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
Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [(3)H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [(3)H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M3 selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M2-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP effect indicating that the M3AChR is the main receptor target of cGMP. Interestingly, these cGMP effects were potentiated by an activator (Sp-8-pCPT-cGMPS), and diminished by an inhibitor (Rp-8-pCPT-CGMPS), of cGMP-dependent protein kinase (PKG-II), which was detected by Western blotting using specific PKG II antibodies. Finally, plasma membrane M3AChRs were phosphorylated in a cGMP-dependent manner and this novel post-translational reversible modification at M3AChRs may act as a feedback mechanism to terminate the cGMP dependent muscarinic signal transduction cascades at the sarcolema of BTSM.
Assuntos
GMP Cíclico/metabolismo , Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Traqueia/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteína Quinase Dependente de GMP Cíclico Tipo II/imunologia , Retroalimentação Fisiológica , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/metabolismo , Piperidinas/metabolismo , Processamento de Proteína Pós-Traducional , Quinuclidinil Benzilato/metabolismoRESUMO
Estudos mostram que anticorpos IgG agonistas muscarínicos, de pacientes chagásicos, alteram a atividade elétrica de células cardíacas in vitro. Outros consideram sua presença, e a da síndrome do nódulo sinusal, conseqüências da lesão cardíaca progressiva. Objetivou-se avaliar a relação entre os anticorpos e as disfunções nodal e ventricular esquerda, em 65 pacientes chagásicos crônicos divididos em grupo I, composto de 31 pacientes portadores da síndrome do nódulo sinusal, e grupo II, de não portadores. A análise dos dados, pelo modelo log linear, mostrou uma interdependência entre a disfunção do nódulo sinusal e os anticorpos (p=0,0021) e entre a disfunção nodal e a ventricular (p=0,0005), mas não houve relação entre esta última e os anticorpos. Idade e sexo não tiveram influência sobre as outras variáveis. Chagásicos crônicos com a síndrome do nódulo sinusal têm maior prevalência de anticorpos agonistas muscarínicos, independentemente da presença de disfunção miocárdica.
Studies have shown that muscarinic agonist IgG antibodies from Chagas disease patients alter the electrical activity of cardiac cells in vitro. Others have considered their presence, along with sinus node dysfunction, to be consequences of progressive cardiac lesions. The aim of this study was to evaluate the relationship between these antibodies and sinus node and left ventricular dysfunction in 65 chronic Chagas disease patients. These patients were divided into group I, composed of 31 patients with sinus node dysfunction, and group II, composed of the patients without this syndrome. Data analysis using the log linear model showed interdependence between sinus node dysfunction and the antibodies (p = 0.0021) and between nodal and ventricular dysfunction (p = 0.0005). However, no relationship was found between the antibodies and ventricular function. Age and sex did not influence any other variables. The chronic Chagas disease patients with sinus node dysfunction had higher prevalence of muscarinic agonist antibodies, independent of the presence of myocardial dysfunction.
Assuntos
Adulto , Idoso , Humanos , Pessoa de Meia-Idade , Cardiomiopatia Chagásica/imunologia , Imunoglobulina G/sangue , Agonistas Muscarínicos/sangue , /sangue , Nó Sinoatrial/fisiopatologia , Disfunção Ventricular Esquerda/fisiopatologia , Autoanticorpos/sangue , Doença Crônica , Cardiomiopatia Chagásica/fisiopatologia , Eletrocardiografia , Imunoglobulina G/metabolismo , Modelos Lineares , Agonistas Muscarínicos/metabolismo , /agonistasRESUMO
Previous studies have demonstrated that antibodies against cholinoreceptors of exocrine glands correlate with dry mouth in persons with primary Sjögren syndrome (pSS). The aim of the present investigation was to establish if serum IgG antibodies (pSS IgG) were able to interact with cholinoreceptors in rat submandibular gland-dependent stimulation of cyclooxygenase 2 (COX-2) mRNA expression and PGE(2) production. Our findings indicated that pSS IgG-stimulating M(3), M(4), and M(1) cholinoreceptors exerted an increase in COX-2 mRNA without affecting COX-1 mRNA expression and increased PGE(2) production. Inhibitors of phospholipase A(2), COX- s, L-type calcium channel currents, and Ca(2+)-ATPase from sarcoplasmic reticulum prevented the pSS IgG effect on PGE(2) production. An ionophore of calcium mimicked pSS IgG action, suggesting a crucial role of calcium homeostasis in the cholinoreceptor-stimulated increase in PGE(2) production. Moreover, the amounts of PGE(2) in saliva and in sera from persons with pSS were significantly higher than in pre- or post-menopausal women. These findings illustrate the importance of autoantibodies to cholinoreceptors in the generation of chronic inflammation of target tissues in SS.
Assuntos
Autoanticorpos/fisiologia , Receptores Muscarínicos/imunologia , Síndrome de Sjogren/imunologia , Adulto , Animais , Autoanticorpos/isolamento & purificação , Cálcio/metabolismo , Estudos de Casos e Controles , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Feminino , Humanos , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Agonistas Muscarínicos/metabolismo , Pós-Menopausa , Ratos , Glândula Submandibular/imunologia , Glândula Submandibular/metabolismoRESUMO
Studies have shown that muscarinic agonist IgG antibodies from Chagas disease patients alter the electrical activity of cardiac cells in vitro. Others have considered their presence, along with sinus node dysfunction, to be consequences of progressive cardiac lesions. The aim of this study was to evaluate the relationship between these antibodies and sinus node and left ventricular dysfunction in 65 chronic Chagas disease patients. These patients were divided into group I, composed of 31 patients with sinus node dysfunction, and group II, composed of the patients without this syndrome. Data analysis using the log linear model showed interdependence between sinus node dysfunction and the antibodies (p = 0.0021) and between nodal and ventricular dysfunction (p = 0.0005). However, no relationship was found between the antibodies and ventricular function. Age and sex did not influence any other variables. The chronic Chagas disease patients with sinus node dysfunction had higher prevalence of muscarinic agonist antibodies, independent of the presence of myocardial dysfunction.
Assuntos
Cardiomiopatia Chagásica/imunologia , Imunoglobulina G/sangue , Agonistas Muscarínicos/sangue , Receptor Muscarínico M2/sangue , Nó Sinoatrial/fisiopatologia , Disfunção Ventricular Esquerda/fisiopatologia , Adulto , Idoso , Autoanticorpos/sangue , Cardiomiopatia Chagásica/fisiopatologia , Doença Crônica , Eletrocardiografia , Humanos , Imunoglobulina G/metabolismo , Modelos Lineares , Pessoa de Meia-Idade , Agonistas Muscarínicos/metabolismo , Receptor Muscarínico M2/agonistasRESUMO
Male Holtzman rats weighting 200-250 g were anesthetized with zoletil 50 mg/Kg (tiletamine chloridrate 125.0 mg and zolazepan chloridrate 125.0 mg) into quadriceps muscle and stainless steel cannulas were implanted into their supraoptic nucleus (SON). We investigated the effects of the injection into the supraoptic nucleus (SON) of FK 409, a nitric oxide donor, and NW-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (NOS), on the salivary secretion, arterial blood pressure, sodium excretion and urinary volume induced by pilocarpine, which was injected into SON. The drugs were injected in 0.5 microl volume over 30-60 s. Controls was injected with a similar volume of 0.15 M NaCl. FK 409 and L-NAME were injected at doses of 20 microg/0.5 microl and 40 microg/0.5 microl respectively. The amount of saliva secretion was studied over a five-minute period after injection of pilocarpine into SON. Injection of pilocarpine (10, 20, 40, 80, 160 microg/microl) into SON produced a dose-dependent increase in salivary secretion. L-NAME was injected into SON prior to the injection of pilocarpine into SON, producing an increase in salivary secretion due to the effect of pilocarpine. FK 409 injected into SON attenuating the increase in salivary secretion induced by pilocarpine. Mean arterial pressure (MAP) increase after injections of pilocarpine into the SON. L-NAME injected into the SON prior to injection of pilocarpine into SON increased the MAP. FK 409 injected into the SON prior to pilocarpine attenuated the effect of pilocarpine on MAP. Pilocarpine (0.5 micromol/0.5 microl) injected into the SON induced an increase in sodium and urinary excretion. L-NAME injected prior to pilocarpine into the SON increased the urinary sodium excretion and urinary volume induced by pilocarpine. FK 409 injected prior to pilocarpine into the SON decreased the sodium excretion and urinary volume induced by pilocarpine. All these roles of pilocarpine depend on the release of nitric oxide into the SON. In summary the present results show: a) SON is involved in pilocarpine-induced salivation; b) that mechanism involves increase in MAP, sodium excretion and urinary volume.
