RESUMEN
Abstract Background: Different immune mechanisms of myocardial damage involved in the pathophysiology of Chagas disease coexist with high titers of autoantibodies induced by T. cruzi . There are few studies in the literature about the adaptive role of anti-β1 and anti-M2 antibodies in chronic Chagas cardiomyopathy (CCC). Objectives: To evaluate the association between anti-β1 and anti-M2 antibodies with heart rate variability (HRV) parameters on 24h Holter monitoring and the rate-pressure product (RPP) on cardiopulmonary exercise testing (CPET). Methods: Anti-β1 and anti-M2 antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) in 64 patients affected by CCC. Analysis of HRV was performed through the time-domain indices NNs, mean NN, SDNN, SDANN, SDNN index, NNNs, RMSSD, and pNN50. Spearman's correlation coefficient was used to assess the association between antibody titers and numerical variables. The Mann-Whitney test was used for comparison between two groups. Multiple linear regression was used to identify independent variables capable of explaining anti-β1 and anti-M2 antibody titers at the 5% significance level. Results: On 24h Holter, during the period of greatest parasympathetic activation (2:00-6:00 a.m.), an inverse association was found between anti-β1 titers and SDNN (rs=-0.13, p =0.041, n=43), as well as a direct association between anti-M2 titers and SDANN ( r s=0.317, p =0.039, n=43). Regarding CPET variables, anti-β1 titers were directly associated with RPP (rs=0.371, p =0.005, n=56). The subgroup of patients with a normal chronotropic response showed higher anti-β1 titers than the subgroup with an impaired response (p=0.023). RPP was an independent explanatory variable for anti-β1 titers, although with a low coefficient of determination (R2=0.147). Conclusion: The findings of this study suggest that, in patients with CCC, anti-β1 and anti-M2 antibodies may affect HRV parameters. RPP was directly associated with higher anti-β1 titers.
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Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Adulto Joven , Sistema Nervioso Autónomo/fisiología , Cardiomiopatía Chagásica/fisiopatología , Receptores Adrenérgicos beta 1/fisiología , Receptor Muscarínico M2/fisiología , Enfermedad Crónica , Estudios Transversales , Anticuerpos Biespecíficos , Prueba de EsfuerzoRESUMEN
Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.
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Acetilcolina/farmacología , Agonistas del Receptor de Adenosina A1/farmacología , Adenosina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canales de Potasio/metabolismo , Receptor de Adenosina A1/metabolismo , Animales , Femenino , Cobayas , Masculino , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismoRESUMEN
Chagas' disease (ChD) is a parasitic disease endemic to regions of Latin America and with an increasingly global reach. Up to 30% of patients with ChD develop severe dilated cardiomyopathy, ventricular arrhythmias, conduction disorders and/or sudden cardiac death. Autoantibodies against M2 muscarinic acetylcholine receptors (M2 mAChR) have been implicated in the pathogenesis of ChD. We sought to understand whether there was an association between anti-M2 mAChR autoantibody titers in patients with chronic ChD and the presence of distal cardiac conduction disorders or cardiac arrhythmias. We conducted a cross-sectional study in 79 patients from Argentina and Bolivia with chronic ChD without evident structural heart disease. Autoantibody titers were measured using indirect enzyme-linked immunosorbent assay. Elevated anti-M2 mAChR autoantibody titers were associated with the presence of distal conduction disease but not with cardiac arrhythmias. High anti-M2 mAChR autoantibody levels could assist with identifying early structural heart disease in patients with chronic ChD.
