RESUMEN
1 The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChR) on DNA synthesis and CD40 expression in human fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with carbachol in presence or absence of specific antagonists and the following parameters were measured: identification of mAChR subtypes, DNA synthesis, inositol phosphates (InsP) production and CD40 expression. 2 Human fibroblasts express mAChR with Kd 0.47 +/- 0.11 nm and Bmax 236 +/- 22 fmol mg protein(-1). Carbachol stimulates DNA synthesis, InsP and the expression of CD40. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine but not by AF-DX 116 and tropicamide, indicating that M3 and M1 mAChR are implicated in carbachol action. The relative Ki of the antagonists obtained by competition binding assay was in parallel to the relative potency for blocking both carbachol-stimulated InsP accumulation and DNA synthesis. 3 The intracellular pathway leading to carbachol-induced biological effects involved phospholipase C and calcium/calmodulin, as U-73122 and trifluoroperazine blocked carbachol effects, respectively. Calphostin C, a protein kinase C inhibitor, had no effect, indicating that this enzyme does not participate in the system. 4 These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on normal human fibroblast function.
Asunto(s)
Antígenos CD40/biosíntesis , ADN/biosíntesis , Fibroblastos/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Atropina/farmacología , Calmodulina/antagonistas & inhibidores , Calmodulina/metabolismo , Carbacol/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Fosfatos de Inositol/metabolismo , Antagonistas Muscarínicos/farmacología , Pirenzepina/farmacología , Pirrolidinonas/farmacología , Quinuclidinil Bencilato , Ensayo de Unión Radioligante , Receptor Muscarínico M1/análisis , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/análisis , Receptor Muscarínico M3/efectos de los fármacos , Receptor Muscarínico M3/metabolismo , Receptores Muscarínicos/análisis , Receptores Muscarínicos/metabolismo , Trifluoperazina/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.
Asunto(s)
Óxido Nítrico Sintasa de Tipo I/metabolismo , Receptor Muscarínico M3/fisiología , Receptor Muscarínico M4/fisiología , Vejiga Urinaria/enzimología , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Masculino , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Músculo Liso/fisiología , Piperidinas/farmacología , Quinuclidinil Bencilato/farmacología , Ratas , Ratas Wistar , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Tritio , Tropicamida/farmacología , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiologíaRESUMEN
Previously we described a decrease in beta-adrenergic receptor expression in B lymphocytes as a consequence of in vivo alloimmunization. This decrease correlates with the highest response of alloantibody production by B cells. In the present report we examined the participation of intracellular signals elicited after alloimmune stimulation. We showed that in vitro stimulation of B cells with mitomycin C-treated allogenic cells induced a reduction in the number of beta-adrenoceptors. This downregulation correlated to changes in basal and in isoproterenol-stimulated intracellular cAMP levels. We found that calcium mobilization and protein kinase C activation triggered after direct allogenic stimulation and/or by the action of T cell-soluble factors induced the reduction in beta-adrenoceptor sites. These findings could be of interest to understand the neuroendocrine mechanisms involved in the regulation of B cell activation.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Regulación hacia Abajo/inmunología , Isoantígenos/inmunología , Activación de Linfocitos/inmunología , Receptores Adrenérgicos beta/biosíntesis , Transducción de Señal/inmunología , Animales , Linfocitos B/enzimología , Calcio/fisiología , Sistema Libre de Células/inmunología , Medios de Cultivo Condicionados/farmacología , Femenino , Isoanticuerpos/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Proteína Quinasa C/fisiología , Receptores Adrenérgicos beta/metabolismo , Solubilidad , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To assess whether exposure of cardiac muscarinic acetylcholine receptors (mAChR) to activating chagasic antimyocardial immunoglobulins results in bradycardia and other dysautonomic symptoms associated with the regulation of heart rate. METHODS: Trypanosoma cruzi infected patients with bradycardia and other abnormalities in tests of the autonomic nervous system were studied and compared with normal subjects. Antipeptide antibodies in serum were demonstrated by an enzyme linked immunosorbent assay using a synthetic 24-mer-peptide corresponding antigenically to the second extracellular loop of the human heart M(2) mAChR. The functional effect of affinity purified antipeptide IgG from chagasic patients on spontaneous beating frequency and cAMP production of isolated normal rat atria was studied. RESULTS: There was a strong association between the finding of antipeptide antibodies in chagasic patients and the presence of basal bradycardia and an altered Valsalva manoeuvre (basal bradycardia: chi(2) = 37.5, p < 0. 00001; Valsalva manoeuvre: chi(2) = 70.0, p < 0.00001). The antipeptide autoantibodies also showed agonist activity, decreasing the rate of contraction and cAMP production. The effects on rat atria resembled the effects of the authentic agonist and those of the total polyclonal chagasic IgG, being selectively blunted by atropine and AF-DX 116, and neutralised by the synthetic peptide corresponding in amino acid sequence to the second extracellular loop of the human M(2) mAChR. CONCLUSIONS: There is an association between circulating antipeptide autoantibodies in chagasic patients and the presence of bradycardia and other dysautonomic symptoms. Thus these autoantibodies are a marker of autoimmune cardiac autonomic dysfunction. The results support the hypothesis that autoimmune mechanisms play a role in the pathogenesis of chagasic cardioneuromyopathy.
