RESUMEN
Thromboxane A(2) (TXA(2)) and 8-iso-PGF(2alpha) are two prostanoid agonists of the thromboxane A(2) receptor (TP), whose activation has been involved in platelet aggregation and atherosclerosis. Agents able to counteract the actions of these agonists are of great interest in the treatment and prevention of cardiovascular events. Here, we investigated in vitro and in vivo the pharmacological profile of BM-520, a new TP antagonist. In our experiments, this compound showed a great binding affinity for human washed platelets TP receptors, and prevented human platelet activation and aggregation induced by U-46619, arachidonic acid and 8-iso-PGF(2alpha). The TP receptor antagonist property of BM-520 was confirmed by its relaxing effect on rat aorta smooth muscle preparations precontracted with U-46619 and 8-iso-PGF(2alpha). Further, its TP antagonism was also demonstrated in vivo in guinea pig after a single intravenous injection (10 mg kg(-1)). We conclude that this novel TP antagonist could be a promising therapeutic tool in pathologies such as atherosclerosis where an increased production of TXA(2) and 8-iso-PGF(2alpha), as well as TP activation are well-established pathogenic events.
Asunto(s)
Aorta/metabolismo , Dinoprost/análogos & derivados , Difenilamina/análogos & derivados , Músculo Liso Vascular/metabolismo , Compuestos de Sulfonilurea/química , Tromboxano A2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/química , Animales , Aorta/efectos de los fármacos , Plaquetas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Dinoprost/química , Difenilamina/química , Difenilamina/farmacología , Ácidos Grasos Insaturados , Cobayas , Humanos , Hidrazinas/química , Masculino , Modelos Químicos , Ratas , Ratas Wistar , Compuestos de Sulfonilurea/farmacologíaRESUMEN
The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Nitrocompuestos/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Sulfonamidas/farmacología , Tromboxano-A Sintasa/antagonistas & inhibidores , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Diuresis/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Humanos , Relajación Muscular , Inhibidores de Agregación Plaquetaria/farmacología , Ratas , Ratas Wistar , EstereoisomerismoRESUMEN
The aim of this study was to characterize the effects of BM-573 [N-terbutyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl] urea], a novel dual thromboxane A2 receptor antagonist and thromboxane synthase inhibitor, on myocardial infarction induced by topical ferric chloride (FeCl3) application to the left anterior descending (LAD) coronary artery in anesthetized pigs. All control animals (n = 6) developed an occlusive thrombus in the LAD coronary artery. The mean infarct size, revealed by triphenyl tetrazolium chloride (TTC), and the area at risk, evidenced by Evans blue, corresponded to 35.3 +/- 2.2 and 36.9 +/- 2.1% of the left ventricular mass, respectively. In the BM-573-treated group (n = 6), a drug infusion (10 mg. kg-1. h-1) started 30 min before FeCl3 application and continued throughout the experimentation. Among the BM-573-treated group, four pigs did not develop coronary artery thrombus and their myocardium appeared healthy. Histopathological examination of FeCl3-injured coronary artery revealed an occlusive and adherent thrombus in control group, while pretreatment with BM-573 prevented thrombus formation. In infarcted zones, lack of desmin staining and muscle structure disorganization were obvious. Depletion of myocardial ATP content was observed in the myocardial necrotic region of the control group, but not in myocardial samples of BM-573-treated pigs that did not develop myocardial infarction. When BM-573 prevented LAD artery occlusion, the area under the curve of plasmatic troponin T was reduced by 77% over 6 h. These data suggest that BM-573 could be useful for the prevention of myocardial infarction.
Asunto(s)
Trombosis Coronaria/complicaciones , Inhibidores Enzimáticos/farmacología , Infarto del Miocardio/prevención & control , Receptores de Tromboxanos/antagonistas & inhibidores , Compuestos de Sulfonilurea/uso terapéutico , Tromboxano-A Sintasa/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Infarto del Miocardio/etiología , Receptores de Tromboxanos/metabolismo , Porcinos , Tromboxano-A Sintasa/metabolismoRESUMEN
The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.