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Arritmias Cardíacas/epidemiología , Autoanticuerpos/inmunología , Trastorno del Sistema de Conducción Cardíaco/epidemiología , Enfermedad de Chagas/epidemiología , Receptor Muscarínico M2/inmunología , Anciano , Argentina/epidemiología , Arritmias Cardíacas/sangre , Arritmias Cardíacas/etiología , Arritmias Cardíacas/inmunología , Autoanticuerpos/sangre , Bolivia/epidemiología , Trastorno del Sistema de Conducción Cardíaco/sangre , Trastorno del Sistema de Conducción Cardíaco/etiología , Trastorno del Sistema de Conducción Cardíaco/inmunología , Enfermedad de Chagas/sangre , Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/inmunología , Enfermedad Crónica , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , PrevalenciaRESUMEN
Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Agonistas Colinérgicos/administración & dosificación , Paclitaxel/administración & dosificación , Receptor Muscarínico M2/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Administración Metronómica , Animales , Arecolina/administración & dosificación , Arecolina/análogos & derivados , Carbacol/administración & dosificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , ARN Interferente Pequeño/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It has been reported that muscarinic type-2 receptors (M2R) are voltage sensitive in an agonist-specific manner. In this work, we studied the effects of membrane potential on the interaction of M2R with the superagonist iperoxo (IXO), both functionally (using the activation of the ACh-gated K+ current (IKACh) in cardiomyocytes) and by molecular dynamics (MD) simulations. We found that IXO activated IKACh with remarkable high potency and clear voltage dependence, displaying a larger effect at the hyperpolarized potential. This result is consistent with a greater affinity, as validated by a slower (τ = 14.8 ± 2.3 s) deactivation kinetics of the IXO-evoked IKACh than that at the positive voltage (τ = 6.7 ± 1.2 s). The voltage-dependent M2R-IXO interaction induced IKACh to exhibit voltage-dependent features of this current, such as the 'relaxation gating' and the modulation of rectification. MD simulations revealed that membrane potential evoked specific conformational changes both at the external access and orthosteric site of M2R that underlie the agonist affinity change provoked by voltage on M2R. Moreover, our experimental data suggest that the 'tyrosine lid' (Y104, Y403, and Y426) is not the previously proposed voltage sensor of M2R. These findings provide an insight into the structural and functional framework of the biased signaling induced by voltage on GPCRs.
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Activación del Canal Iónico/efectos de los fármacos , Isoxazoles/farmacología , Simulación de Dinámica Molecular , Compuestos de Amonio Cuaternario/farmacología , Receptor Muscarínico M2/fisiología , Acetilcolina/farmacología , Animales , Gatos , Células Cultivadas , Estimulación Eléctrica , Femenino , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Modelos Moleculares , Agonistas Muscarínicos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Conformación Proteica , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Xenopus laevisRESUMEN
Central cholinergic activation stimulates water intake, but also NaCl intake when the inhibitory mechanisms are blocked with injections of moxonidine (α2 adrenergic/imidazoline agonist) into the lateral parabrachial nucleus (LPBN). In the present study, we investigated the involvement of central M1 and M2 muscarinic receptors on NaCl intake induced by pilocarpine (non-selective muscarinic agonist) intraperitoneally combined with moxonidine into the LPBN or by muscimol (GABAA agonist) into the LPBN. Male Holtzman rats with stainless steel cannulas implanted bilaterally in the LPBN and in the lateral ventricle were used. Pirenzepine (M1 muscarinic antagonist, 1 nmol/1 µl) or methoctramine (M2 muscarinic antagonist, 50 nmol/1 µL) injected intracerebroventricularly (i.c.v.) reduced 0.3 M NaCl and water intake in rats treated with pilocarpine (0.1 mg/100 g of body weight) injected intraperitoneally combined with moxonidine (0.5 nmol/0.2 µL) into the LPBN. In rats treated with muscimol (0.5 nmol/0.2 µL) into the LPBN, methoctramine i.c.v. also reduced 0.3 M NaCl and water intake, however, pirenzepine produced no effect. The results suggest that M1 and M2 muscarinic receptors activate central pathways involved in the control of water and sodium intake that are under the influence of the LPBN inhibitory mechanisms.
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Ingestión de Líquidos/efectos de los fármacos , Núcleos Parabraquiales/metabolismo , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Cloruro de Sodio/metabolismo , Animales , Diaminas/farmacología , Conducta de Ingestión de Líquido/efectos de los fármacos , Imidazoles/farmacología , Masculino , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Muscimol/farmacología , Núcleos Parabraquiales/efectos de los fármacos , Pilocarpina/farmacología , Pirenzepina/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M2/efectos de los fármacos , Sodio en la DietaRESUMEN
Autoantibodies against the M2 subtype of muscarinic acetylcholine receptors with functional activities have been found in the sera of patients with dilated cardiomyopathy (DCM), and the second extracellular loop has been established as the predominant epitope. However, it has been shown that the third intracellular loop is recognized by Chagas disease patients with severe cardiac dysfunction. In this work, BALB/c mice were immunized with plasmids encoding these two epitopes, and a control group received the empty plasmid (pcDNA3 vector). Serum from these DNA-immunized animals had elevated and persistent titres of antibodies against respective antigens. Heart echocardiography indicated diminished left ventricular wall thickness and reduced ejection fraction for both epitope-immunized groups, and ergospirometry tests showed a significant decrease in the exercise time and oxygen consumption. Transfer of serum from these immunized mice into naïve recipients induced the same alterations in cardiac structure and function. Furthermore, electron microscopy analysis of donor-immunized animals revealed several ultrastructural alterations suggestive of autophagy and mitophagy, suggesting novel roles for these autoantibodies. Overall, greater functional and structural impairment was observed in the donor and recipient epitope groups, implicating the third intracellular loop epitope in the pathological effects for the first-time. Therefore, the corresponding peptides could be useful for autoimmune DCM diagnosis and targeted therapy.