Asunto(s)
Autoanticuerpos/sangre , Bradicardia/inmunología , Cardiomiopatía Chagásica/inmunología , Receptores Muscarínicos/inmunología , Animales , Cardiomiopatía Chagásica/fisiopatología , Técnicas de Cultivo , AMP Cíclico/biosíntesis , Atrios Cardíacos/fisiopatología , Frecuencia Cardíaca , Humanos , Inmunoglobulina G/inmunología , Masculino , RatasRESUMEN
Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has a large number of immunologic and nonimmunologic functions. We have described that IFN-gamma could activate muscarinic cholinergic receptors (mAchR) of rat intestine, stimulating ileal motility. We also observed that mAchR activation induced inhibition of cAMP levels and stimulation of cGMP formation. The objectives of our work were to clarify the signal transduction pathways involved in regulation of ileal motility through mAchR activation by IFN-gamma. Our results demonstrate that this cytokine produces an ileal cholinergic response through tyrosine kinase activity. The activation of tyrosine kinase mediates ileal contractility, phosphoinositide hydrolysis by phospholipase C, nitric oxide synthase via protein kinase C, and cGMP synthesis. The increment in ileal motility is probably due to hyperproduction of prostaglandin E2 (PGE2) by ileal tissue. This prostanoid is an important mediator because it stimulates ileal motility. We conclude that IFN-gamma not only immunomodulates the gut microenvironment but also exerts a local nonimmunologic regulation on intestinal motility.
Asunto(s)
Íleon/efectos de los fármacos , Interferón gamma/farmacología , Agonistas Muscarínicos/farmacología , Proteínas Tirosina Quinasas/metabolismo , Animales , GMP Cíclico/metabolismo , Activación Enzimática , Motilidad Gastrointestinal/efectos de los fármacos , Hidrólisis , Íleon/enzimología , Fosfatos de Inositol/metabolismo , Masculino , Óxido Nítrico Sintasa/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Estimulación QuímicaRESUMEN
Primary Sjögren Syndrome is a chronic autoimmune disease characterized by exocrine gland dysfunction. Here we present evidence of the activation of nitric oxide signaling cascade by circulating antibodies of patients with Sjögren Syndrome in rat submandibular glands. Constitutive nitric oxide synthase and cyclic GMP levels are modulated by Sjögren IgGs through the activation of muscarinic acetylcholine receptors on the glands. The effects are similar to those produced by the agonist carbachol and blocked by the antagonist atropine. The involvement of M1 subtype of muscarinic receptors is proposed since both a synthetic peptide homologous to an extracellular domain of M1 receptor and pirenzepine, a selective M1 antagonist, partially blocked the effects. We conclude that Sjögren Syndrome antibodies can activate nitric oxide signaling in submandibular glands by interacting with muscarinic acetylcholine receptors.
