have been cloned so far and a sixth one must still be isolated. In this review we will show that they differ by their preference for adenine versus uracil nucleotides and triphospho versus diphospho nucleotides, as well as by their transduction mechanisms and cell expression.
Assuntos
Nucleotídeos/fisiologia , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/fisiologia , Adenilil Ciclases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Exocitose , Espaço Extracelular/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Células HL-60/efeitos dos fármacos , Humanos , Canais Iônicos/metabolismo , Ligantes , Nucleotídeos/química , Fosfatidilinositol Diacilglicerol-Liase , Proteínas Quinases/fisiologia , Receptores Purinérgicos P2/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismoRESUMO
Through the cAMP signaling pathway, TSH stimulates thyroid follicular cell proliferation, differentiation, and function. Although the autocrine production of IGF-I in the thyroid gland suggests an important physiological function for this factor in these processes, the exact role of the IGF-I/IGF-I receptor system in vivo remains unclear. Although the mitogenic action of TSH requires the presence of IGF-I or insulin in primary culture of dog and human thyroid cells, IGF-I has an effect equal to and independent of the effect of TSH on cell proliferation in rat thyroid cell lines and may even be the main growth regulator in this case. To investigate the in vivo function of the IGF-I/IGF-I receptor system, transgenic mice overexpressing human IGF-I, IGF-I receptor, or both in the thyroid were generated. Adult transgenic mice did not present external signs of thyroid dysfunction, but mice overexpressing both transgenes had significantly increased gland weight and follicular lumen area. A decreased TSH level together with a slightly increased serum T(4) concentration and increased thyroidal iodine uptake were also observed, suggesting that IGF-I and IGF-I receptor stimulate thyroid function to some extent in vivo.
Assuntos
Bócio/etiologia , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Glândula Tireoide/fisiopatologia , Tireotropina/fisiologia , Animais , Bócio/genética , Bócio/patologia , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Iodo/metabolismo , Camundongos , Camundongos Transgênicos/genética , Tamanho do Órgão , Receptor IGF Tipo 1/genética , Valores de Referência , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tireotropina/sangue , Tiroxina/sangueRESUMO
The cytoskeleton undergoes dramatic changes during apoptosis and many cytoskeletal proteins are known to be degraded during this process. The number of proteases found to be involved in apoptosis is growing but the role of the proteolysis they cause remains poorly understood. This report describes for the first time that myosin heavy chain is cleaved in aortic endothelial cell apoptosis induced either by tumour necrosis factor-alpha or okadaic acid. The cleavage was specific since a well-defined major 97 kDa fragment of myosin heavy chain was produced. The intermediate filament component vimentin was also cleaved into well-defined fragments (31, 28 and 23 kDa). Kinetic studies showed that proteolysis occurred concomitantly with the morphological changes associated with apoptosis, i.e. cellular condensation and fragmentation in apoptotic bodies. These data suggest that the degradation of myosin and vimentin could be involved in the execution of the morphological alterations observed during apoptotic cell death.
Assuntos
Apoptose/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Cadeias Pesadas de Miosina/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Animais , Aorta/citologia , Apoptose/efeitos dos fármacos , Western Blotting , Bovinos , Células Cultivadas , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Eletroforese em Gel Bidimensional , Endotélio Vascular/química , Inibidores Enzimáticos/farmacologia , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/química , Ácido Okadáico/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Vimentina/análise , Vimentina/químicaRESUMO
HL-60 cells are human promyelocytic cells expressing two ATP receptors: the P2Y(2) and P2Y(11) subtypes. Our Northern blotting experiments have shown that P2Y(2) and P2Y(11) messengers were up-regulated in these cells, rapidly and independently of protein synthesis, following treatment with granulocytic differentiating agents such as retinoic acid, dimethylsulfoxide, granulocyte-colony stimulating factor, dibutyryl cyclic AMP and ATP. AR-C67085 and adenosine 5'-O-(3-thiotriphosphate), two potent agonists of the recombinant P2Y(11) receptor, increased intracellular cAMP concentration in HL-60 cells more potently than ATP itself. These observations support the conclusion that the effect of ATP on HL-60 cell differentiation is mediated by the P2Y(11) receptor.