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Autoanticuerpos , Autofagia/inmunología , Cardiomiopatía Dilatada/inmunología , Miocardio/inmunología , Receptor Muscarínico M2/inmunología , Animales , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Miocardio/patología , Miocardio/ultraestructura , Péptidos/genética , Péptidos/inmunología , Plásmidos/genética , Receptor Muscarínico M2/genéticaRESUMEN
The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.
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Potenciales de Acción , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Miocitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animales , Gatos , Células Cultivadas , Femenino , Atrios Cardíacos/citología , Magnesio/metabolismo , Masculino , Miocitos Cardíacos/fisiología , Poliaminas/metabolismoRESUMEN
Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. We investigate the role of toll-like receptor 4 (TLR4) on voiding dysfunction and inflammation in the cyclophosphamide (CYP)-induced mouse cystitis. Male C57BL/6 [wild-type, (WT)] and/or TLR4 knockout (TLR4-/-) mice were treated with an injection of CYP (300 mg/kg, 24 h) or saline (10 ml/kg). The pharmacological blockade of the TLR4 by resatorvid (10 mg/kg) was also performed 1 h prior CYP-injection in WT mice. Urodynamic profiles were assessed by voiding stain on filter paper and filling cystometry. Contractile responses to carbachol were measured in isolated bladders. In CYP-exposed WT mice, mRNA for TLR4, myeloid differentiation primary response 88, and TIR-domain-containing adapter-inducing interferon-ß increased by 45%, 72%, and 38%, respectively ( P < 0.05). In free-moving mice, CYP-exposed mice exhibited a higher number of urinary spots and smaller urinary volumes. Increases of micturition frequency and nonvoiding contractions, concomitant with decreases of intercontraction intervals and capacity, were observed in the filling cystometry of WT mice ( P < 0.05). Carbachol-induced bladder contractions were significantly reduced in the CYP group, which was paralleled by reduced mRNA for M2 and M3 muscarinic receptors. These functional and molecular alterations induced by CYP were prevented in TLR4-/- and resatorvid-treated mice. Additionally, the increased levels of inflammatory markers induced by CYP exposure, myeloperoxidase activity, interleukin-6, and tumor necrosis factor-alpha were significantly reduced by resatorvid treatment. Our findings reveal a central role for the TLR4 signaling pathway in initiating CYP-induced bladder dysfunction and inflammation and thus emphasize that TLR4 receptor blockade may have clinical value for IC/BPS treatment.
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Antiinflamatorios/farmacología , Ciclofosfamida , Cistitis Intersticial/prevención & control , Sulfonamidas/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/deficiencia , Vejiga Urinaria/efectos de los fármacos , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Cistitis Intersticial/inducido químicamente , Cistitis Intersticial/genética , Cistitis Intersticial/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Peroxidasa/metabolismo , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/metabolismo , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatología , Micción/efectos de los fármacos , Urodinámica/efectos de los fármacosRESUMEN
Cryptome is as a subset of a given proteome containing bioactive cryptides embedded in larger peptides or proteins. We pinpointed a striking sequence similarity between two peptides from the Tityus serrulatus venom: Ts10 (KKDGYPVEYDRAY) and the N-terminal of Ts3 (KKDGYPVEYDNCAY). Ts3 (former Tityustoxin or TsIV) is an α-neurotoxin acting on voltage-gated sodium channels while Ts10 (former Peptide T) is a bradykinin-potentiating peptide and was originally reported as inhibitor of the angiotensin-converting enzyme (ACEi). Thus, the goal of this study was to evaluate whether such peptide hidden in the N-terminal of Ts3 (Ts31-14[C12S]) was able to mimic known effects of Ts10 as well as to expand the current knowledge of the vascular effects and molecular targets of these peptides. Similar to Ts10, Ts31-14[C12S] was able to potentiate the hypotensive effect of bradykinin (BK). However, none of these peptides was able to induce a long-lasting BK-potentiating effect, suggesting that this effect may not be their main biological outcome. On the other hand, we report that Ts10 and mainly Ts31-14[C12S] induced a strong vasodilation effect depending on the presence of functional endothelium and nitric oxide (NO) production. Unlike previously reported, Ts10 was not able to inhibit ACE activity (similar result was observed for Ts31-14[C12S]). On the other hand, we report that Ts31-14[C12S] induces vasodilation via the activation of muscarinic acetylcholine receptors (mAChRs) M2 and M3 while only the activation of mAChR M2 seems to be required for Ts10-induced vasodilation.