Asunto(s)
Plaquetas/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Compuestos de Sulfonilurea/farmacología , Tromboxano-A Sintasa/metabolismo , Plaquetas/fisiología , Humanos , Tromboxano B2/biosíntesisRESUMEN
The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.
Asunto(s)
Plaquetas/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Receptores de Tromboxanos/antagonistas & inhibidores , Tromboxano-A Sintasa/antagonistas & inhibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Antihipertensivos/farmacología , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/metabolismo , Ácido Araquidónico/antagonistas & inhibidores , Plaquetas/enzimología , Plaquetas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Relación Dosis-Respuesta a Droga , Ácidos Grasos Insaturados , Cobayas , Humanos , Hidrazinas/farmacología , Concentración 50 Inhibidora , Modelos Químicos , Músculo Liso/enzimología , Músculo Liso/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Ratas , Ratas Wistar , Sulfonamidas/farmacología , Torasemida , Tráquea/efectos de los fármacos , Tráquea/enzimología , Tráquea/metabolismo , Vasoconstrictores/farmacologíaRESUMEN
BM-531 (N-tert-butyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA2 receptors labeled with [3H]SQ-29548 (IC50 = 0.0078 microM) is higher than sulotroban (IC50 = 0.93 microM) and SQ-29548 (IC50 = 0.021 microM). Moreover, BM-531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet-rich plasma BM-531 prevents platelet aggregation induced by arachidonic acid (600 microM) (ED100 = 0.125 microM), U-46619, a stable TXA2 agonist (1 microM) (ED50 = 0.482 microM) or collagen (1 microgram/mL) (percentage of inhibition: 42.9% at 10 microM) and inhibits the second wave of ADP (2 microM)-induced aggregation. On the other hand, when BM-531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100) is significantly prolonged. In addition, at the concentrations of 10 and 1 microM, BM-531 totally prevents the production of TXB2 by human platelets activated by arachidonic acid. Finally, at 10 microM, BM-531 significantly prevents rat fundus contractions induced by U-46619 but not by prostacyclin. These results suggest that BM-531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Tromboxanos/antagonistas & inhibidores , Compuestos de Sulfonilurea/farmacología , Tromboxano A2/metabolismo , Tromboxano-A Sintasa/antagonistas & inhibidores , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Diuresis/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/fisiología , Humanos , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Inhibidores de Agregación Plaquetaria/metabolismo , Ensayo de Unión Radioligante , Receptores de Tromboxanos/metabolismo , Sulfonamidas/farmacología , Compuestos de Sulfonilurea/metabolismo , Tromboxano A2/biosíntesis , TorasemidaRESUMEN
The synthesis and the structure of N-isopropyl-N'-[2-(3'-methylphenylamino)-5-nitrobenzenesulfonyl] urea (14) was drawn from two thromboxane A2 receptor antagonists structurally related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A2 (TXA2) receptor of human washed platelets. Compound 14 prevented platelet aggregation induced by arachidonic acid (0.6 mM) and U-46619 (1 microM) with an IC50 value of 0.45 and 0.15 microM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea precontracted by U-46619, a TXA2 agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively. Finally, 14 (1 microM) completely inhibited TXA2 synthase of human platelets. The pKa value and the crystallographic data of 14 were determined and used to propose an interaction model between the TXA2 antagonists related to torasemide and their receptor.
Asunto(s)
Receptores de Tromboxanos/antagonistas & inhibidores , Compuestos de Sulfonilurea/síntesis química , Compuestos de Sulfonilurea/farmacología , Tromboxano-A Sintasa/antagonistas & inhibidores , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Ácido Araquidónico/farmacología , Cobayas , Masculino , Agregación Plaquetaria/efectos de los fármacos , Ratas , Ratas Wistar , Tromboxano-A Sintasa/metabolismo , Tráquea/efectos de los fármacos , Tráquea/fisiologíaRESUMEN
BM567, a sulfonylurea compound whose crystal structure is here discussed and terbogrel, are both thromboxane receptor antagonists and thromboxane synthase inhibitors. In this paper, their crystallographic and electronic structures are compared and lead to new synthesis prospects among the sulfonylurea series.
Asunto(s)
Piridinas/química , Piridinas/farmacología , Receptores de Tromboxanos/antagonistas & inhibidores , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/farmacología , Tromboxano-A Sintasa/antagonistas & inhibidores , Cristalografía , Enlace de Hidrógeno , Conformación Molecular , Estructura Molecular , Espectrofotometría Atómica/métodos , Electricidad EstáticaRESUMEN
The effects of the synthetic glucocorticoid dexamethasone on the cAMP content of murine T lymphocyte cell lines has been investigated. Incubation of the 3B4.15 T cell hybrids with dexamethasone results in an average 5-fold increase in intracellular cyclic AMP levels after 6 h of treatment. This phenomenon is abolished in the presence of RU486 and of cycloheximide, indicating that it requires binding of the drug to the intracellular glucocorticoids receptor and de novo protein synthesis. Dexamethasone-induced elevation of intracellular cyclic AMP correlates with both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity in T cell hybrids. This modulation of cyclic AMP metabolism is independent of serum-derived factors, suggesting that it is not secondary to transmembrane receptor stimulation by an extracellular ligand. We propose that glucocorticoids interfere with the homeostatic control of intracellular cAMP concentration, leading to a sustained increase in the content of this important second messenger in murine T lymphocyte cell lines. This study suggests that elevation of cAMP levels may represent one way by which glucocorticoids modulate the immune response.
Asunto(s)
AMP Cíclico/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Medio de Cultivo Libre de Suero , Cicloheximida/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP Heterotriméricas/efectos de los fármacos , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Células Híbridas , Ratones , Mifepristona/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/efectos de los fármacos , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismoRESUMEN
In this study we examined the thromboxane A(2)(TXA(2)) receptor antagonist property of BM-531 (N-tert -butyl- N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on platelet function. The drug affinity for human washed platelet TXA(2)receptors labelled with [(3)H]SQ-29,548 has been determined (IC50: 0.0078 microM) and demonstrated to be higher than sulotroban (IC50: 0.93 microM) and SQ-29,548 (IC50: 0.021 microM). The antiaggregatory potency has been confirmed since we demonstrated that BM-531 prevented platelet aggregation in human citrated platelet-rich plasma induced by arachidonic acid (600 microM) (ED100: 0.125 microM), U-46619, a stable TXA(2)agonist (1 microM) (ED50: 0.482 microM) and collagen (1 microg mL(-1)) (% of inhibition: 42.9% at 10 microM) and inhibited the second wave of ADP (2 microM). Moreover, when BM-531 was incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100(trade mark)) was significantly prolonged. These results suggest that BM-531 can be regarded as a novel non-carboxylic TXA(2)antagonist with a powerful antiplatelet potency.
Asunto(s)
Agregación Plaquetaria/efectos de los fármacos , Receptores de Tromboxanos/antagonistas & inhibidores , Sulfonamidas/farmacología , Compuestos de Sulfonilurea/farmacología , Adenosina Difosfato/farmacología , Unión Competitiva , Plaquetas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Relación Dosis-Respuesta a Droga , Ácidos Grasos Insaturados , Hemostasis/efectos de los fármacos , Humanos , Hidrazinas/metabolismo , Concentración 50 Inhibidora , Estructura Molecular , Inhibidores de Agregación Plaquetaria/farmacología , Sulfonamidas/química , Sulfonamidas/metabolismo , Factores de Tiempo , TorasemidaRESUMEN
Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress-activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol-specific phospholipase-C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector.
Asunto(s)
Sangre/parasitología , Proteínas/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismo , Equilibrio Ácido-Base , Adaptación Fisiológica , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Dimetadiona/metabolismo , Activación Enzimática , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Protones , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/enzimología , Tripsina/metabolismo , Moscas Tse-Tse/parasitología , Fosfolipasas de Tipo C/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
Torasemide, a new sulfonylurea high ceiling loop diuretic, has been demonstrated to induce a concentration dependent relaxation of canine coronary precontracted with thromboxane A2 (TXA2). With the aim to develop more potent TXA2 receptor antagonists, we investigated a series of torasemide derivatives. This pharmacomodulation led to the discovery of a sulfonyl-cyanoguanidine (BM 144) which presents the same pharmacological profile as sulotroban, a TXA2 receptor antagonist used as reference.
Asunto(s)
Receptores de Tromboxanos/antagonistas & inhibidores , Sulfonamidas/química , Sulfonamidas/farmacología , Humanos , Técnicas In Vitro , Agregación Plaquetaria/efectos de los fármacos , Sulfonamidas/síntesis química , TorasemidaRESUMEN
In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27 degrees C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37 degrees C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC-) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC+).
Asunto(s)
Proteínas Protozoarias , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/crecimiento & desarrollo , Ácido Aconítico/farmacología , Animales , Ácido Cítrico/farmacología , Medios de Cultivo , Dihidrolipoamida Deshidrogenasa/metabolismo , Genes Protozoarios , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Concentración de Iones de Hidrógeno , Glicoproteínas de Membrana/biosíntesis , Ratones , Mutación , Fosfatidilinositol Diacilglicerol-Liasa , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/análisisRESUMEN
In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.
Asunto(s)
Glicosilfosfatidilinositoles/fisiología , Parasitemia/enzimología , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/enzimología , Fosfolipasas de Tipo C/fisiología , Animales , Modelos Animales de Enfermedad , Glicosilfosfatidilinositoles/genética , Ratones , Ratones Endogámicos , Mutagénesis Insercional , Parasitemia/genética , Parasitemia/parasitología , Fenotipo , Eliminación de Secuencia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/parasitología , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/genéticaRESUMEN
Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, beta-galactosidase and beta-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Proteínas Protozoarias , Trypanosoma brucei brucei/genética , Trypanosoma brucei gambiense/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Marcación de Gen , Genes Protozoarios , Glicoproteínas de Membrana/biosíntesis , Microscopía Electrónica , Oligosacáridos/metabolismo , Unión Proteica , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.
Asunto(s)
Adenilil Ciclasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Estrés Fisiológico/enzimología , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Activación Enzimática , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Proteína Quinasa C/metabolismo , Trypanosoma brucei brucei/fisiología , Tripsina/farmacología , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoAsunto(s)
Adenilil Ciclasas/metabolismo , Sistema Digestivo/parasitología , Insectos Vectores/parasitología , Trypanosoma brucei brucei/enzimología , Moscas Tse-Tse/parasitología , Animales , Sistema Digestivo/química , Interacciones Huésped-Parásitos , Insectos Vectores/química , Masculino , Moscas Tse-Tse/anatomía & histología , Moscas Tse-Tse/químicaRESUMEN
We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender forms are induced to transform under the same conditions.
Asunto(s)
ARN Protozoario/biosíntesis , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/genética , Animales , Northern Blotting , División Celular/genética , ADN Protozoario/biosíntesis , Electroforesis en Gel de Poliacrilamida , Cinética , Proteínas Protozoarias/metabolismoRESUMEN
Pleomorphic bloodstream forms of Trypanosoma brucei differentiate synchronously into procyclic forms when cultivated at 27 degrees C in the presence of citrate/cis-aconitate. The activity of adenylate cyclase was monitored during this process. Two phases of transient stimulation were observed. The first phase occurred 6-10 h after the triggering of differentiation, a period which immediately follows the release of the bulk of the VSG and immediately precedes both the first cell division and the loss of the bloodstream-specific ESAG 4 transmembrane adenylate cyclase. The second phase occurred between 20 and 40 h, when the cells that emerged from the first division began to proliferate. These observations suggest that cAMP may be involved in differentiation/proliferation of the parasite.
Asunto(s)
Adenilil Ciclasas/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/crecimiento & desarrollo , Animales , División Celular , Activación Enzimática , Citometría de Flujo , Cinética , Ratones , Ratas , Tripanosomiasis/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.