Assuntos
Granulócitos/citologia , Granulócitos/metabolismo , Receptores Purinérgicos P2/biossíntese , Diferenciação Celular , Células HL-60 , Humanos , RNA Mensageiro/biossíntese , Regulação para CimaRESUMO
1 The human P2Y11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways. A pharmacological characterization of the recombinant human P2Y11 receptor has been conducted following stable expression in two different cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO-K1 cells for cyclic AMP assays. The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPgammaS approximately BzATP > dATP > ATP > ADPbetaS > 2MeSATP. 2 ADPbetaS, AMPalphaS and A3P5PS behaved as partial agonists of the human P2Y11 receptor. At high concentrations, these three nucleotides were able to partially inhibit the ATP response. 3 Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was completely inactive. The P2Y11 receptor proved to be sensitive to suramin in a competitive way with an apparent Ki value of 0.82+/-0. 07 microM. 4 The ATP derivative AR-C67085 (2-propylthio-beta, gamma-dichloromethylene-D-ATP), a potent inhibitor of ADP-induced platelet aggregation, was the most potent agonist of the P2Y11 receptor, among the various nucleotides tested. 5 The pharmacological profile of the recombinant human P2Y11 receptor is closely similar to that of the cyclic AMP-coupled P2 receptor recently described in HL-60 cells, suggesting that it is the same receptor.
Assuntos
Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Fosfoadenosina Fosfossulfato/farmacologia , Receptores Purinérgicos P2/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Suramina/farmacologia , Teofilina/análogos & derivados , Teofilina/farmacologia , Tionucleotídeos/farmacologia , Fatores de Tempo , Triazinas/farmacologia , Células Tumorais CultivadasRESUMO
The P2Y6 receptor is a recently cloned P2 receptor which displays a high sensitivity for diphosphonucleotides. In 1321N1 astrocytoma cells stably expressing this receptor, UDP induced a slow and sustained accumulation of inositol trisphosphate via a pertussis toxin-insensitive G-protein: the maximal level was only reached after 15 min and a significant response was maintained for at least 3 h. A full second response to UDP was obtained after the first 45-min stimulation, but was lost after 165 min. This slow and sustained time-course and the lack of desensitization was reproduced with ADP. UTP was unable to restimulate the P2Y4 receptor, another recently cloned P2 receptor with a preference for UTP, after the first 5-min stimulation. The P2Y4 receptor is thus rapidly desensitized whereas desensitization of the P2Y6 receptor is delayed. The rank order of potency of various diphosphonucleotides at the P2Y6 receptor was: UDP > TDP > IDP > GDP > ADP >> CDP. The activity of three non-specific antagonists of P2 receptors was characterized by the following rank order of potency: reactive blue 2 > pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) > suramin. In conclusion, the most impressive features of the human P2Y6 receptor revealed by this study are the slow and sustained time-course of its activation and its high resistance to desensitization.
Assuntos
Receptores Purinérgicos P2/metabolismo , Difosfato de Adenosina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/biossíntese , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Suramina/farmacologia , Triazinas/farmacologia , Células Tumorais Cultivadas , Difosfato de Uridina/farmacologiaRESUMO
To isolate the mouse P2Y4 receptor gene, a mouse genomic library was screened with a human P2Y4 probe. An open reading frame encoding a protein of 361 amino acids was isolated. This protein showed 82% and 95% amino acid identity with the human and rat P2Y4 receptors, respectively. By reverse transcription and polymerase chain reaction (RT-PCR), the P2Y4 messenger RNA was detected in mouse liver, intestine, stomach, bladder and lung among the 16 mouse tissues tested. In 1321N1 transfected cells, the mouse P2Y4 receptor was equally activated by UTP and ATP, and was antagonized by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and Reactive Blue 2, and not by suramin. Moreover, when expressed in 1321N1 cells, the rat P2Y4 is also antagonized by PPADS. Thus, when compared in the same expression system, the mouse P2Y4 is closer to the rat ortholog in terms of agonist stimulation, while in terms of antagonist profile, the three P2Y4 receptor orthologs are similar.
Assuntos
Clonagem Molecular , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Inibidores da Agregação Plaquetária/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Receptores Purinérgicos P2/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Suramina/farmacologia , Transfecção , Triazinas/farmacologia , Células Tumorais Cultivadas , Uridina Trifosfato/farmacologiaRESUMO
The circadian control of cell Proliferation and Differentiation has been studied principally in rat liver. The comparison between the differentiation by hepatic enzymes and the division by the cell cycle under various experimental conditions (postnatal maturation, regeneration after partial hepatectomy, adrenalectomy, corticosterone treatments etc.) leads to the following conclusions: Under physiological conditions, proliferation and differentiation activities present a mutually exclusive relationship with a specific circadian rhythm. For both functions, the circadian variation of corticosterone plays the role of synchronizer, each evening (peak) it induces the synthesis of tissue specific enzymes in G0 cells and simultaneously inhibits the DNA synthesis in cycling cells. The same parameters have been studied during the different stages of hepatocarcinogenesis induced by Diethylnitrosamine (DEN). After initiation alone, (DEN for 2 weeks) circadian control is unchanged and precancerous cells are not able to reach malignancy. Promotion (DEN for 6 weeks) consists of disturbing the circadian synchronization to liberate the selective growth of initiated precancerous cells. This proliferation advantage favours the accumulation of chromosomal aberrations including those implicated in malignant transformation: i.e. activation of oncogenes or inhibition of antioncogenes.
Assuntos
Neoplasias Hepáticas/fisiopatologia , Fígado/citologia , Animais , Divisão Celular , Células , Fenômenos Cronobiológicos , Ritmo Circadiano , Humanos , Cinética , Fígado/fisiologia , RatosRESUMO
BACKGROUND/OBJECTIVES: We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor and P2Y(12) receptor deficiency in vivo. METHODS: Deep vein thrombosis was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor or vehicle and in P2Y(12-/-) mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles. RESULTS: Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25 min. Sham-operated mice, enoxaparin- and ticagrelor-pretreated wild-type and P2Y(12-/-) mice developed only small thrombi. After injection of the contrast agent, growing thrombi were delineated clearly as negative contrast on CEUS. Thrombus size on CEUS after 25 min was significantly smaller in enoxaparin- (0.3 ± 0.1 mm(2)) and ticagrelor-treated (0.5 ± 0.1 mm(2)) wild-type and in P2Y(12-/-) mice (0.4 ± 0.1 mm(2)) as compared with vehicle-treated wild-type mice (2.0 ± 0.3 mm(2)) in the maximal sagittal plane (P < 0.001, n = 5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT. CONCLUSIONS: Contrast-enhanced ultrasound allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y(12) receptor in early DVT formation.
Assuntos
Anticoagulantes/farmacologia , Enoxaparina/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/química , Trombose Venosa/diagnóstico por imagem , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Cloretos/química , Meios de Contraste/farmacologia , Compostos Férricos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbolhas , Microscopia , Transdução de Sinais , Hexafluoreto de Enxofre/química , Trombose/tratamento farmacológico , Ticagrelor , Ultrassonografia , Veia Cava Inferior/patologia , Trombose Venosa/induzido quimicamenteRESUMO
RATIONALE: Extracellular nucleotides have widespread effects and various cell responses. Whereas the effect of a purine nucleotide (ATP) and a pyrimidine nucleotide (UTP) on myocardial infarction has been examined, the role of different purine and pyrimidine nucleotides and nucleosides in cardioprotection against hypoxic stress has not been reported. OBJECTIVE: To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia. METHODS AND RESULTS: Rat cultured cardiomyocytes were treated with various extracellular nucleotides and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y2 -/- , P2Y4 -/-, and P2Y2 -/-/P2Y4 -/- receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. CONCLUSIONS: Extracellular tri/di-phosphates are apparently the molecule responsible for cardioprotection against hypoxic damage, probably by preventing free radicals formation.
Assuntos
Líquido Extracelular/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia , Nucleotídeos de Purina/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Nucleotídeos de Pirimidina/farmacologia , Animais , Antioxidantes/farmacologia , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quelantes/farmacologia , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Nucleosídeos de Purina/metabolismo , Nucleotídeos de Purina/metabolismo , Antagonistas Purinérgicos/farmacologia , Nucleosídeos de Pirimidina/metabolismo , Nucleotídeos de Pirimidina/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y2/genética , Estresse Fisiológico , Uridina Trifosfato/fisiologiaRESUMO
Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na(+) channel (ENaC)-mediated Na(+) absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y(2) and a luminal P2Y(4) receptor, stimulate K(+) secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na(+) absorption in distal colonic mucosa of mice treated on a low Na(+) diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V (te)): -13.7 +/- 1.9 mV (lumen negative), transepithelial resistance (R (te)): 24.1 +/- 1.8 Omega cm(2), equivalent short circuit current (I (sc)): -563.9 +/- 63.8 microA/cm(2) (n = 21). Amiloride completely inhibited I (sc) to -0.5 +/- 8.5 microA/cm(2). Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I (sc) by 160.7 +/- 29.7 microA/cm(2) (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC(50) values of 10 microM and 3 microM respectively. In P2Y(2) knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na(+) absorption was absent. In contrast, in P2Y(4) KO mice the inhibitory effect of luminal UTP on Na(+) absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na(+) diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y(2) and P2Y(4) receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y(2) receptor mediates inhibition of electrogenic Na(+) absorption.
Assuntos
Colo/metabolismo , Absorção Intestinal/fisiologia , Receptores Purinérgicos P2/metabolismo , Sódio/metabolismo , Trifosfato de Adenosina/metabolismo , Amilorida/farmacologia , Animais , Separação Celular , Colo/citologia , Dieta Hipossódica , Cultura em Câmaras de Difusão , Diuréticos/farmacologia , Hipoxantina Fosforribosiltransferase/genética , Técnicas In Vitro , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Nucleotídeos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/farmacologia , Especificidade da Espécie , Uridina Trifosfato/metabolismoRESUMO
Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K+ secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP-stimulated K+ secretion was still present in P2Y2 receptor knock-out mice, but significantly reduced (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 2.08 +/- 0.52 mV, n = 9). In P2Y4 receptor knock-out mice the UTP-induced K+ secretion was similarly reduced. Luminal UTP-stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.
Assuntos
Colo/metabolismo , Potássio/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Colo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Knockout , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2Y2RESUMO
Using the arachidonic acid release as a probe of phospholipase A2 activity, we tested the involvement of this enzyme in the TNF-triggered apoptotic cell death in the bovine aortic endothelial cells. We observed that TNF induced a liberation of arachidonic acid from these cells which was comparable to that obtained from the L929 cells. An augmentation of the amount of released arachidonic acid or the reduction of the TNF-stimulated phospholipase A2 activity did not modify the TNF-induced DNA fragmentation in the endothelial cells. We suggest that these events are not required for TNF apoptotic cytotoxicity in the endothelial cells.
Assuntos
Apoptose/fisiologia , DNA/metabolismo , Endotélio Vascular/enzimologia , Fosfolipases A/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Ácido Araquidônico/metabolismo , Bovinos , Fosfolipases A2RESUMO
ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).
Assuntos
Nucleotídeos de Adenina/fisiologia , Endotélio Vascular/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Aorta , Bradicinina/farmacologia , Calcimicina/farmacologia , Cálcio/fisiologia , Bovinos , Toxina da Cólera/farmacologia , Colina/metabolismo , Regulação para Baixo , Espaço Extracelular/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Técnicas In Vitro , Toxina Pertussis , Proteína Quinase C/fisiologia , Receptores Purinérgicos/fisiologia , Relação Estrutura-Atividade , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The establishement of the circadian rhythm of cholesterol 7alpha-hydroxylase activity requires protein and RNA synthesis. The spontaneous decrease of the enzymic activity, at the end of the night, allows us to evaluate a half-life time of about two hours. The half-life time goes up to about four hours when the enzymatic activity decay is measured following cycloheximide administration. This difference suggests that an active mechanism is involved in the control of the enzyme degradation. The daily variation of the enzyme activity is regulated via the hypothalamo-hypophysis-adrenal axis. At the cellular level glucocorticoids are the most likely responsible agent. The hepatic cholesterol 7alpha-hydroxylase variations always parallel the plasmatic corticosterone concentration fluctuations, the latter being by far the most important adrenocortical excretion product. These two rhythms are modified in a similar manner under different physio-pathological conditions, such as the inversion of lighting in the animal room or the inversion of feeding time. Of these two parameters, the moment of food intake is the most important and accounts for the synchronisation of the rhythm in the animals. The rhythm is retained after several days of starvation but its amplitude decreases and the individual variations among the animals increase significantly at each time point.
Assuntos
Colesterol 7-alfa-Hidroxilase/fisiologia , Ritmo Circadiano , Fígado/enzimologia , Esteroide Hidroxilases/fisiologia , Animais , Ritmo Circadiano/efeitos dos fármacos , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Escuridão , Luz , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Fatores de TempoRESUMO
The pattern of protein expression and phosphorylation after an apoptotic stimulus has been studied in two systems. Bovine aortic endothelial cells were induced to undergo apoptotic cell death by a combination of a cytokine (tumor necrosis factor, TNF) and inhibitors of protein synthesis, like cycloheximide. Two-dimensional (2-DE) electrophoresis of proteins from such cells revealed specific proteolysis of distinct proteins, some at an early stage of apoptosis and some at a later stage. These proteins may have antiapoptotic properties. In rat IPC-81 promyelocytic leukemia cells, cAMP induced apoptosis. 2-DE of such cells pulse-labeled with [35S]methionine revealed two "novel" protein spots (of 30 kDa and 46 kDa, respectively), induced very rapidly by a posttranscriptional mechanism. It is proposed that "dysphosphorylation" may accompany apoptosis in general, since both endothelial cells treated with TNF/cycloheximide and IPC-81 cells treated with cAMP analog or the apoptosis-inducing phosphatase inhibitors okadaic acid or calyculin A all showed altered protein phosphorylation patterns, as revealed by 2-DE electrophoresis of proteins from cells prelabeled with 32Pi.
Assuntos
Apoptose/fisiologia , Eletroforese em Gel Bidimensional/métodos , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fosfoproteínas/metabolismo , Biossíntese de Proteínas , Animais , Aorta , Apoptose/efeitos dos fármacos , Autorradiografia/métodos , Bovinos , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Endopeptidases/metabolismo , Endotélio Vascular/efeitos dos fármacos , Fluorescência , Leucina/metabolismo , Fosfatos/metabolismo , Fosfoproteínas/isolamento & purificação , Radioisótopos de Fósforo , Fosforilação , Proteínas/isolamento & purificação , Tionucleotídeos/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis.