RESUMEN
AIM: The aim of the study was to investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) activity and expression in experimentally induced inflammation of rat dental pulp tissue. METHODOLOGY: Inflammation was induced by application of bacterial lipopolysaccharide (LPS) to the pulp. Extirpated pulp-tissue samples were incubated in saline solution until the various experiments were performed. Saline-treated pulp and healthy pulp tissues were used as controls. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. Nitrite/nitrate assay was evaluated by the conversion of nitrate to nitrite in the presence of nicotinamide adenine dinucleotide phosphate. i-nos, e-nos and n-nos mRNA levels were measured using reverse-transcriptase polymerase chain reaction by co-amplification of target cDNA with a single set of primers. RESULTS: Application of LPS to the pulp increased NOS activity and nitrate production (P < 0.001), generated by iNOS over-activity and expression. Pilocarpine acting on mAChRs triggered a biphasic action on NOS activity and NO accumulation. At low concentrations, pilocarpine induced a negative effect associated with a decrease in i-nos mRNA level, whilst at high concentration, it produced a positive effect associated with increased e-nos and n-nos mRNA levels. In control pulp tissue, only the positive effect of pilocarpine was observed. CONCLUSIONS: Irreversible pulpitis changes mAChR conformation increasing its efficiency of coupling to transducing molecules that in turn induce activate iNOS. The capacity of pilocarpine to prevent NO accumulation and iNOS activity, by acting on mAChR mutation induced by pulpitis, might be useful therapeutically as a local treatment.
Asunto(s)
Óxido Nítrico Sintasa de Tipo II/metabolismo , Pulpitis/enzimología , Receptores Muscarínicos/metabolismo , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Pulpa Dental/efectos de los fármacos , Pulpa Dental/enzimología , Masculino , Agonistas Muscarínicos/uso terapéutico , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Pilocarpina/uso terapéutico , Conformación Proteica , Pulpitis/tratamiento farmacológico , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores Muscarínicos/efectos de los fármacos , Estadísticas no ParamétricasRESUMEN
BACKGROUND AND OBJECTIVE: Autoimmune mechanisms may contribute to the pathogenesis of periodontal disease. Autoantibodies with the potential to bind and activate beta(1)-adrenoceptors (beta(1)-AR) of human gingival fibroblasts were studied to provide evidence of altered humoral immune response in chronic periodontal disease. MATERIAL AND METHODS: Flow cytometry and enzyme-linked immunosorbent assay using cell culture-adherent gingival fibroblasts and/or their purified membranes and/or a synthetic peptide corresponding to the second extracellular loop of human beta(1)-AR were used to detect serum antibodies. The effects of antibodies from chronic periodontal disease patients on PGE(2) generation and CD40 expression were also tested. RESULTS: Circulating immunoglobulin G (IgG) from chronic periodontal disease patients (but not from normal individuals) interacted with the fibroblast surface, activating beta(1)-AR. Atenolol or CGP 20712 (beta 1-AR antagonists) and beta(1) synthetic peptide inhibited the interaction of IgG with beta(1)-AR. Immunoglobulin G from chronic periodontal disease patients also displayed agonist-like activity associated with specific beta(1)-AR activation, increasing PGE(2) generation and CD40 overexpression. The corresponding affinity-purified anti-beta(1)-AR peptide IgG mimicked these effects. Both effects were prevented by inhibition of cyclo-oxygenase. CONCLUSION: This article supports the participation of humoral immune alterations in chronic periodontal disease resulting in postsynaptic functional deregulation. Overproduction of proinflammatory mediators (PGE(2) and CD40 expression) is induced as a consequence of antibody-beta(1)-AR interaction. The PGE(2)-CD40-IgG axis may play a part in the pathophysiological mechanisms underlying the inflammatory process in chronic periodontal disease.
Asunto(s)
Autoanticuerpos/inmunología , Antígenos CD40/biosíntesis , Periodontitis Crónica/inmunología , Dinoprostona/metabolismo , Receptores Adrenérgicos beta 1/inmunología , Formación de Anticuerpos , Biopelículas , Membrana Celular/inmunología , Células Cultivadas , Periodontitis Crónica/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Encía/citología , Encía/inmunología , Humanos , Inmunoglobulina G/inmunología , Indometacina/farmacología , Masculino , Persona de Mediana Edad , Imitación Molecular/inmunología , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Autoanticuerpos/fisiología , Receptores Muscarínicos/inmunología , Síndrome de Sjögren/inmunología , Adulto , Animales , Autoanticuerpos/aislamiento & purificación , Calcio/metabolismo , Estudios de Casos y Controles , Ciclooxigenasa 2/biosíntesis , Dinoprostona/biosíntesis , Femenino , Humanos , Inmunoglobulina G/inmunología , Persona de Mediana Edad , Agonistas Muscarínicos/metabolismo , Posmenopausia , Ratas , Glándula Submandibular/inmunología , Glándula Submandibular/metabolismoRESUMEN
AIM: To investigate the role of muscarinic acetylcholine receptor (mAChR) subtype activity in the regulation of endothelial- (e) and neuronal- (n) nitric oxide synthase (NOS) expression and activity. METHODOLOGY: Rat dental pulp tissue was used throughout the study. The e-nos and n-nos mRNA levels were specifically measured using reverse transcriptase polymerase chain reaction procedures that involve simultaneous co-amplification of both target cDNA and a reference template with a single set of primers. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. RESULTS: Stimulation of M(1)/M(2) and M(3)/M(4) mAChRs with pilocarpine caused an increase in e-nos and n-nos mRNA levels and NOS activity in the dental pulp. The specific mAChR subtype antagonists, L-NMMA, l-NIO and N(2)-propyl-L-arginine but not aminoguanidine attenuated all these effects. Inhibitors of phospholipase C (PLC), protein kinase C (PKC) and calcium/calmodulin (CaM) prevented the pilocarpine-dependent increase in n-nos and e-nos mRNA levels and NOS activity. CONCLUSIONS: Activation of mAChR subtypes stimulated NOS activity by increasing the production of NO through e-nos and n-nos gene expression and NOS activity. The mechanism appears to occur secondarily to stimulation of CaM and PKC enzymatic activity.
Asunto(s)
Pulpa Dental/metabolismo , Neurotransmisores/análisis , Óxido Nítrico Sintasa/análisis , Óxido Nítrico/análisis , Receptores Muscarínicos/fisiología , Transducción de Señal/fisiología , Animales , Arginina/farmacología , Calmodulina/antagonistas & inhibidores , Pulpa Dental/enzimología , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Masculino , Agonistas Muscarínicos/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/análisis , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/análisis , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Ornitina/análogos & derivados , Ornitina/farmacología , Pilocarpina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Wistar , Receptores Muscarínicos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Fosfolipasas de Tipo C/antagonistas & inhibidores , omega-N-Metilarginina/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Agonists of the M(2) muscarinic acetylcholine receptor (mAChR) increase mRNA for this receptor and mRNA for endothelial and neuronal isoforms of NO synthase (eNOS or nNOS). Here we examine the different signalling pathways involved in such events in rat cardiac atria. EXPERIMENTAL APPROACH: In isolated atria, the effects of carbachol on mRNA for M(2) receptors, eNOS and nNOS were measured along with changes in phosphoinositide (PI) turnover, translocation of protein kinase C (PKC), NOS activity and atrial contractility. KEY RESULTS: Carbachol increased mRNA for M(2) receptors, activation of PI turnover, translocation of PKC and NOS activity and decreased atrial contractility. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), NOS and PKC prevented the carbachol-dependent increase in mRNA for M(2) receptors. These inhibitors also attenuated the carbachol induced increase in nNOS- and eNOS-mRNA levels. Inhibition of nNOS shifted the dose response curve of carbachol on contractility to the right, whereas inhibition of eNOS shifted it to the left. CONCLUSIONS AND IMPLICATIONS: From our results, activation of M(2) receptors induced nNOS and eNOS expression and activation of NOS up-regulated M(2) receptor gene expression. The signalling pathways involved included stimulation of PI turnover via PLC activation, CaM and PKC. nNOS and eNOS mediated opposing effects on the negative inotropic effect in atria, induced by stimulation of M(2) receptors. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with cardiac neuromyopathy.
Asunto(s)
Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , ARN Mensajero/genética , Receptor Muscarínico M2/genética , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Estrenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/fisiología , Atrios Cardíacos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Naftalenos/farmacología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositoles/metabolismo , Pirrolidinonas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor Muscarínico M2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trifluoperazina/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , omega-N-Metilarginina/farmacologíaRESUMEN
OBJECTIVE: To define the capacity of different bracket materials to modify the growth and adherence of microorganisms. METHODS: Three types of brackets from the right upper central incisor were used: metallic, ceramic, and composite. Streptococcus mutans and Candida albicans were studied. The association of both species was also evaluated. The brackets were placed in flat-bottomed vials containing basal medium with 20% sucrose added; the flasks were inoculated with each of the microbial suspensions. The samples were incubated at 37 degrees C for 48 hours, after which the brackets were removed. The supernatant was removed from the flasks, the cells adhering to the glass were counted, and the brackets were studied with electron microscopy. RESULTS: The adherence of Streptococcus mutans was not modified by the different brackets. The adherence of Candida albicans was increased by the composite bracket, whereas the use of metallic brackets decreased the number of colony-forming units (CFUs). By electron microscopy we demonstrated that the adherence of Streptococcus mutans plus Candida albicans together varied according to the bracket materials with composite > ceramic > metallic. CONCLUSIONS: Orthodontic appliances serve as different impact zones and modify microbial adherence and colonization, acting as foreign reserves and possible sources of infection.
Asunto(s)
Candida albicans/fisiología , Soportes Ortodóncicos/microbiología , Streptococcus mutans/fisiología , Resinas Acrílicas/química , Adhesión Bacteriana , Candida albicans/crecimiento & desarrollo , Adhesión Celular , Cerámica/química , Resinas Compuestas/química , Metales/química , Microscopía Electrónica de Rastreo , Poliuretanos/química , Streptococcus mutans/crecimiento & desarrolloRESUMEN
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
In this paper, we have determined the effect of both muscarinic acetylcholine receptor (mAChR) and exogenous prostaglandin E(2) (PGE(2)) on PGE(2) production and cyclooxygenases (COX) mRNA gene expression on rat cerebral frontal cortex. Carbachol and PGE(2) increase endogenous PGE(2) production and the COX-1 mRNA levels by activation of PLA(2)s. The COX-1 and COX-2 activity participated in the production of PGE(2) triggered by exogenous PGE(2). While in carbachol-PGE(2) only COX-1 activity is affected. The specific inhibition of PGE(2) receptor was able to impair the increase of endogenous PGE(2) production triggered by both carbachol and exogenous PGE(2). These results suggest that carbachol-activation mAChR increased PGE(2) production that in turn interacting with its own receptor triggers an additional production of PGE(2). Both mechanisms appear to occur by using PLA(2) signaling system. This data should be able to contribute to understand the involvement of PGE(2) in normal brain function and its participation in neuroinflammatory processes.
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Corteza Cerebral/metabolismo , Dinoprostona/biosíntesis , Lóbulo Frontal/metabolismo , Transducción de Señal , Animales , Carbacol/metabolismo , Carbacol/farmacología , Corteza Cerebral/citología , Ciclooxigenasa 1/metabolismo , Relación Dosis-Respuesta a Droga , Lóbulo Frontal/citología , Proteínas de la Membrana/metabolismo , Fosfolipasas A/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Muscarínicos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
AIM: In this study, we have determined signalling pathways involved in adenosine A(1) receptor (A(1) receptor)-dependent stimulation of amylase release in rat parotid gland. METHODS: Amylase release, binding and cyclic adenosine monophosphate (cAMP) assays, inositol phosphates (IPs) production and nitric oxide synthase (NOS) activity in the presence of cyclopentyl-1,3-dipropylxanthine (CPA) alone or in the presence of different inhibitory drugs were performed. RESULTS: The binding parameters of specific A(1) antagonist [(3)H]-cyclopentyl 1,3-dipropilxanthine ([(3)H]-DPCPX) in parotid gland membranes show a population of high affinity sites with K(d) (nm) 0.53 +/- 0.06 and B(max) (fmol mg(-1) protein) 122.6 +/- 10.2. CPA stimulation of A(1) receptor exerts an increase in amylase release, IPs accumulation, cAMP production and NOS activity. All these A(1) agonist effects were blocked by the A(1) receptor antagonist DPCPX. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), and adenylate cyclase, but not NOS, activities attenuated the CPA stimulatory effect on amylase release. The effect of CPA on amylase release significantly correlated with its action either on cAMP or on IPs accumulation. CONCLUSION: These results suggest that CPA activation of parotid gland A(1) receptor induces a stimulatory effect on amylase release associated with increased production of cAMP and IPs accumulation. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving CaM and PKC. The CPA stimulation of NOS does not appear to participate in amylase release.
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Amilasas/metabolismo , Glándula Parótida/fisiología , Receptor de Adenosina A1/fisiología , Transducción de Señal/fisiología , Antagonistas del Receptor de Adenosina A1 , Animales , Calcio/metabolismo , AMP Cíclico/análisis , Femenino , Fosfatos de Inositol/análisis , Óxido Nítrico Sintasa/metabolismo , Glándula Parótida/enzimología , Ratas , Ratas Wistar , Receptores Purinérgicos P1/metabolismo , Xantinas/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
It is known that nitric oxide modulates the prostaglandin generation. However, little is known about the regulatory action of prostaglandin on nitric oxide production. There is a molecular cross-talk between nitric oxide and prostaglandin. Here, we examined biochemical signalling pathways coupled to the prostaglandin E(2) (PGE(2)) receptor related to nitric oxide synthase stimulation in rat submandibular gland. PGE(2) through the stimulation of its own receptor, triggered activation of phosphoinositide turnover (IPs), translocation of protein kinase C (PKC), stimulation of nitric oxide synthase activity (NOS) and increased production of cyclic GMP (cGMP). PGE(2) stimulation of NOS and cGMP production was blunted by agents interfering with calcium influx, calcium/calmodulin and phospholipase C (PLC) activities; while PKC inhibitor was able to stimulate PGE(2) effects. PGE(2) did not evoke amylase release, indicating that NOS/ cGMP pathway were not associated with this enzyme secretion. Our results suggest that this prostanoid could act as vasoactive chemical mediator through its ability to activate NOS-cGMP pathway via own gland membrane receptor.
Asunto(s)
Óxido Nítrico Sintasa/metabolismo , Prostaglandinas E/farmacología , Transducción de Señal/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/metabolismo , Amilasas/metabolismo , Animales , GMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Óxido Nítrico/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Glándula Submandibular/enzimologíaRESUMEN
BACKGROUND AND AIMS: Gastrointestinal disorders is one of the clinical manifestations of chronic Chagas' disease. The pathogenesis seems to be associated with autonomic dysfunction. Here, we consider the muscarinic cholinoceptor mediated alteration in distal colon function in chagasic megacolon. PATIENTS: Patients were divided into four groups: group I, chronic chagasic patients with megacolon; group II, chronic chagasic patients without megacolon; group III, non-chagasic patients with megacolon; and group IV, normal healthy volunteers (control). METHODS: Binding assay and immunoblot of cholinoceptors from human and rat colon and enzyme immunoassay (ELISA) using a synthetic 24mer peptide corresponding to the second extracellular loop of human M2 muscarinic acetylcholine receptors (mAChR) were used to detect the presence of serum antibodies. The effect of antibodies on basal tone and 3',5'-cyclic monophosphate (cAMP) production of human and rat distal colon strips were also tested. RESULTS: Group I but not the other groups had circulating antibodies capable of interacting with human colon activating M2 mAChR, as they competed with binding of specific radioligand to mAChR and interacted with the second extracellular loop of human M2 mAChR. Moreover, affinity purified anti-M2 peptide IgG from group I, in common with monoclonal antihuman M2 mAChR, recognised bands with a molecular weight corresponding to colon mAChR. This antibody also displayed an agonist-like activity, increasing basal tone and decreasing cAMP accumulation. Both effects were blunted by AF-DX 116 and neutralised by the synthetic peptide. CONCLUSIONS: In chagasic patients with megacolon there are antibodies that can recognise and activate M2 mAChR. The implications of these autoantibodies in the pathogenesis of chagasic megacolon is discussed.
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Enfermedad de Chagas/inmunología , Inmunoglobulina G/fisiología , Megacolon/inmunología , Receptores Colinérgicos/fisiología , Adulto , Anciano , Análisis de Varianza , Animales , Anticuerpos Monoclonales/fisiología , Autoanticuerpos/fisiología , Western Blotting/métodos , Estudios de Casos y Controles , Enfermedad de Chagas/complicaciones , AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Megacolon/etiología , Persona de Mediana Edad , Músculo Liso/metabolismo , Ratas , Ratas WistarRESUMEN
Isolated congenital heart block may be associated with autoimmune disorder such as Sjögren Syndrome and systemic lupus erythematosus. In this work we demonstrate circulating autoantibodies against neonatal heart M1 muscarinic acetylcholine receptor (mAChR) in the sera of children with congenital heart block. This antibody were able to react with the second extracellular loop of the human M1 mAChR as demonstrated using a synthetic peptide in enzyme immune assay and binding assay. Affinity purified anti-peptide IgG as well as total IgG from children with congenital heart block, interfered with the specific radioligand occupancy from neonatal heart M1 mAChR, interacting irreversibly. The antipeptide antibodies also displayed an 'agonist-like' activity, i.e. decreased contractility, activated nitric oxide synthase activity and increased production of cyclic GMP. All of these effects were selectively blunted by pirenzepine and neutralized by the synthetic M1 peptide. Both binding and biological effects were obtained using neonatal rat heart instead adult heart and were independent of Ro/SS-A and La/SS-B antibodies and were also absent in the sera of normal children. A clinical relevance of these findings is demonstrated by a strong association between the existence of circulating M1 mAChR antipeptide antibodies and the presence of isolated congenital heart block, making these antibodies a proper marker of this disease.
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Autoanticuerpos/inmunología , Autoantígenos/inmunología , Bloqueo Cardíaco/inmunología , Receptores Muscarínicos/inmunología , Secuencia de Aminoácidos , Animales , Autoanticuerpos/sangre , Niño , Preescolar , GMP Cíclico/inmunología , Corazón , Bloqueo Cardíaco/sangre , Bloqueo Cardíaco/complicaciones , Cardiopatías Congénitas/sangre , Cardiopatías Congénitas/inmunología , Humanos , Lactante , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/inmunología , Péptidos/inmunología , Ratas , Receptor Muscarínico M1RESUMEN
PURPOSE: The authors investigated whether circulating autoantibodies against M(3) muscarinic acetylcholine receptors (mAChRs) could be a new marker for diagnosis for primary and secondary Sjögren syndrome (SS) dry eye. METHODS: Enzyme-linked immunosorbent assay (ELISA) using both rat exorbital lacrimal gland acinar cell membranes and synthetic 25-mer peptide as antigens was used to determine autoantibodies against acinar cells and M(3) mAChRs. Also, nitric oxide synthase (NOS) activity was assessed to determine the biological effect of these autoantibodies in relation to the M(3) mAChR. RESULTS: Sera from dry eye primary SS (pSS) or secondary SS (sSS) patients tested by ELISA recognized membrane lacrimal gland acinar cells antigens and the synthetic 25-mer peptide, corresponding to the second extracellular loop of human M(3) mAChRs. Moreover, the IgG fraction and the corresponding affinity-purified anti-M(3) peptide autoantibodies from the same patients were able to activate NOS coupled to lacrimal gland M(3) mAChRs. As controls, IgG and sera from women without dry eye with or without rheumatoid arthritis and from normal control subjects gave negative results on ELISA and biological assay; thus demonstrating the specificity of the reaction. CONCLUSIONS: Autoantibodies against mAChR may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes and could be a new marker to differentiate SS dry eyes from non-SS dry eyes.
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Autoanticuerpos/sangre , Receptores Muscarínicos/inmunología , Síndrome de Sjögren/inmunología , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Autoanticuerpos/aislamiento & purificación , Autoantígenos/inmunología , Biomarcadores , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/análisis , Aparato Lagrimal/enzimología , Persona de Mediana Edad , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Péptidos/inmunología , Ratas , Ratas Wistar , Receptor Muscarínico M3 , Síndrome de Sjögren/diagnósticoRESUMEN
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
Cardiac tissue from autoimmune myocarditis mice was studied to evaluate the expression and biological activity of mRNA encoding H(1) receptor and iNOS. BALB/c inbred mice were immunized with heart protein and sacrificed at 20, 45 and 50 days post immunization. Heart contractility studies and RT-PCR assays were performed. Heart from autoimmune myocarditis mice show mRNA iNOS-related dysfunction with a decrease in heart contractility. This effect was accompanied with an increase production of cyclic GMP and was improved by treating autoimmune mice with an inhibitor of iNOS activity. In addition, autoimmune myocardium expressed an active histamine H(1) receptor mRNA coupled to phospholipase C. The activation of H(1) receptor by ThEA stimulated both phosphoinositide hydrolysis and heart contractility. Normal myocardium did not expressed neither iNOS mRNA nor H(1) receptor mRNA. In conclusions: the development of autoimmune cardiac dysfunction was associated with the expression of iNOS mRNA, cyclic GMP accumulation and the expression of an active histamine H(1) receptor mRNA with increase production of inositol phosphates. These protein emergence during the course of autoimmune myocarditis may be involved a distinct compensatory mechanism operating in this disease.
Asunto(s)
Miocarditis/metabolismo , Óxido Nítrico Sintasa/metabolismo , ARN Mensajero/metabolismo , Receptores Histamínicos H1/metabolismo , Animales , GMP Cíclico/metabolismo , Fosfatos de Inositol/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Contracción Miocárdica/inmunología , Miocarditis/enzimología , Miocarditis/inmunología , Radioinmunoensayo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
beta-Adrenoceptor (betaAR) expression and function as well as its modulation via intracellular transduction signals, were analyzed on the T cell lymphoma BW5147. Independently to the kinetic of proliferation and relative to the number of receptors displayed in normal T lymphocytes, BW5147 cells displayed a decreased number of betaAR, uncoupled to adenylate cyclase, but coupled to protein kinase C stimulation. This last effect was impaired by a beta-antagonist and by blockers of the enzymatic pathways involved in T lymphocyte proliferation, inducing a recovery of betaAR sites. Down-regulation of betaAR would implicate the loss of a negative neuroimmune control mechanism for lymphocyte proliferation. The coupling of the remaining sites to a positive signal for cellular activation, would contribute to establish an hyperproliferative state.
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Proteína Quinasa C/metabolismo , Receptores Adrenérgicos beta/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenilil Ciclasas/metabolismo , Agonistas Adrenérgicos beta/farmacología , División Celular/efectos de los fármacos , División Celular/inmunología , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Indoles/farmacología , Isoproterenol/farmacología , Linfoma de Células T , Maleimidas/farmacología , Neuroinmunomodulación/inmunología , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
The effect of fluoxetine on mitogen-induced B-cell proliferation was studied. In particular, we analyzed the influence of fluoxetine on the signal transduction pathways triggered after stimulation with lipopolysaccharide (LPS) and anti-immunoglobulin M antibodies (anti-IgM). We showed that fluoxetine had a dual effect on anti-IgM-stimulated B-cell proliferation: at optimal anti-IgM concentration, fluoxetine inhibited proliferation, whereas at suboptimal anti-IgM concentration, the drug enhanced proliferation. Fluoxetine exerted only an inhibitory effect on LPS-induced B-cell proliferation. Calcium influx seemed to be involved in these effects.
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Anticuerpos Antiidiotipos/farmacología , Linfocitos B/efectos de los fármacos , Fluoxetina/farmacología , Lipopolisacáridos/farmacología , Animales , Antidepresivos de Segunda Generación/farmacología , Linfocitos B/citología , Transporte Biológico , Calcio/metabolismo , División Celular/efectos de los fármacos , Interacciones Farmacológicas , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB CRESUMEN
Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.
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Óxido Nítrico Sintasa/metabolismo , Receptores Muscarínicos/fisiología , Glándulas Salivales/metabolismo , Animales , Activación Enzimática/fisiología , Fosfatidilinositoles/fisiología , Proteína Quinasa C/metabolismo , Ratas , Transducción de Señal/fisiologíaRESUMEN
The aim of this study was to evaluate whether the antidepressant drug fluoxetine could modify rat vas deferens response to norepinephrine (NE), and to compare its effect with that of desipramine and cocaine. Results showed that 10(-5)m fluoxetine produced a super-sensibility of vas deferens to NE. This result was the same as those obtained for 10(-6)m desipramine or cocaine. Since the effect was Na(+)- and Cl(-)-dependent, an inhibitory mechanism of neuronal NE transport was suggested. Fluoxetine did not modify [(3)H]prazosin K(d) or B(max) in rat vas deferens, reinforcing the hypothesis of a pre-synaptic site of action. On the other hand fluoxetine inhibited NE maximal effect. This inhibitory effect could be related to an antagonism of calcium entry through the voltage-dependent calcium channel, since it was partially reverted by increasing calcium concentration and, besides, the drug was able to inhibit the calcium concentration-response curve also. Contractions induced by 5-hydroxytryptamine (5-HT) were not modified in the presence of fluoxetine. It is concluded that fluoxetine modulates rat vas deferens response to low NE concentrations in the same manner as the selective inhibitor of NE neuronal uptake desipramine. This peripheral effect could participate in the modulation of the male reproductive tract observed by these drugs when used in clinical trials.