Asunto(s)
Autoanticuerpos/farmacología , Óxido Nítrico/metabolismo , Receptores Muscarínicos/inmunología , Receptores Muscarínicos/metabolismo , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Glándula Submandibular/inmunología , Glándula Submandibular/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Autoanticuerpos/sangre , Autoantígenos/genética , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/farmacología , Persona de Mediana Edad , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Transducción de SeñalRESUMEN
The purpose of this study was to explore the pharmacological and biochemical mechanisms involved in diabetic cardiomyopathy, with particular interest in the abnormal function of cholinergic neurotransmission at the onset of the pathology. The muscarinic acethylcholine agonist carbachol showed a negative inotropic response on both normal and diabetic isolated atria, but the latter showed a supersensitive response. No changes were found in muscarinic acethylcholine receptor (mAChR) expression. Measurements of mAChR-associated second messengers indicated no significant differences between normal and diabetic rat atria in the stimulatory effect of carbachol on protein kinase C activity and the production of inositol phosphates, or in the inhibitory effect induced by carbachol on cyclic AMP (cAMP) production. On the contrary, nitric oxide (NO) synthase activity and cyclic GMP production were higher in diabetic cardiac preparations than in normal ones. Moreover, in diabetic atria, nitric oxide synthase and guanylate cyclase inhibitors shifted the carbachol concentration-response curve on contractility to the right, reaching values similar to those of normal atria. These results suggest an early alteration in the mACh system during the diabetic state, associated with increased production of nitric oxide and cyclic GMP (cGMP). This, in turn, could increase the biological mechanical activity of the mAChR agonist, inducing in this way a higher pharmacological response, without changes in mAChR expression.
Asunto(s)
Carbacol/farmacología , Cardiomiopatías/metabolismo , GMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Agonistas Muscarínicos/farmacología , Óxido Nítrico Sintasa/metabolismo , Receptores Muscarínicos/metabolismo , Sistemas de Mensajero Secundario , Animales , Cardiomiopatías/enzimología , Cardiomiopatías/etiología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/enzimología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Óxido Nítrico Sintasa/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Muscarínicos/efectos de los fármacosRESUMEN
The mechanisms by which beta adrenergic stimulation regulates phagocytosis of Candida albicans by human peripheral monocytes (HPM) are characterized. Isoproterenol (ISO) inhibits phagocytosis in a concentration-dependent manner. This effect was blunted by propranolol, inhibitors of phospholipase A2 (PLA2), cyclooxygenase and verapamil, pointing to a participation of arachidonic acid (AA) metabolites and calcium in the phenomenon. Prostaglandin E2 (PGE2) and dibutyryl cyclic AMP (db-cAMP) also exerted the same inhibitory effect on phagocytosis. ISO interacts with beta adrenergic receptors of HPM increasing PGE2 and cAMP. We conclude that the mechanisms by which beta adrenergic stimulation regulates phagocytosis of Candida albicans by HPM appear to be secondary to beta adrenoceptor-mediated hydrolysis of AA accompanied by an increase in PGE2 generation and cAMP production. Both PGE2 and cAMP could act as mediators of the inhibitory action of beta agonists on the HPM-phagocytosis process.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Ácido Araquidónico/fisiología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Fagocitosis/efectos de los fármacos , Acetofenonas/administración & dosificación , Acetofenonas/farmacología , Agonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/farmacología , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Bucladesina/administración & dosificación , Bucladesina/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/inmunología , Candida albicans/metabolismo , AMP Cíclico/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/administración & dosificación , Dinoprostona/metabolismo , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Humanos , Indometacina/administración & dosificación , Indometacina/farmacología , Isoproterenol/administración & dosificación , Isoproterenol/farmacología , Oxitócicos/administración & dosificación , Oxitócicos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Propranolol , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Verapamilo/administración & dosificación , Verapamilo/farmacologíaRESUMEN
IgG obtained from sera of primary Sjögren's syndrome (pSS-IgG) patients and its interaction with M3 muscarinic cholinoceptors of rat exorbital lacrimal glands were studied by indirect immunofluorescence (IFI) and binding assay. Primary Sjögren's syndrome IgG stained epithelial cells with a continuous fluorescence pattern. The IFI imagen was attenuated by incubating the pSS-IgG with a synthetic peptide corresponding to the second extracellular loop of M3 muscarinic cholinoceptor. Primary SS-IgG was also able to bound irreversibly to muscarinic acetylcholine receptors (mAChRs) displacing the specific cholinergic antagonist QNB. Moreover, these antibodies triggered intracellular signals coupled to M3 muscaric cholinoceptors such as nitric oxide synthase (NOS) activation and cGMP production. Both primary Sjögren's syndrome IgG effects mimicked carbachol action and were abrogated by specific muscarinic antagonist 4-DAMP. The nitric oxide pathway through muscarinic cholinoceptors activation by pSS-IgG on rat exorbital lacrimal gland is also described. We proposed that chronic interaction of these autoantibodies on lacrimal gland muscarinic acetylcholine receptors could lead to tissue damage through nitric oxide release after immunological stimulation.