Assuntos
Pressão Sanguínea/fisiologia , Agonistas Muscarínicos/metabolismo , Natriurese/fisiologia , Óxido Nítrico/metabolismo , Pilocarpina/metabolismo , Salivação/fisiologia , Núcleo Supraóptico/metabolismo , Animais , Inibidores Enzimáticos/metabolismo , Masculino , Agonistas Muscarínicos/farmacologia , NG-Nitroarginina Metil Éster/metabolismo , Doadores de Óxido Nítrico/metabolismo , Nitrocompostos/metabolismo , Pilocarpina/farmacologia , Ratos , Ratos Endogâmicos , Núcleo Supraóptico/anatomia & histologia , Núcleo Supraóptico/efeitos dos fármacosRESUMO
Peripheral treatment with cholinergic or adrenergic agonists results in salivation and the possibility of synergy between cholinergic and adrenergic efferent mechanisms in the control of salivation has been proposed. Central injections of the cholinergic agonist pilocarpine also induce salivation, while the effects of central injections of noradrenaline (norepinephrine) are not known. Here (a) the effects of intracerebroventricular (i.c.v.) injection of noradrenaline on the salivation induced by i.c.v. or intraperitoneal (i.p.) injection of pilocarpine and (b) the receptors involved in the effects of central noradrenaline on pilocarpine-induced salivation were investigated. Male Holtzman rats with a stainless-steel guide cannula implanted into the lateral ventricle were used. Rats were anaesthetized with tribromoethanol (200mg/kg body weight) and saliva was collected on small, preweighed cotton balls inserted into the animal's mouth. Noradrenaline (40, 80 and 160 nmol/1 microl) injected i.c.v. reduced the salivary secretion induced by pilocarpine (0.5 micro mol/1 microl) injected i.c.v.. Noradrenaline (80 and 160 nmol/1 microl) injected i.c.v. also reduced the salivation induced by pilocarpine (4 micromol/kg) injected i.p. Previous treatment with the alpha(2)-adrenergic receptor antagonists RX 821002 (40, 80 and 160 nmol/1 microl) or yohimbine (160 and 320 nmol/1 microl) abolished the inhibitory effect produced by i.c.v. injection of noradrenaline on pilocarpine-induced salivation in rats. Prazosin (alpha(1)-adrenergic receptor antagonist) injected icv did not change the effect of noradrenaline on pilocarpine-induced salivation. Prior icv injection of only RX 821002 (80 or 160 nmol/1 microl) or yohimbine (320 nmol/1 microl) increased pilocarpine-induced salivation. The results show that (1) contrary to its peripheral effects, noradrenaline acting centrally inhibits cholinergic-induced salivation in rats; (2) central mechanisms involving alpha(2)-adrenergic receptors inhibit pilocarpine-induced salivation.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Idazoxano/análogos & derivados , Agonistas Muscarínicos/metabolismo , Norepinefrina/farmacologia , Pilocarpina/antagonistas & inibidores , Salivação/efeitos dos fármacos , Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/administração & dosagem , Antagonistas Adrenérgicos alfa/farmacologia , Análise de Variância , Animais , Idazoxano/farmacologia , Injeções Intraperitoneais , Injeções Intraventriculares , Masculino , Agonistas Muscarínicos/administração & dosagem , Norepinefrina/administração & dosagem , Pilocarpina/administração & dosagem , Prazosina/farmacologia , Ratos , Ratos Sprague-Dawley , Saliva/efeitos dos fármacos , Saliva/metabolismo , Estatística como Assunto , Ioimbina/farmacologiaRESUMO
A soluble fraction from human frontal cortex with molecular weight less than 10 kD was tested for the presence of endogenous substances capable of modulating the [3H]-QNB binding to crude P1 + P2 fractions from the same region. The soluble fraction was able to decrease [3H]-QNB binding in a dose-response manner with an IC50 of about 30 micrograms/ml. The effect appeared to be noncompetitive in nature, since Bmax but not Kd was significantly affected; however, in some specimens a biphasic profile, with an initial inhibition of 88-90% of [3H]-QNB binding and 50-60% ulterior binding recuperation was also found. The modulator appeared to have a molecular weight less than 10,000 Daltons and was heat and trypsin resistant. These results point out the existence of an endogenous factor, which could be heterogeneous in regard to its molecular nature or to its action sites.