RESUMO
Prostaglandin (PG) D2, the major cyclooxygenase metabolite generated from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases due to its various inflammatory effects. However, since no DP receptor antagonist has been developed as an antiallergic drug, the role of PGD2 in the pathogenesis of allergic diseases remains uncertain. Here, we report the in vivo efficacy of our newly established DP receptor antagonist, S-5751 [((Z)-7-[(1R,2R,3S,5S)-2-(5-hydroxy benzo[b]thiophen-3-ylcarbonylamino)-10-norpinan-3-yl]hept-5- enoic acid)], using various allergic inflammation guinea pig models. In allergic rhinitis models, oral administration of S-5751 dramatically inhibited not only early nasal responses, as assessed by sneezing, mucosal plasma exudation, and nasal blockage, but also late responses such as mucosal plasma exudation and eosinophil infiltration. Even when S-5751 was administered after recovery from the early responses, these late phase responses were almost completely suppressed. In addition, S-5751 alleviated allergen-induced plasma exudation in the conjunctiva in an allergic conjunctivitis model and antigen-induced eosinophil infiltration into the lung in an asthma model. These findings provide evidence for the crucial role of PGD2 as a mediator of allergic inflammation in guinea pigs and suggest that DP receptor antagonists may be useful in the treatment of allergic diseases triggered by mast cell activation.
Assuntos
Hipersensibilidade/prevenção & controle , Inflamação/prevenção & controle , Antagonistas de Prostaglandina/farmacologia , Receptores de Prostaglandina/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Conjuntivite/tratamento farmacológico , Conjuntivite/patologia , AMP Cíclico/sangue , Cobaias , Células HL-60 , Humanos , Hipersensibilidade/complicações , Técnicas In Vitro , Inflamação/etiologia , Ligantes , Masculino , Doenças Respiratórias/tratamento farmacológico , Doenças Respiratórias/patologia , Rinite Alérgica Sazonal/tratamento farmacológico , Rinite Alérgica Sazonal/patologiaRESUMO
1. TxA2 receptor (TP-receptor) antagonists such as S-1452 and Bay u 3405 have been shown to be effective in alleviating nasal blockage in patients with allergic rhinitis as well as guinea-pig allergic rhinitis models. The present study was conducted to examine the existence and localization of the TP-receptor in human and guinea-pig nasal mucosa by in vitro receptor binding autoradiography using radiolabelled (+)-S-145, which is a potent and specific TP-receptor antagonist with an extremely slow dissociation rate. 2. We ascertained the binding specificity of [3H]-(+ )-S-145 in human and guinea-pig platelet membranes by comparing the ability of four TP-receptor ligands of U-46619, (+)-S-145, I-(+)-S-145 and Bay u 3405 to displace the specific binding of [3H]-(+)-S-145 and [3H]-U-46619. The rank order of potency (Ki) for the displacement was correlated highly with that determined from [3H]-U-46619 binding to the same preparations. 3. Quantitative autoradiography using a radioluminographic imaging plate system, in which the radioactivity of [3H]-(+)-S-145 is expressed as photostimulated luminescence (PSL) per area (mm2), revealed that specific binding of [3H]-(+)-S-145 to human and guinea-pig nasal mucosa was saturable. Scatchard analysis showed about three fold higher affinity and two fold greater maximal binding to the nasal mucosa of humans than that of guinea-pigs: the KD and Bmax values in human mucosa were 2.82+/-0.35 nM and 6.47+/-0.33 PSL mm(-2) and those in guinea-pig mucosa were 8.23+/-1.93 nM and 3.37+/-0.66 PSL mm(-2), respectively. 4. Specific [3H]-(+)-S-145 binding to cryostat sections of human and guinea-pig nasal mucosa was displaced by another TP-receptor antagonist, Bay u 3405, and a TP-receptor agonist, U-46619. The order of potency (Ki value: nM) was (+)-S-145 (2.5) > Bay u 3405 (15.4) > > U-46619 (359.6) in human nasal mucosa and (+)-S-145 (22.8) > U-46619 (49.8) approximately Bay u 3405 (62.1) in guinea-pig nasal mucosa. These rank orders showed rather good correlation with those obtained for the respective platelet membranes. 5. Autoradiographs of human nasal mucosa demonstrated that specific [125I]-(+)-S-145 binding sites mainly exist on the smooth muscle layers of venous sinusoids and arterioles in the lamina propria, with few or no binding sites in the epithelium and nasal gland. 6. We concluded that radiolabelled (+)-S-145 can be used as a TP-receptor ligand for autoradiographic study, and that the TP-receptor is exclusively located on smooth muscle layers of venous sinusoids and arterioles in the nasal mucosa. The potent vasoconstrictive activity of TxA2 may cause reduction of local blood flow followed by mucosal oedema probably through mechanisms of vascular injury such as ischaemia-reperfusion.
Assuntos
Compostos Bicíclicos com Pontes/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Mucosa Nasal/metabolismo , Receptores de Tromboxanos/metabolismo , Tromboxano A2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Animais , Autorradiografia , Ligação Competitiva , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cobaias , Humanos , Técnicas In Vitro , Radioisótopos do Iodo , Mucosa Nasal/efeitos dos fármacos , Ensaio Radioligante , Receptores de Tromboxanos/agonistas , Receptores de Tromboxanos/antagonistas & inibidoresRESUMO
Gas6, a product of a growth arrest-specific gene 6, potentiates proliferation of vascular smooth muscle cells and prevents cell death of vascular smooth muscle cells. It has been also demonstrated that Gas6 is a ligand of receptor tyrosine kinases Axl, Sky, and Mer. Gas6 contains gamma-carboxyglutamic acid residues, which are found in some blood coagulation factors and mediate the interaction of the coagulation factors with negatively charged phospholipid. In this study, we clarified that Gas6 specifically bound to phosphatidylserine and the binding was dependent on Ca2+ and gamma-carboxyglutamic acid residues. Furthermore, we found that U937 cells, which express Gas6 receptor on their surfaces, adhered to phosphatidylserine-coated enzyme-linked immunosorbent assay (ELISA) plate only in the presence of Gas6 and Ca2+. U937 cells also bound to ELISA plate coated with phosphatidylinositol, but the binding was independent of Gas6 and Ca2+. On the other hand, U937 cells did not adhere to phosphatidylcholine- or phosphatidylethanolamine-coated ELISA plate even in the presence of Gas6 and Ca2+. These findings suggest that Gas6 may play a role in recognition of cells exposing phosphatidylserine on their surfaces by phagocytic cells, which is supposed to be one of the mechanisms for clearing dying cells.
Assuntos
Adesão Celular , Peptídeos e Proteínas de Sinalização Intercelular , Fosfatidilserinas/metabolismo , Proteínas/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Ensaio de Imunoadsorção Enzimática , Cinética , Magnésio/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Células Tumorais Cultivadas , Receptor Tirosina Quinase AxlAssuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Norbornanos/farmacologia , Prostaglandina D2/metabolismo , Receptores Imunológicos , Receptores de Prostaglandina/administração & dosagem , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/química , Plaquetas/efeitos dos fármacos , Compostos Bicíclicos com Pontes/administração & dosagem , Compostos Bicíclicos com Pontes/química , Cobaias , Humanos , Norbornanos/administração & dosagem , Norbornanos/química , Receptores de Prostaglandina/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Gas6 (encoded by growth-arrest-specific gene 6) is a gamma-carboxyglutamic acid (Gla)-containing protein which is released from growth-arrested vascular smooth muscle cells (VSMCs) and potentiates VSMC proliferation induced by Ca2+-mobilizing growth factors, but not that induced by receptor tyrosine kinases. In this study we examined the importance of Gla residues for the biological activities of Gas6 and tried to assess the importance of endogenous Gas6 in VSMC proliferation. We demonstrated that Gla-deficient Gas6 lacked receptor-binding and growth-potentiating activities. Therefore the vitamin K-dependent modification of Gas6 appeared to be essential for its biological activities. Next we used warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, to estimate the contribution of endogenous Gas6 to VSMC proliferation. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Therefore the inhibition seems to be due to prevention of the vitamin K-dependent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing antibody against Gas6 gave a similar result, i.e. it inhibited thrombin-induced VSMC proliferation but not that induced by epidermal growth factor. These results indicate that endogenously produced Gas6 is very important for VSMC proliferation induced by Ca2+-mobilizing growth factors.
