RESUMO
Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [(3)H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [(3)H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[(32)P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [(32)P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [(32)P]-labeling. The [(32)P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [(32)P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356-369) and M3mAChR (480-493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain ((480)MSLIKEKK(485)), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Asma/etiologia , Asma/fisiopatologia , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Retroalimentação Fisiológica , Humanos , Técnicas In Vitro , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Quinuclidinil Benzilato/metabolismo , Quinuclidinil Benzilato/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Tionucleotídeos/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/metabolismoRESUMO
In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), can act as an inducer of m1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M1 synthetic peptide (with identical aminoacid sequence to human M1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M1 mAChR. The corresponding affinity-purified anti M1 peptide IgG (anti M1 peptide IgG) from schizophrenic patients by stimulation of M1 mAChR exerted an increase in m1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (alpha = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.
Assuntos
Autoanticorpos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Receptor Muscarínico M1/metabolismo , Esquizofrenia/imunologia , Adulto , Análise de Variância , Animais , Autoanticorpos/química , Northern Blotting/métodos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Cromatografia de Afinidade/métodos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Fosfatos de Inositol/metabolismo , Masculino , Pessoa de Meia-Idade , Antagonistas Muscarínicos/farmacocinética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Quinuclidinil Benzilato/farmacocinética , Ensaio Radioligante/métodos , Ratos , Ratos Wistar , Receptor Muscarínico M1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Trítio/farmacocinéticaRESUMO
It has been proposed that anti-myocardial antibodies (Ab) against neurotransmitter (NT) receptors are involved in the immunopathology of chronic Chagas' heart disease. We demonstrated that an anti-Trypanosoma cruzi monoclonal Ab (mAb), CAK20.12, binds to murine cardiac beta-adrenergic and muscarinic acetyl choline (mACh) receptors eliciting abnormal physiological responses on normal heart. No cross-linking requirement for mAb actions was demonstrated using Fab fragment derived from CAK20.12. mAb binding to synthetic peptides from the second extracellular loop of both beta1-adrenergic and mACh receptors, demonstrated by ELISA, identified the region of NT receptors involved. Cross-reactivity between these peptides and T. cruzi antigen was confirmed by binding inhibition assays. These results support the existence of cross-reactivity due to molecular mimicry between a parasite antigen and the major antigenic epitopes present on both beta1-adrenergic and M2-ACh receptors. Its possible relationship with cardiac dysfunction during chronic stage of Chagas' disease is also discussed.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antiprotozoários/farmacologia , Contração Miocárdica/efeitos dos fármacos , Pindolol/análogos & derivados , Receptor Muscarínico M2/imunologia , Receptores Adrenérgicos beta 1/imunologia , Trypanosoma cruzi/imunologia , Antagonistas Adrenérgicos beta , Análise de Variância , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/metabolismo , Epitopos/farmacologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Técnicas In Vitro , Isótopos de Iodo/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Antagonistas Muscarínicos/farmacocinética , Contração Miocárdica/fisiologia , Pindolol/farmacocinética , Quinuclidinil Benzilato/farmacocinética , Radioimunoensaio/métodos , Ensaio Radioligante/métodos , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 1/química , Titulometria/métodos , Trypanosoma cruzi/químicaRESUMO
The administration of convulsant drugs has proven a powerful tool to study experimental epilepsy. We have already reported that the administration of convulsant 3-mercaptopropionic acid (mp) at 150 mg/kg enhances binding affinity of muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) to certain rat CNS membranes during seizure and postseizure without affecting site number. Results obtained with a 100-mg/kg dose of mp have shown reversible increases in [3H]QNB binding to cerebellum and hippocampus, whereas a delayed response has been found in striatum. Neither a subconvulsant dose nor in vitro addition modifies binding. In order to evaluate preseizure, seizure as well as early (30 min) and late (24 h) postseizure stages, we employed a 50 mg/kg dose and tested [3H]QNB binding to CNS membranes. Changes in binding were as follows (in %): in cerebellum, +37, +86, and +40 at preseizure, seizure and early postseizure stages, respectively, but there was a decrease at late postseizure; in hippocampus, +27 at pre- and seizure stages, but a decrease at early and late postseizure. No changes were found in striatum or cerebral cortex membranes at any stage studied. Saturation curves analysed by Scatchard plots indicated that changes in [3H]QNB binding to cerebellar membranes are attributable to an increase in ligand affinity at seizure, followed by a decrease in binding site number at postseizure. A similar profile was observed for hippocampus except that the decrease in binding site number, though lower than at postseizure, was already evident at seizure stage. Results confirm a region-specific response to the convulsant and transient changes provide an example of neuronal plasticity.