Asunto(s)
Autoanticuerpos/farmacología , Aparato Lagrimal/efectos de los fármacos , Óxido Nítrico/metabolismo , Receptores Muscarínicos/metabolismo , Síndrome de Sjögren/inmunología , Adulto , Animales , Autoanticuerpos/aislamiento & purificación , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/farmacología , Aparato Lagrimal/metabolismo , Persona de Mediana Edad , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Óxido Nítrico Sintasa/metabolismo , Fragmentos de Péptidos/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptor Muscarínico M3RESUMEN
Taking into account that the activation of different subtypes of ileal muscarinic acetylcholine receptors (mAChR) regulate gut functions such as tone, motility, and electrolyte secretion, we characterized the expression of mAChR in ileal-purified membranes. We also studied intracellular signals triggered by mAChR activation. Binding parameters obtained from saturation assays with the nonselective tritiated muscarinic antagonist, quinuclidynil benzilate ([3H]-QNB), were maximal number of binding sites (Bmax): 30 +/- 2 fmol/mg prot and dissociation constant (Kd): 0.2 +/- 0.03 nM. The competitive inhibition of [3H]-QNB specific binding by various nonlabelled muscarinic antagonists was measured and the rank order of potency was: atropine (ATROP) > 4-DAMP > AF-DX 116 > pirenzepine (PZ). The activation of mAChR by carbachol (CARB) increased ileal motility in a concentration-dependent manner (EC50 2 x 10[-7] M). The antagonists' order of potency to displace dose-response curve of CARB was: ATROP > 4-DAMP > AF-DX116 > PZ. Optimal concentration of CARB on ileal strips increased phosphoinositide turnover and cGMP levels by activating ml receptor subtype and decreased isoproterenol (ISO) stimulated levels of cAMP due to M2 receptor activation. We can conclude that the activation of different mAchR subtypes triggers different intracellular signals that could regulate intestinal tone and motility.
Asunto(s)
Íleon/metabolismo , Receptores Muscarínicos/metabolismo , Transducción de Señal , Animales , Membrana Celular/metabolismo , AMP Cíclico , GMP Cíclico , Activación Enzimática , Íleon/citología , Técnicas In Vitro , Fosfatos de Inositol , Cinética , Antagonistas Muscarínicos/farmacología , Quinuclidinil Bencilato/farmacología , Ratas , Receptores Muscarínicos/biosíntesis , Análisis de Regresión , Transducción de Señal/fisiologíaRESUMEN
Chronic Chagas' disease is associated with pathologic changes of the cardiovascular, digestive, and autonomic nervous system, culminating in autonomic denervation and congestive heart failure. Previously, circulating autoantibodies that activate signaling by cardiac muscarinic acetylcholine receptors (mAChRs) have been described. However, it remains unclear whether the chagasic IgGs directly interact with the m2 mAChRs (predominant cardiac subtype), and, if so, whether chronic exposure of the mAChRs to such activating IgGs would result in receptor desensitization. Here we performed studies with purified and reconstituted hm2 mAChRs and demonstrate that IgGs from chagasic serum immunoprecipitated the mAChRs in a manner similar to an anti-m2 mAChR monoclonal antibody tested in parallel. The chagasic antibodies did not directly interact with the ligand binding site, because the binding of radiolabeled antagonist was unchanged by the addition of the chagasic IgG. In intact cells stably expressing the hm2 mAChR, the chagasic IgGs, but not normal IgGs, mimicked the ability of the agonist acetylcholine to induce two effects associated with agonist-induced receptor desensitization: a decrease in affinity for agonist binding to m2 mAChR and sequestration of the hm2 mAChRs from the cell surface. The results demonstrate that the chagasic IgGs can directly interact with and desensitize m2 mAChRs and provide support for the hypothesis of autoimmune mechanisms having a role in the pathogenesis of Chagas' cardioneuromyopathy.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedad de Chagas/inmunología , Receptores Muscarínicos/inmunología , Animales , Autoanticuerpos/metabolismo , Autoinmunidad , Células CHO , Cricetinae , Humanos , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , TransfecciónRESUMEN
In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M1 and M2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.