Assuntos
Ácido 1-Carboxiglutâmico/análise , Peptídeos e Proteínas de Sinalização Intercelular , Músculo Liso Vascular/citologia , Proteínas/química , Ácido 1-Carboxiglutâmico/metabolismo , Animais , Anticoagulantes/farmacologia , Western Blotting , Células CHO , Cálcio/metabolismo , Divisão Celular , Células Cultivadas , Cricetinae , Fator de Crescimento Epidérmico/farmacologia , Músculo Liso Vascular/metabolismo , Proteínas/metabolismo , Ratos , Relação Estrutura-Atividade , Trombina/farmacologia , Vitamina K/farmacologia , Varfarina/farmacologiaRESUMO
We have purified a novel growth-potentiating factor and demonstrated that the factor is coded by the gas6 gene. Moreover, we have suggested the presence of a Gas6 receptor on rat vascular smooth muscle cells. In this study, we further analyzed the binding of Gas6 to its receptor. Tissue and cellular distribution of the binding activity of (125)I-labeled Gas6 showed that the binding site existed in many but a limited range of tissues and cell types. Further characterization of the binding of [(125)I]Gas6 using HOS cells demonstrated that the specific binding was dependent on the presence of Ca(2+). Chemical cross-linking of [(125)I]Gas6 to HOS cells resulted in the formation of a high-molecular-mass complex, suggesting the presence of a high-molecular-weight receptor.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Músculo Liso Vascular/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Cálcio/farmacologia , Células Cultivadas , Ácido Edético/farmacologia , Humanos , Ligantes , Músculo Liso Vascular/citologia , Ratos , Succinimidas/farmacologia , Células Tumorais CultivadasRESUMO
Proliferation of vascular smooth muscle cells (VSMC) is triggered by two types of growth factors. One activates tyrosine kinase-type receptors and the other activates G-protein-coupled receptors. We found that a conditioned medium of rat VSMC contained a growth-potentiating activity for the latter type of growth factor, and we purified a 70-kDa growth-potentiating factor (GPF) from the conditioned medium. Analyses of GPF and its cDNA revealed GPF to be a gamma-carboxyglutamic acid-containing protein encoded by a growth arrest-specific gene, gas6, which related to protein S. GPF specifically potentiated cell proliferation mediated by Ca(2+)-mobilizing receptors. The presence of a specific binding site suggests that the effect of GPF is mediated by a receptor. Thus, GPF may be a new type of extracellular factor regulating VSMC proliferation.
Assuntos
Cálcio/metabolismo , DNA/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Músculo Liso Vascular/citologia , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia em Gel , Cromatografia por Troca Iônica , Meios de Cultivo Condicionados , Fator de Crescimento Epidérmico/farmacologia , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Proteínas/isolamento & purificação , Proteínas/metabolismo , Ratos , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Trombina/farmacologia , UltrafiltraçãoRESUMO
Our earlier studies have shown that mammalian pancreatic group I phospholipase A2 (PLA2-I) has its specific receptor (PLA2 receptor) on a wide range of mammalian cells and that the receptor-binding capability of PLA2-I is a property of this molecule separable from its enzymatic activity. To clarify whether PLA2 activity is required for eliciting a biological response via the receptor or not, we examined the enzymatic activity of PLA2-I/PLA2 receptor complex and the inducibility of prostaglandin (PG) E2 production in rat mesangial cells by mutant PLA2s-I. Using a recombinant soluble PLA2 receptor, we first found that PLA2-I could not hydrolyze a phospholipid substrate when complexed with the receptor. In the next experiment using various mutant porcine PLA2s-I, we found that PGE2 production in rat mesangial cells could be induced by a mutant PLA2-I which retained the receptor-binding activity but had almost completely lost its enzymatic activity. These findings indicate that the enzyme action of PLA2-I is not required for a PLA2-I-induced biological response, i.e., the augmentation of PGE2 production in rat mesangial cells.
Assuntos
Dinoprostona/metabolismo , Mesângio Glomerular/metabolismo , Pâncreas/enzimologia , Fosfolipases A/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Ligação Competitiva , Células CHO , Cricetinae , Dinoprostona/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Cinética , Mutagênese Sítio-Dirigida , Fosfolipases A/biossíntese , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Mutação Puntual , Ratos , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/isolamento & purificação , Receptores da Fosfolipase A2 , Proteínas Recombinantes/metabolismo , Suínos , TransfecçãoRESUMO
We determined the nucleotide sequence of a mouse cDNA encoding the receptor for pancreatic group I phospholipase A2 (PLA2-I). Interspecies structural comparison of the mouse receptor with bovine PLA2-I receptor, whose structure had been clarified, revealed that the fourth carbohydrate-recognition domain (CRD)-like domain (CRD-like 4) was the most conserved among the domains in the PLA2-I receptor, suggesting the functional importance of CRD-like 4. A transient expression experiment with a truncated form of the receptor consisting of three CRD-like domains, from the third to the fifth, demonstrated that the PLA2-I-binding site of the receptor is constituted from these three CRD-like domains, supporting the functional indispensability of CRD-like 4 in the receptor. Since the PLA2-I-binding region was thus assigned to be CRD-like domains 3-5, we further analyzed the structures of the PLA2-I-binding regions in the PLA2-I receptors from the rat, rabbit and human. Furthermore, the obtained PLA2-I receptor cDNA fragments from these animals made it possible to examine the tissue expression patterns of this receptor in various mammals. The results, together with the results of the genomic structural analysis of this gene, indicated that a PLA2 receptor recently characterized by Lambeau et al. [Lambeau, G., Ancian, P., Barhanin, J. & Lazdunski, M. (1994) J. Biol. Chem. 269, 1575-1578] is a rabbit counterpart of the PLA2-I receptor although these two PLA2 receptors have distinctive PLA2-binding specificities.
Assuntos
Fosfolipases A/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Primers do DNA , Feminino , Feto , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Pâncreas/metabolismo , Fosfolipases A2 , Gravidez , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Coelhos , Ratos , Receptores de Superfície Celular/biossíntese , Receptores da Fosfolipase A2 , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We have recently reported that mammalian pancreatic group I phospholipase A2 (PLA2-I) has its specific receptor (PLA2 receptor) on a variety of mammalian cells and that various biological responses are elicited by PLA2-I via this receptor. In this study, we cloned cDNAs encoding a protein corresponding to the bovine PLA2 receptor purified from the corpora lutea on the basis of its partial amino acid sequences. The identity of a protein encoded by the cloned cDNA with the bovine PLA2 receptor was verified by a transient expression experiment using COS-7 cells. Interestingly, the deduced primary structure of the PLA2 receptor (1,463 amino acid residues) exhibits a close relatedness throughout the molecule to that of the macrophage mannose receptor, a unique member of Ca(2+)-dependent (C-type) animal lectin family, in spite of their functional diversity. Based on this sequence similarity between these two receptors, the domain organization of the PLA2 receptor could be tentatively assigned as follows; 10 extracellular domains including 8 tandem repeats homologous to C-type carbohydrate-recognition domains (CRDs) and a single transmembrane region followed by a short cytoplasmic tail. The results of transient expression experiments for mutant PLA2 receptors supported this assignment and furthermore suggested the region responsible for PLA2-I binding corresponds to CRDs in the mannose receptor.
Assuntos
Lectinas Tipo C , Lectinas de Ligação a Manose , Pâncreas/metabolismo , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Complementar , Receptor de Manose , Dados de Sequência Molecular , Mutação , RNA/análise , Receptores da Fosfolipase A2 , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
The effect of pancreatic group I phospholipase A2 (PLA2-I) on receptor-mediated expression of arthritic group II phospholipase A2 (PLA2-II) and its correlation with prostaglandin E2 (PGE2) synthesis were examined in cultured rat mesangial cells. Scatchard analysis using 125I-PLA2-I revealed the existence of a single class of specific binding sites for PLA2-I in rat mesangial cells with an equilibrium dissociation constant (Kd) of 1.6 nM and a maximum binding capacity of 10.1 fmol/10(6) cells. The mammalian mature type of PLA2-I specifically recognized this binding site, whereas its inactive zymogen and mammalian PLA2-II showed much lower affinities. PLA2-I markedly increased PLA2-II mRNA levels as well as PLA2-II secretion from the cells in a time- and dose-dependent manner that was closely correlated with PGE2 production. Both PLA2-II expression and PGE2 synthesis were completely suppressed by pretreatment of the cells with actinomycin D, cycloheximide, or dexamethasone. These results strongly suggest that there may be crosstalk between PLA2-I and PLA2-II via the specific PLA2-I receptor that elicits PGE2 synthesis.
Assuntos
Dinoprostona/biossíntese , Mesângio Glomerular/metabolismo , Isoenzimas/metabolismo , Isoenzimas/farmacologia , Pâncreas/enzimologia , Fosfolipases A/metabolismo , Fosfolipases A/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Venenos de Abelha/farmacologia , Sítios de Ligação , Ligação Competitiva , Células Cultivadas , Citosol/enzimologia , Venenos Elapídicos/farmacologia , Expressão Gênica , Mesângio Glomerular/enzimologia , Immunoblotting , Isoenzimas/biossíntese , Cinética , Masculino , Fosfolipases A/biossíntese , Fosfolipases A2 , Ratos , Ratos Wistar , SuínosRESUMO
Mammalian pancreatic phospholipase A2 (PLA2-I) has its specific receptor through which PLA2-I induces a variety of biological responses. In this study, a fundamental relationship between the enzymatic and the receptor-binding activities of PLA2-I was investigated. The specific binding of PLA2-I to the receptor was found to be independent of Ca2+ which is requisite for the PLA2 activity. On the basis of this observation, we designed and produced mutant PLA2-Is without Ca(2+)-binding abilities in order to demonstrate that the structural requirement for the enzymatic activity of PLA2-I is not identical with that for its receptor-binding reaction. These mutant PLA2-Is lost almost all enzymatic activity through a disturbance at the Ca(2+)-binding site, as expected, but still retained a substantial affinity to the receptor, allowing us to conclude that the receptor-binding reaction of PLA2-I is separable from its catalytic action.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Fosfolipases A/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Cães , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Pâncreas/enzimologia , Fosfolipases A2 , Ligação Proteica , Receptores da Fosfolipase A2 , Relação Estrutura-AtividadeRESUMO
The effects of three forms of endothelin (ET), ET-1, -2 and -3, on intracellular free Ca2+ concentration ([Ca2+]i), their receptor binding activities, and DNA synthesis were investigated in cultured porcine basilar arterial smooth muscle cells. Endothelin-1 and ET-2 induced cytosolic free calcium elevations that did not return to the basal level. The maximum levels of [Ca2+]i induced by the three points were ET-1 approximately equal to ET-2 > ET-3. The cells display specific binding of 125I-labelled endothelin-1 which was displaced by three endothelins; ET-1, -2, and -3, in a concentration-dependent manner. The order of potency in inhibiting 125I-labeled endothelin-1 binding was ET-1 approximately equal to ET-2 > ET-3. Endothelin-1, ET-2, and ET-3 stimulated [3H]thymidine incorporation in a dose-dependent manner, the maximum order of potency (100 nM) was again ET-1 approximately equal to ET-2 > ET-3. These results indicate that ET-1 and ET-2 are more potent in their biological activities than ET-3 in cultured cerebral arterial smooth muscle cells.
Assuntos
Endotelinas/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Artéria Basilar/citologia , Artéria Basilar/metabolismo , Ligação Competitiva/fisiologia , Cálcio/metabolismo , Células Cultivadas , Endotelinas/metabolismo , Feminino , Masculino , Músculo Liso Vascular/citologia , Suínos , Timidina/metabolismoRESUMO
Previously, we have reported a novel proliferative action of pancreatic group I phospholipase A2 (PLA2-I) via a specific binding site in Swiss 3T3 fibroblasts, vascular smooth muscle cells, and chondrocytes. In this study, we characterized the PLA2-I specific binding site in osteoblastic cell line (MC3T3-E1 cells) with an equilibrium binding constant (Kd) value of 1.13 nM and maximum binding capacity of 40.1 fmol/10(6) cells. PLA2-I stimulated prostaglandin E2 (PGE2) production in a concentration-dependent manner in MC3T3-E1 cells, and its EC50 value was similar to the Kd value for PLA2-I binding. This effect of PLA2-I was type-specific and did not depend on its hydrolytic activity. PLA2-I increased the activity of prostaglandin endoperoxide synthase (PES), and PLA2-I-stimulated PGE2 synthesis was inhibited by cycloheximide. Northern blot analysis showed the increase in both type-1 and type-2 PES mRNAs. These findings indicated that PLA2-I stimulated PGE2 synthesis by induction of PES via a specific binding site in osteoblastic cells.
Assuntos
Dinoprostona/biossíntese , Osteoblastos/metabolismo , Fosfolipases A/fisiologia , Células 3T3 , Animais , Ácido Araquidônico/metabolismo , Sítios de Ligação , Bradicinina/farmacologia , Calcimicina/farmacologia , Indução Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Camundongos , Ácido Oleico , Ácidos Oleicos/metabolismo , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
To understand the molecular mechanism of the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage, we analysed the effect of cerebrospinal fluid from patients with subarachnoid haemorrhage on DNA synthesis and cytosolic-free calcium elevation in cultured porcine cerebral smooth muscle cells. Cerebrospinal fluid from patients on day 2 after subarachnoid haemorrhage induced transient elevation in cytosolic-free calcium levels. In contrast, the maximal elevation of cytosolic-free calcium levels induced by cerebrospinal fluid from control patients (without subarachnoid haemorrhage) was significantly lower than that induced by cerebrospinal fluid from patients with subarachnoid haemorrhage. In cultured porcine cerebral arterial smooth muscle cells, cerebrospinal fluid from patients with subarachnoid haemorrhage promoted levels of [3H]-thymidine incorporation (DNA synthesis) more than 2.5-fold higher than that promoted by cerebrospinal fluid from control patients without subarachnoid haemorrhage. However, in cultured aortic smooth muscle cells, there was no significant difference in [3H]-thymidine incorporation between cerebrospinal fluid from patients with subarachnoid haemorrhage and that by control cerebrospinal fluid. From these results in cerebral arterial smooth muscle cells, cerebrospinal fluid from patients following subarachnoid haemorrhage may play not only constrictive functions, evidenced by cytosolic-free calcium elevations, but also proliferative functions, demonstrated by promotion of [3H]-thymidine incorporation. The relevance of these factors to vasospasm will be discussed.
Assuntos
Cálcio/metabolismo , Artérias Cerebrais/citologia , Replicação do DNA/efeitos dos fármacos , Ataque Isquêmico Transitório/etiologia , Músculo Liso Vascular/efeitos dos fármacos , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Animais , Fatores Biológicos/líquido cefalorraquidiano , Fatores Biológicos/farmacologia , Células Cultivadas , Feminino , Substâncias de Crescimento/líquido cefalorraquidiano , Substâncias de Crescimento/farmacologia , Humanos , Líquido Intracelular/metabolismo , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Músculo Liso Vascular/metabolismo , Hemorragia Subaracnóidea/complicações , SuínosRESUMO
We studied the presence of specific binding sites for pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, and a PLA2-I action on the DNA synthesis of rat chondrocytes. Rat chondrocytes, derived from the xiphisternum of adult rats, had a single class of PLA2-I binding site with an equilibrium binding constant value of 0.9 nM and a maximum binding capacity of 53.9 fmol/10(6) cells. PLA2-I alone did not show any proliferative effect, however, PLA2-I dose-dependently stimulated thymidine incorporation in DNA in the presence of basic fibroblast growth factor (bFGF). The mammalian mature type of PLA2s-I specifically recognized the binding sites in these cells and had a synergistic effect on DNA synthesis with bFGF, whereas its inactive zymogen and group II PLA2 showed much lesser activities. The type-specific action of PLA2s implicated the involvement of PLA2-I specific binding sites in this activation process.
Assuntos
Cartilagem/citologia , Replicação do DNA/efeitos dos fármacos , Fosfolipases A/metabolismo , Fosfolipases A/farmacologia , Animais , Sítios de Ligação , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Indometacina/farmacologia , Cinética , Fosfolipases A2 , Ratos , Ratos Endogâmicos , SuínosRESUMO
Porcine pancreatic group I phospholipase A2 (PLA2-I) induced contraction of guinea pig parenchyma in a concentration-dependent manner. Its EC50 value was similar to the Kd value calculated from the specific binding of 125I-labeled porcine PLA2-I in the membrane fraction of guinea pig lung. Type-specific action of PLA2's and homologous desensitization strongly implicated the involvement of PLA2-I-specific sites in the activation process. Thromboxane A2 was found to be the main product from lung tissue by PLA2-I action and the contractile response by PLA2-I was specifically suppressed by thromboxane A2 receptor antagonists and cyclooxygenase inhibitor, but not by leukotriene receptor antagonist and H1 blocker. These findings indicate that PLA2-I-induced contractile response may depend on the secondarily produced thromboxane A2, thus providing a new aspect of PLA2-I from the pathophysiological standpoint.