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Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistas , Venenos de Escorpión/farmacología , Vasodilatadores/farmacología , Secuencia de Aminoácidos , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Humanos , Masculino , Modelos Animales , Peptidil-Dipeptidasa A/efectos de los fármacos , Ratas , Ratas Wistar , Vasodilatación/efectos de los fármacos , Canales de Sodio Activados por Voltaje/efectos de los fármacosRESUMEN
Recently, it has been shown that G protein-coupled receptors (GPCRs) display intrinsic voltage sensitivity. We reported that the voltage sensitivity of M2 muscarinic receptor (M2R) is also ligand specific. Here, we provide additional evidence to understand the mechanism underlying the ligand-specific voltage sensitivity of the M2R. Using ACh, pilocarpine (Pilo), and bethanechol (Beth), we evaluated the agonist-specific effects of voltage by measuring the ACh-activated K(+) current (I KACh) in feline and rabbit atrial myocytes and in HEK-293 cells expressing M2R-Kir3.1/Kir3.4. The activation of I KACh by the muscarinic agonist Beth was voltage insensitive, suggesting that the voltage-induced conformational changes in M2R do not modify its affinity for this agonist. Moreover, deactivation of the Beth-evoked I KACh was voltage insensitive. By contrast, deactivation of the ACh-induced I KACh was significantly slower at -100 mV than at +50 mV, while an opposite effect was observed when I KACh was activated by Pilo. These findings are consistent with the voltage affinity pattern observed for these three agonists. Our findings suggest that independent of how voltage disturbs the receptor binding site, the voltage dependence of the signaling pathway is ultimately determined by the agonist. These observations emphasize the pharmacological potential to regulate the M2R-parasympathetic associated cardiac function and also other cellular signaling pathways by exploiting the voltage-dependent properties of GPCRs.
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Acetilcolina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Canales de Potasio/metabolismo , Potasio/metabolismo , Receptor Muscarínico M2/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Gatos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Conejos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M(2)AChR pharmacological profile. Therefore, a novel Ca(2+)/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58 kDa) being the 58 kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [(3)H]cGMP and [(3)H]cAMP exhibiting a higher affinity as Km (µM) for cGMP than cAMP but being close values with V(max) cAMP/cGMP ratio of 1.5. The co-factor Mg(2+) showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC(50) of 4.9 and 4.6 µM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M(2)AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.
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Membrana Celular/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Tráquea/metabolismo , Animales , Atropina/farmacología , Western Blotting , Calmodulina/metabolismo , Bovinos , Membrana Celular/efectos de los fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Soluciones Hipotónicas , Concentración 50 Inhibidora , Cinética , Músculo Liso/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Alcaloides de la Vinca/farmacologíaRESUMEN
Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7(-/-) mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7(-/-) mice showed an attenuated production. Despite this, WT and P2×7(-/-) showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7(-/-) mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3(+)CD25(-) subset. M2AChR WT immunized mice showed an increase in IL-1ß, IFNγ and IL-17 levels in the heart, while P2×7(-/-) group produced lower amounts of IL-1ß and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM.
