RESUMO
We demonstrate that serum IgG in chagasic patients interacting with the second extracellular loop of human cardiac M(2) muscarinic acetylcholine receptors (M(2) mAChR) trigger the production of PGE(2) and NO, that in turn induces COX-2/iNOS mRNA expression. An association between serum anti-M(2) peptide IgG, anti-cardiac membrane IgG and PGE(2) levels (p<0.05) in chagasic dysautonomic patients was observed. Thus, we establish that serum anti-mAChR autoantibodies and PGE(2) might be considered as early markers of Chagas' associated dysautonomia. Affinity purified anti-M(2) peptide IgG from chagasic sera, while stimulating myocardial M(2) mAChR, it exerts an increase on PGE(2) generation and NOS activity, as well as COX-2/iNOS isoforms mRNA expression. The expression of these genes is related with phosphoinositides (PIs), cGMP accumulation and PKC activity. Inhibition of these enzymes shows that chagasic autoantibodies up-regulation of COX-2/iNOS mRNA level is under the control of endogenous iNO/cGMP signaling system. These results provide a novel insight into the role that cholinoceptor antibodies play in the development of myocardial inflammation. To our knowledge, there has been no previous report showing that an antibody interacting with heart mAChR can act as expression inducer of proinflammatory mediators.
Assuntos
Anticorpos Antiprotozoários/sangue , Cardiomiopatia Chagásica/imunologia , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico/metabolismo , Adulto , Ácido Araquidônico/metabolismo , Autoanticorpos/sangue , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/fisiopatologia , Ciclo-Oxigenase 2/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Imunoglobulina G/sangue , Mediadores da Inflamação/imunologia , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Disautonomias Primárias/imunologia , Disautonomias Primárias/parasitologia , RNA Mensageiro/metabolismo , Receptor Muscarínico M2/imunologia , Receptor Muscarínico M2/metabolismoRESUMO
In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects.
Assuntos
GMP Cíclico/metabolismo , Frequência Cardíaca/fisiologia , Contração Miocárdica/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Frequência Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo III , Fenoxiacetatos/farmacologia , Fenoxipropanolaminas/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The goal of this study was to investigate whether cruzipain, a Trypanosoma cruzi immunodominant antigen, was able to induce antibodies reactive to the cardiac M(2) muscarinic acetylcholine receptor (M(2) mAChR). Immunization with cruzipain that was devoid of enzyme activity triggered IgG antibodies against cardiac M(2) mAChR. By radioligand competition assay we proved that the anti-cruzipain IgG fraction, purified from serum, inhibited binding of the specific M(2) mAChR radioligand [(3)H]quinuclidinyl benzilate. We also demonstrated that anti-cruzipain IgG reacted against the second extracellular loop of the M(2) mAChR. The corresponding affinity-purified serum anti-M(2)e2 IgG (reacting against a synthetic peptide corresponding to this loop in humans) displayed agonist-like activity associated with specific M(2) mAChR activation - increase of cGMP, inositol phosphate accumulation and nitric oxide synthase activity - triggering a decrease in myocardial contractility. Moreover, the same IgG fraction decreased heart frequency, related to inhibition of adenylate cyclase activity. These results imply that cruzipain plays a role in the production of antibodies against M(2) mAChR, which have been related to the pathogenesis of dysautonomic syndrome described in Chagas' disease.
Assuntos
Autoanticorpos/imunologia , Cisteína Endopeptidases/imunologia , Miocárdio/imunologia , Receptores Muscarínicos/imunologia , Animais , Doença de Chagas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários , Trypanosoma cruzi/imunologiaRESUMO
Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the beta(1)-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Trypanosoma cruzi/classificação , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Sequência de Bases , Glicoproteínas , Humanos , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
Previously, we have demonstrated that plasma membranes from the parasite Trypanosoma cruzi (T. cruzi) recognize and adhere to host cells through parasite surface attachment molecules that have affinity for beta(1)-adrenergic receptors (beta(1)-ARs) on target organs. In this report we identify a parasite protein that not only interacts with beta(1)-ARs, but also displays beta-agonist-like activity. We demonstrate that a recombinant maltose binding protein fusion of Tc13 Tul (MBP-Tc13 Tul), a member of the T. cruzi antigen 13 family of surface antigen proteins, competes for binding sites with the beta-adrenergic receptor antagonist [125I]-CYP on membranes purified both from CHO cells expressing human beta(1)-ARs and from rat atria. The competition is prevented by pre-treating MBP-Tc13 Tul with antibodies directed against the EPKSA repeat domain of Tc13 Tul, implicating this portion of the molecule in binding to the beta(1)-AR. Furthermore, MBP-Tc13 Tul activates rat myocardial beta(1)-ARs, resulting in synthesis of cyclic adenosine monophosphate (cAMP) and an increase in cardiac contractility. These biological effects are selectively suppressed by the beta(1)-AR antagonist atenolol, by a synthetic peptide corresponding to the second extracellular loop of the human beta(1)-AR, and by the anti-EPKSA repeat antibodies. These results imply that the Tc13 Tul cell-surface antigen of T. cruzi plays a central role in misregulating the beta(1)-AR following parasite infection, and may be a causative factor of dysautonomic syndrome described in Chagas' disease.
Assuntos
Antígenos de Protozoários/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Trypanosoma cruzi/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Antígenos de Protozoários/farmacologia , Atenolol/farmacologia , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Propranolol/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
Therapeutic use of a peptide corresponding to the aminoacid sequence of the second extracellular loop of human M2 muscarinic acetylcholine receptor (M2 mAChR peptide) was studied. Expression and biological activity of M2 mAChR in association with circulating M2 mAChR-related antibodies in cardiac tissue from chagasic mice were evaluated. Mice infected or not with trypomastigotes Tulahuen strain either treated or not treated with M2 mAChR peptide were sacrificed at 8-9 weeks post-infection. Morphological, binding and contractility studies were performed on all animal groups. Hearts from infected mice showed a mAChR-related dysfunction, with a decrease in heart contractility, impaired response to exogenous mAChR agonist (carbachol) and a significant reduction of mAChR binding sites. Treating infected mice with M2 mAChR peptide reversed those effects. Moreover, autoantibodies from infected mice recognized the M2 mAChR peptide. In addition, serum from infected mice and the corresponding affinity purified IgG was capable of interacting with cardiac mAChR, reducing the number of binding sites and inhibiting the contractile response to exogenous agonist. In conclusion, (1) the development of alterations in mAChR related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) the chronic treatment with M2 mAChR peptide prevented infected mice heart dysfunction. The mechanism could be explained by the ability of the M2 mAChR peptide to inhibit the chronic interaction of autoantibodies specific to mAChR. The implication of M2 mAChR peptide treatment in the host's immune response is discussed.
Assuntos
Cardiomiopatia Chagásica/prevenção & controle , Fragmentos de Peptídeos/uso terapêutico , Receptores Muscarínicos/química , Sequência de Aminoácidos , Animais , Função Atrial/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Carbacol/farmacologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/fisiopatologia , Coração/fisiopatologia , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Quinuclidinil Benzilato/metabolismo , Quinuclidinil Benzilato/farmacologia , Receptor Muscarínico M2 , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M(1) mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M(1) mAChR radioligand [(3)H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M(1) mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M(1) mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.