Asunto(s)
Corazón/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Contracción Miocárdica/fisiología , Receptores Muscarínicos/fisiología , Transducción de Señal/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Carbacol/farmacología , Atrios Cardíacos/efectos de los fármacos , Fosfatos de Inositol/metabolismo , Contracción Miocárdica/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Wistar , Receptor Muscarínico M1 , Receptor Muscarínico M2RESUMEN
Experimental autoimmune myocarditis obtained in mice by immunization with heart antigens is characterized by the presence of lymphomononuclear infiltrates in atria and ventricles. Here we show the ability of soluble factors released by immune cells from mice immunized with heart antigens to decrease heart contractility in a similar way to a muscarinic agonist. These effects appear to be mediated by IFN-gamma since all of them could be blocked by an anti-IFN-gamma monoclonal antibody. Moreover, the negative inotropic effect induced by immune cell-conditioned media was blocked by atropine, confirming previous findings that IFN acts as a muscarinic agonist on isolated atria. The role of locally released cytokines and especially of IFN-gamma was also evaluated in infiltrated autoimmune myocarditis hearts; thus, the addition of monoclonal anti-IFN-gamma antibody reversed the decreased contractility characteristics of this model. We conclude that IFN released both systemically and locally by autoreactive T cells may contribute to the impaired cardiac function in this experimental model of autoimmune myocarditis.
Asunto(s)
Enfermedades Autoinmunes/complicaciones , Modelos Animales de Enfermedad , Corazón/fisiopatología , Miocarditis/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Medios de Cultivo Condicionados/farmacología , Interferón gamma/fisiología , Ratones , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/inmunología , Miocardio/inmunología , Receptores Muscarínicos/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
IL-2 is known to have various effects outside the frame of the immune system and it could have a role as an autocrine modulatory agent in certain cardiac dysfunctions associated to inflammatory cell infiltration of the heart. In this study, by means of binding assays on cardiac membranes or by evaluating contractility and cAMP levels of isolated rat atria in the presence of IL-2, we demonstrate that IL-2 may indirectly stimulate beta-adrenergic-mediated function by triggering the presynaptic release of norepinephrine (NE) on isolated rat atria. Thus it reacts with cardiac tissue inducing a positive inotropic response that can be blocked by the beta-adrenergic antagonist propranolol but not by phentolamine. Binding of the beta-adrenergic antagonist CGP 12177 is not affected by IL-2. Furthermore, chemical sympathectomy with 6-hydroxydopamine (6-OHDA) prevents the inotropic effect of IL-2, while cocaine treatment sensitizes atria to IL-2, indicating that NE secretion is required, and that the beta-adrenergic effect of IL-2 is presynaptic. In addition, IL-2 mimics beta-adrenergic stimulation increasing cAMP synthesis. Colchicine inhibited cAMP stimulation as well as positive inotropism, and both effects of IL-2 were additive with isoproterenol. However, cAMP synthesis could only be partially blocked by propranolol, suggesting that IL-2 stimulated cAMP synthesis both by a direct, beta-adrenergic independent mechanism and by a beta-adrenergic dependent pathway. We conclude that IL-2 induces a positive inotropic response in isolated rat atria through two different pathways: an indirect activation of myocardial beta-adrenergic receptors by releasing catecholamines and a direct action by increasing the production of the second messenger cAMP.
Asunto(s)
Atrios Cardíacos/efectos de los fármacos , Interleucina-2/farmacología , Transducción de Señal/fisiología , Animales , Función Atrial , AMP Cíclico/biosíntesis , Corazón/efectos de los fármacos , Corazón/fisiología , Atrios Cardíacos/metabolismo , Humanos , Masculino , Miocardio/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos beta/metabolismo , Sistema Nervioso Simpático/fisiologíaRESUMEN
The mitogenic effect of recombinant human erythropoietin (rHuEpo) on primary cultures of neonatal rat cardiac myocytes was observed. rHuEpo triggered a dose-dependent increase in myocyte proliferation. The hormone effect over optimally grown control culture 1 day after addition was maximum with 0.5 U/ml and was inhibited with anti-rHuEpo. Inhibitors of enzymatic pathways known to be involved in the cytokines intracellular mechanism such as genistein (tyrosine kinase inhibitor), 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (phospholipase C [PLC] inhibitor), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (protein kinase C [PKC] inhibitor) prevented the mitogenic action of rHuEpo. Also the inhibition of Na(+)-K(+)-ATPase activity by ouabain blunted the stimulatory action of rHuEpo on cell proliferation. The mitogenic action of the hormone was correlated with cardiac membrane paranitrophenylphosphatase (pNPPase) and PKC activity, since concentrations of rHuEpo that stimulate DNA synthesis increased pNPPase and PKC activity. Moreover, the enzymatic inhibition of tyrosine kinase, PLC, and PKC attenuated the stimulatory action of rHuEpo upon cardiac pNPPase activity. In this paper we demonstrate a non-hematopoietic action of rHuEpo showing both mitogenic and enzymatic effect upon primary myocyte cell culture and on pNPPase activity of neonatal rat heart. These effects are related to the capacity of rHuEpo to stimulate Na(+)-K(+)-ATPase activity and appear to be secondary to the activation of tyrosine kinase and PKC, indicating that in the rHuEpo mediated mitogenic action on cardiomyocytes involves the activation of the same enzymatic pathways that have been described by other cytokines in different tissues.
Asunto(s)
Eritropoyetina/farmacología , Mitógenos/farmacología , Miocardio/metabolismo , Transducción de Señal/genética , 4-Nitrofenilfosfatasa/antagonistas & inhibidores , 4-Nitrofenilfosfatasa/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Genisteína , Humanos , Isoflavonas/farmacología , Miocardio/citología , Ouabaína/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Evidence accumulated over the last decade gives adequate proof for the existence of circulating antibodies in Chagas' disease which bind to beta adrenergic and muscarinic cholinergic receptors of myocardium. The interaction of antibodies with cardiac neurotransmitter receptors behaving as an agonist, triggers intracellular signal transductions in the cells that alter the physiological behaviour of the heart. These events convert the normal to pathologically active cells. The interaction of antibodies against heart beta adrenergic and cholinergic receptors triggers physiologic, morphologic, enzymatic and molecular alterations, leading to cardiac damage. The analysis of the prevalence and distribution of these antibodies shows a strong association with seropositive asymptomatic patients with autonomic dysfunction in comparison with those asymptomatic without alteration of the heart autonomic disorders. The presence of these antibodies may thus partially explain the cardiomyoneuropathy of Chagas' disease, in which the sympathetic and parasympathetic systems are affected. The deposit of autoantibodies on the myocardial neurotransmitter receptors, behaving like an agonist, induced desensitization and/or down regulation of the receptors. This in turn, could lead to a progressive blockade of myocardium neurotransmitter receptors, with sympathetic and parasympathetic dennervation, a phenomenon that has been described in the course of Chagas cardioneuropathy.
Asunto(s)
Cardiomiopatía Chagásica , Receptores Adrenérgicos beta/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Muscarínicos/metabolismo , Receptores de Neurotransmisores/metabolismo , Cardiomiopatía Chagásica/inmunología , Cardiomiopatía Chagásica/fisiopatología , Humanos , Miocardio/inmunología , Miocardio/metabolismo , Receptores Adrenérgicos beta/inmunología , Receptores Colinérgicos/inmunología , Receptores Muscarínicos/inmunología , Receptores de Neurotransmisores/inmunologíaRESUMEN
We have previously shown that myocardium from experimental autoimmune myocarditis expresses H1 receptors not present in normal mice heart. ThEA acting via H1 receptors, augments cyclic AMP production in atria from autoimmune myocarditis mice without any effect on atria from control mice. Addition of mepyramine before ThEA caused cyclic AMP levels to fall to a level similar to basal, confirming the H1 receptor participation. Histamine at low concentrations mimicked the ThEA action on H1 receptor-stimulation of cyclic AMP production by autoimmune myocardium. The fact that the inhibition of phospholipase C blocked the cyclic AMP stimulation by ThEA, supports the assumption that this action is secondary to receptor-mediated hydrolysis of phosphoinositides, generating some oxidative metabolites (IP3-DAG), which in turn may be responsible for the cyclic AMP effect. So, the inhibition of protein kinase C and calcium/calmodulin partially prevented the stimulatory action of ThEA on cyclic AMP levels in autoimmune myocardium, suggesting that both pathways are implicated in this effect. Data shows that the stimulation of H1 receptors by specific agonist in atria from autoimmune myocarditis mice, augments the cyclic AMP, requiring the hydrolysis of phosphoinositide cycle. The role of this cyclic AMP augmentation in myocardium from autoimmune myocarditis mice, will provide a basis to assess the role of this second messenger as an important factor in the regulation and/or modulation of the physiological behaviour of the heart in the course of autoimmune myocarditis.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , AMP Cíclico/biosíntesis , Miocarditis/metabolismo , Receptores Histamínicos H1/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Cimetidina/farmacología , AMP Cíclico/fisiología , Atrios Cardíacos/metabolismo , Histamina/farmacología , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Masculino , Ratones , Fosfatidilinositoles/metabolismo , Pirilamina/farmacología , Receptores Histamínicos H1/efectos de los fármacos , Tiazoles/farmacologíaRESUMEN
The inotropic effect of isoproterenol, as well as the beta-adrenoceptor population, was measured in pregnant uterine tissue from female spontaneous hypertensive rats (SHR) (control group: C) and female SHR that were grafted with skin from Holtzman male rats (eclamptic group: E). The Kd value of the concentration-response curve of isoproterenol was higher for uteri from E rats than C rats. This phenomenon was not accompanied by a modification in the expression of beta-adrenoceptors. Inhibition of the synthesis of prostaglandins prevented the hyporeactivity to isoproterenol during eclampsia. Moreover, uteri from E rats generated and released greater amounts of prostaglandin E2 (PGE2) than uteri from C rats, even in the presence or absence of isoproterenol. In addition, whereas isoproterenol administered alone increased basal cyclic AMP (cAMP) production from C uteri, PGE2 administered alone enhanced cAMP production in E uterine tissue. These results suggest that the decrease in beta-adrenergic response to the agonist in E rats is ascribed to PGE2 production. The abnormal reactivity to the beta-agonist could be associated with a heterologous desensitization of uterine beta-adrenoceptors exerted by PGE2 overload in uteri from E rats. These results bear directly on the regulation of uterine motility during pregnancy, since an impaired response to beta-adrenergic innervation could lead to increased uterine motility, impairing the maintenance of pregnancy.
Asunto(s)
Dinoprostona/farmacología , Eclampsia/metabolismo , Receptores Adrenérgicos beta/efectos de los fármacos , Útero/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Metoxamina/farmacología , Embarazo , Ratas , Ratas Endogámicas SHR , Receptores Adrenérgicos beta/metabolismo , Contracción Uterina/efectos de los fármacos , Útero/efectos de los fármacosRESUMEN
We have recently shown that interleukin 2 (IL-2) can increase the contractile strength or rat atrial muscle through the activation of phospholipases and protein kinase C. The results of this study confirm that the reaction of IL-2 with rat atria involves protein kinase C and not the Ca(++)-calmodulin dependent kinases. Phorbol 12-myristate 13-acetate (PMA), a tumor promoter that activates protein kinase C directly (bypassing the phosphoinositide turnover step), has effects that are similar to those of IL-2 on atrial contractility. Furthermore, preincubation of the tissue with PMA prevents the IL-2 effect, suggesting that the kinases activated by the tumor promoter and IL-2 share a common substrate. This effect of IL-2 on atrial contractility opposes the cholinomimetic inhibitory action of IFN gamma. Thus, preincubation of cardiac tissue with IL-2 or PMA eliminates the effects of IFN gamma and viceversa. Apparently, the inhibitory action of IL-2 on IFN gamma involves an impaired response or atria to cholinergic activation, as IL-2 shifts to the right the dose response curve of the tissue to the cholinergic muscarinic agonist carbachol. Protein kinase C is also necessary for the occurrence of the IL-2-IFN gamma interference. The results of this study suggest that, during an inflammatory reaction in the heart, the balance of the two lymphokines can determine changes in the response of the tissue to autonomous nervous system agonists or to the cytokines that mimic the effects of these neurotransmitters.
Asunto(s)
Interferón gamma/farmacología , Interleucina-2/farmacología , Contracción Miocárdica/efectos de los fármacos , Animales , Carbacol/administración & dosificación , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Técnicas In Vitro , Masculino , Fosfolipasas/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas WistarRESUMEN
This review regards the main functional characteristics of hearts subjected to an autoimmune response, focusing especially on the role of T lymphocytes and autoantibodies in the development of cardiac dysfunction. Evidence of a strong association in the onset and time-course of immune response and cardiac dysfunction is presented and the results are viewed comparatively with myocarditis models induced by heart, parasite or virus inoculation. Cardiac damage is evaluated regarding various aspects, namely histologic, immunologic, biochemical, pharmacologic, physiologic. Finally, the model, for its characteristics of resulting from an autoimmune response against the heart with functional consequences, has proved its usefulness to study neuroimmune interaction, mainly the immune to nervous direction, as autoantibodies and T cell-derived factors have a role in cardiac failure.