Asunto(s)
Cardiomiopatía Dilatada/genética , Interleucina-17/inmunología , Miocardio/inmunología , Receptor Muscarínico M2/genética , Receptores Purinérgicos P2X7/genética , Linfocitos T Reguladores/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoantígenos/genética , Autoantígenos/inmunología , Cardiomiopatía Dilatada/inmunología , Cardiomiopatía Dilatada/patología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica , Frecuencia Cardíaca , Inmunización , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/biosíntesis , Interleucina-1beta/biosíntesis , Interleucina-1beta/inmunología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Condicionamiento Físico Animal , Plásmidos/administración & dosificación , Receptor Muscarínico M2/inmunología , Receptores Purinérgicos P2X7/deficiencia , Transducción de Señal , Bazo/inmunología , Bazo/patología , Linfocitos T Reguladores/patología , Remodelación VentricularRESUMEN
SUMMARY Antibodies (Ab) recognizing G-protein coupled receptors, such as ß 1 and ß 2 adrenergic (anti-ß 1-AR and anti-ß 2-AR, respectively) and muscarinic cholinergic receptors (anti-M2-CR) may contribute to cardiac damage, however their role in chronic chagasic cardiomyopathy is still controversial. We describe that Trypanosoma cruzi-infected C3H/He mice show increased P and QRS wave duration, and PR and QTc intervals, while the most significant ECG alterations in C57BL/6 are prolonged P wave and PR interval. Echocardiogram analyses show right ventricle dilation in infected animals of both mouse lineages. Analyses of heart rate variability (HRV) in chronically infected C3H/He mice show no alteration of the evaluated parameters, while C57BL/6 infected mice display significantly lower values of HRV components, suggesting autonomic dysfunction. The time-course analysis of anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR Ab titres in C3H/He infected mice indicate that anti-ß 1-AR Ab are detected only in the chronic phase, while anti-ß 2-AR and anti-M2-CR are observed in the acute phase, diminish at 60 dpi and increase again in the chronic phase. Chronically infected C57BL/6 mice presented a significant increase in only anti-M2-CR Ab titres. Furthermore, anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR, exhibit significantly higher prevalence in chronically T. cruzi-infected C3H/He mice when compared with C57BL/6. These observations suggest that T. cruzi infection leads to host-specific cardiac electric alterations.
Asunto(s)
Antagonistas Adrenérgicos/sangre , Anticuerpos Antiprotozoarios/sangre , Arritmias Cardíacas/fisiopatología , Enfermedad de Chagas/fisiopatología , Colinérgicos/sangre , Disautonomías Primarias/fisiopatología , Trypanosoma cruzi/fisiología , Animales , Cardiomiopatía Chagásica/parasitología , Cardiomiopatía Chagásica/fisiopatología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta 1/metabolismoRESUMEN
BACKGROUND/AIMS: Impaired pancreatic beta cell function and insulin secretion/action are a link between obesity and type 2 diabetes, which are worldwide public health burdens. We aimed to characterize the muscarinic acetylcholine receptor (mAChR) M1-M4 subtypes in isolated pancreatic islets from pre-diabetic obese rats that had been treated neonatally with monosodium L-glutamate (MSG). METHODS: At 90 days of age, both the MSG and the control groups underwent biometric and biochemical evaluation. Anti-muscarinic drugs were used to study mAChR function either in vivo or in vitro. RESULTS: The results demonstrated that atropine treatment reduced insulin secretion in the MSG-treated and control groups, whereas treatment with an M2mAChR-selective antagonist increased secretion. Moreover, the insulinostatic effect of an M3mAChR-selective antagonist was significantly higher in the MSG-treated group. M1mAChR and M3mAChR expression was increased in the MSG-obese group by 55% and 73%, respectively. In contrast, M2mAChR expression decreased by 25% in the MSG group, whereas M4mAChR expression was unchanged. CONCLUSIONS: Functional changes in and altered content of the mAChR (M1-M4) subtypes are pivotal to the demand for high pancreatic beta cell insulin secretion in MSG-obese rats, which is directly associated with vagal hyperactivity and peripheral insulin resistance.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Obesidad/metabolismo , Receptores Muscarínicos/metabolismo , Glutamato de Sodio/farmacología , Animales , Glucemia/análisis , Prueba de Tolerancia a la Glucosa , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Obesidad/patología , Ratas , Ratas Wistar , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M4/metabolismo , Receptores Muscarínicos/químicaRESUMEN
Asthma is a complex disease for which genetic predisposition has been widely documented. Considerable evidence supports the hypothesis that polymorphisms in the muscarinic-cholinergic (CHRM) genes could be involved in asthma pathogenesis, bronchial hyperresponsiveness, and mucus secretion. To determine whether single nucleotide polymorphisms (SNPs) or haplotypes in CHRM1, CHRM2, or CHRM3 are associated with asthma in Mexican pediatric population. We performed a case-control study including 398 pediatric cases with asthma and 450 healthy controls. We analyzed 19 SNPs distributed among these three genes. Two of the seven SNPs located in CHRM2, the 3' untranslated region rs8191992 and rs6962027, differed significantly in allele frequencies between patients with asthma and healthy controls [odds ratio (OR) 1.42, 95 % confidence interval (95 % CI) 1.14-1.77, P = 0.001, and OR 1.50, 95 % CI 1.21-1.87, P = 0.0002, respectively]. Statistical significance remained after multiple comparison corrections (P = 0.003 and P = 0.005, respectively). The haplotypes AA and TT, containing both major and minor alleles from rs8191992 and rs6962027, also differed between cases and controls. The haplotype AA occurred at a lower frequency in cases (OR 0.67, 95 % CI 0.53-0.85, P = 0.001) whereas the haplotype TT was overrepresented in cases compared to controls (28 vs 21 %, respectively; OR 1.46, 95 % CI 1.15-1.85, P = 0.002). No association was observed between CHRM1 or CHRM3 SNPs or haplotypes and asthma. CHRM2 polymorphisms are implicated in the genetic etiology of asthma.