Assuntos
Contratura/metabolismo , Pulmão/metabolismo , Pâncreas/enzimologia , Fosfolipases A/metabolismo , Animais , Sítios de Ligação , Cobaias , Técnicas In Vitro , Pulmão/fisiologia , Masculino , Fosfolipases A2 , Tromboxano A2/metabolismoRESUMO
The effects of cholinergic agents on the K(+)-evoked release of endogenous histamine from hypothalamic slices of rats were examined by a superfusion method in vitro. Acetylcholine and carbamylcholine significantly inhibited K(+)-evoked release of histamine from the slices, and the effect of acetylcholine was antagonized by the muscarinic antagonist atropine. On the other hand, nicotine significantly enhanced the K(+)-evoked release and its effect was antagonized by the nicotinic antagonist hexamethonium. The effects of carbamylcholine and nicotine on histamine release from slices of whole hypothalamus and from slices of the anterior hypothalamic region, which do not contain cell bodies of the histaminergic neurons, were the same. Thus, the K(+)-evoked release of endogenous histamine from histaminergic fibers in the hypothalamic slices was inhibited by muscarinic agents and enhanced by nicotinic agents. These results suggest that muscarinic and nicotinic receptors exert antagonistic effects on the release of hypothalamic histamine from in vitro slice preparations. The cholinergic receptors modulating histamine release might be located presynaptically on the histaminergic terminals.
Assuntos
Estimulantes Ganglionares/farmacologia , Liberação de Histamina/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Parassimpatomiméticos/farmacologia , Potássio/farmacologia , Animais , Hipotálamo/metabolismo , Técnicas In Vitro , Masculino , Ratos , Ratos EndogâmicosRESUMO
1. The mechanism for the stereospecific recognition of the antagonist S-145 by the thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor was examined by ligand-binding techniques in rat vascular smooth muscle cells (VSMCs) and in human platelet membranes. 2. Scatchard analysis revealed the existence of a single class of binding sites with the same maximum number for both [3H]-(+)-S-145 and [3H]-(-)-S-145 in both cell types. The dissociation constants (Kd) for the binding of the (+)-isomer in rat VSMCs and human platelet membranes were, respectively, 0.40 +/- 0.03 and 0.20 +/- 0.02 nM, each value being lower than that for the (-)-isomer (3.57 +/- 0.74 and 2.87 +/- 0.08 nM, respectively). 3. The rank orders of potency (Ki) for a series of TXA2/PGH2 ligands at inhibiting [3H]-(+)-S-145 binding were highly correlated with those determined for [3H]-(-)-S-145 binding in both cell preparations. 4. Kinetic analysis of the binding of both radioligands revealed a much lower dissociation rate constant (k-1) and a slightly greater association rate constant (k1) for the (+)-isomer compared to those for the (-)-isomer. 5. These results suggest that it is at the stage of dissociation from the TXA2/PGH2 receptor that the stereochemistry of the optical isomers of S-145 confers their difference in affinity for these receptors in rat VSMCs and human platelet membranes.
Assuntos
Compostos Bicíclicos com Pontes/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Prostaglandinas H/metabolismo , Receptores de Prostaglandina/metabolismo , Tromboxano A2/metabolismo , Animais , Plaquetas/química , Membrana Celular/química , Células Cultivadas , Humanos , Masculino , Músculo Liso Vascular/química , Ratos , Ratos Endogâmicos , Receptores de Tromboxanos , Receptores de Tromboxano A2 e Prostaglandina H2 , EstereoisomerismoRESUMO
The effects of thromboxane A2 (TXA2) on the synthesis of platelet-activating factor (PAF) and leukotriene B4 (LTB4) were studied using human polymorphonuclear neutrophils (PMN). Scatchard analysis for binding experiments using [3H]S-145, a specific TXA2/prostaglandin H2 (PGH2) receptor antagonist, revealed the existence of a single class of binding sites (Kd = 83.0 +/- 2.8 nM, Bmax = 113.0 +/- 3.1 fmol/2.10(6) cells) in human PMN. Upon stimulation with a combination of U46619, a TXA2 mimetic agonist, and N-formylmethionylleucylphenylalanine (FMLP, 1 microM), the synthesis of PAF was detected, although this was not significantly enhanced by U46619 or FMLP alone. The maximal production of PAF as well as the maximal activity of acetyl-CoA acetyltransferase was observed at approx. 20 min after addition of both stimuli. The effects of U46619 plus FMLP on PAF synthesis showed dose dependence to different concentrations of U46619 (0.1-10 microM), and were completely inhibited by S-145. Contrarily, no significant amounts of LTB4 were detected by radioimmunoassay during the stimulation with U46619 and FMLP. These results suggest that TXA2 and FMLP synergistically activate human PMN to induce PAF synthesis and this effect of TXA2 is mediated through its specific receptor.