Asunto(s)
Asma/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/genética , Adolescente , Alelos , Asma/diagnóstico , Estudios de Casos y Controles , Niño , Femenino , Frecuencia de los Genes , Orden Génico , Estudios de Asociación Genética , Haplotipos , Humanos , Desequilibrio de Ligamiento , Masculino , México , Oportunidad RelativaAsunto(s)
Autoanticuerpos/inmunología , Cardiomiopatía Chagásica/tratamiento farmacológico , Cardiomiopatía Chagásica/inmunología , Nitroimidazoles/farmacología , Receptor Muscarínico M2/inmunología , Receptores Adrenérgicos beta/inmunología , Animales , Autoanticuerpos/sangre , Modelos Animales de Enfermedad , Perros , Humanos , Inmunosupresores/farmacologíaRESUMEN
AIM: The present study was designed to investigate the effects of progesterone-carbachol derivative on perfusion pressure and coronary resistance in rats. An additional aim was to identify the molecular mechanisms involved. METHODS: The Langendorff model was used to measure perfusion pressure and coronary resistance changes in isolated rat heart after progesterone-carbachol derivative alone and after the following compounds; mifepristone (progesterone receptor blocker), yohimbine (α2 adreno-receptor antagonist), ICI 118,551 (selective ß2 receptor blocker), atropine (non-selective muscarinic receptor antagonist), methoctramine (antagonist of M2 receptor) and L-NAME (inhibitor of nitric oxide synthase). RESULTS: The results show that progesterone-carbachol derivative [10(-9) mM] significantly decreased perfusion pressure (P=0.005) and coronary resistance (P=0.006) in isolated rat heart. Additionally, the effect of progesterone-carbachol on perfusion pressure [10(-9) to 10(-4) mM] was only blocked in the presence of methoctramine and L-NAME. CONCLUSIONS: These data suggest that progesterone derivative exert its effect on perfusion pressure via activation of the M2 muscarinic. In addition, this phenomenon involves stimulation of nitric oxide synthase (NOS).
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Carbacol/análogos & derivados , Vasos Coronarios/efectos de los fármacos , Progesterona/análogos & derivados , Receptor Muscarínico M2/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos , Animales , Carbacol/farmacología , Vasos Coronarios/fisiología , Masculino , Progesterona/farmacología , RatasAsunto(s)
Fibrilación Atrial/fisiopatología , Sistema de Conducción Cardíaco/fisiopatología , Corazón/inervación , Animales , Fibrilación Atrial/patología , Fibrilación Atrial/cirugía , Sistema Nervioso Autónomo/anatomía & histología , Sistema Nervioso Autónomo/fisiopatología , Tronco Encefálico/fisiología , Estimulación Cardíaca Artificial , Ablación por Catéter , Cromogranina A/uso terapéutico , Variación Genética , Atrios Cardíacos/inervación , Sistema de Conducción Cardíaco/patología , Humanos , Células Intersticiales de Cajal/fisiología , Modelos Cardiovasculares , Miocitos Cardíacos/fisiología , Fragmentos de Péptidos/uso terapéutico , Venas Pulmonares/anatomía & histología , Receptor Muscarínico M2/fisiología , Receptores Adrenérgicos beta/fisiología , Terapias en Investigación , Vagotomía , Nervio Vago/fisiología , Venas Cavas/anatomía & histologíaRESUMEN
Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation.