RESUMEN
RATIONALE: A higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure. OBJECTIVE: Here, we tested the putatively protective effects of PP1 inhibitor-2 (I2) overexpression using a heart failure model induced by chronic ß-adrenergic stimulation. METHODS AND RESULTS: Transgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7â¯days. I2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TGNaCl and WTNaCl. TGISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WTISO. After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TGISO as compared to WTISO. In contrast to basal contractility, the peak amplitude of [Ca]i and SR Ca load were reduced in TGNaCl as compared to WTNaCl. These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca]i decay kinetics were hastened in TGISO as compared to WTISO, which can be explained by a higher phospholamban phosphorylation at Ser16. Chronic catecholamine stimulation was followed by an enhanced expression of GSK3ß, whereas the phosphorylation at Ser9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I2 phosphorylation that may reactivate PP1. CONCLUSION: Our findings suggest that the basal desensitization of ß-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3ß-dependent phosphorylation of I2.
Asunto(s)
Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Oncogénicas/metabolismo , Proteína Fosfatasa 1/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN , Insuficiencia Cardíaca/metabolismo , Chaperonas de Histonas , Humanos , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Sarcómeros/efectos de los fármacos , Sarcómeros/genética , Cloruro de Sodio/farmacologíaRESUMEN
The cannabinoid CB1 receptors on the noradrenergic neurons in guinea pig hippocampal slices show an endogenous endocannabinoid tone. This conclusion is based on rimonabant, the facilitatory effect of which on noradrenaline release might be due to its inverse CB1 receptor agonism and/or the interruption of a tonic inhibition elicited by endocannabinoids. To examine the latter mechanism, a neutral antagonist would be suitable. Therefore, we studied whether O-2050 is a neutral CB1 receptor antagonist in the guinea pig hippocampus and whether it mimics the facilitatory effect of rimonabant. CB1 receptor affinity of O-2050 was quantified in cerebrocortical membranes, using (3)H-rimonabant binding. Its CB1 receptor potency and effect on (3)H-noradrenaline release were determined in superfused hippocampal slices. Its intrinsic activity at CB1 receptors was studied in hippocampal membranes, using (35)S-GTPγS binding. Endocannabinoid levels in hippocampus were determined by liquid chromatography-multiple reaction monitoring. O-2050 was about ten times less potent than rimonabant in its CB1 receptor affinity, potency and facilitatory effect on noradrenaline release. Although not affecting (35)S-GTPγS binding by itself, O-2050 shifted the concentration-response curve of a CB1 receptor agonist to the right but that of rimonabant to the left. Levels of anandamide and 2-arachidonoyl glycerol in guinea pig hippocampus closely resembled those in mouse hippocampus. In conclusion, our results with O-2050 confirm that the CB1 receptors on noradrenergic neurons of the guinea pig hippocampus show an endogenous tone. To differentiate between the two mechanisms leading to an endogenous tone, O-2050 is not superior to rimonabant since O-2050 may increase the inverse agonistic effect of endocannabinoids.
Asunto(s)
Antagonistas de Receptores de Cannabinoides/farmacología , Dronabinol/análogos & derivados , Hipocampo/efectos de los fármacos , Norepinefrina/metabolismo , Piperidinas/farmacología , Piranos/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Benzoxazinas/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Dronabinol/farmacología , Interacciones Farmacológicas , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Cobayas , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Alcamidas Poliinsaturadas/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , RimonabantRESUMEN
BACKGROUND AND METHODS: Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. The substrate of AF is composed of a complex interplay between structural and functional changes of the atrial myocardium often preceding the occurrence of persistent AF. However, there are only few animal models reproducing the slow progression of the AF substrate to the spontaneous occurrence of the arrhythmia. Transgenic mice (TG) with cardiomyocyte-directed expression of CREM-IbΔC-X, an isoform of transcription factor CREM, develop atrial dilatation and spontaneous-onset AF. Here we tested the hypothesis that TG mice develop an arrhythmogenic substrate preceding AF using physiological and biochemical techniques. RESULTS: Overexpression of CREM-IbΔC-X in young TG mice (<8weeks) led to atrial dilatation combined with distension of myocardium, elongated myocytes, little fibrosis, down-regulation of connexin 40, loss of excitability with a number of depolarized myocytes, atrial ectopies and inducibility of AF. These abnormalities continuously progressed with age resulting in interatrial conduction block, increased atrial conduction heterogeneity, leaky sarcoplasmic reticulum calcium stores and the spontaneous occurrence of paroxysmal and later persistent AF. This distinct atrial remodelling was associated with a pattern of non-regulated and up-regulated marker genes of myocardial hypertrophy and fibrosis. CONCLUSIONS: Expression of CREM-IbΔC-X in TG hearts evokes abnormal growth and development of the atria preceding conduction abnormalities and altered calcium homeostasis and the development of spontaneous and persistent AF. We conclude that transcription factor CREM is an important regulator of atrial growth implicated in the development of an arrhythmogenic substrate in TG mice.
Asunto(s)
Fibrilación Atrial/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/biosíntesis , Regulación de la Expresión Génica , Atrios Cardíacos/metabolismo , Miocardio/metabolismo , Animales , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Ratones , Ratones Transgénicos , Miocardio/patología , Técnicas de Cultivo de Órganos , Factores de TiempoRESUMEN
Different types of presynaptic inhibitory Gα(i/o) protein-coupled receptors usually do not act independently of each other but rather pre-activation of receptor X impairs the effect mediated via receptor Y. It is, however, unknown whether this interaction extends to the cannabinoid CB(1) receptor on cholinergic neurones and hence we studied whether its activation, pharmacological blockade, or genetic inactivation affects the function of other presynaptic inhibitory receptors. The electrically evoked acetylcholine or noradrenaline release was determined in superfused rodent tissues preincubated with (3)H-choline or (3)H-noradrenaline. The muscarinic M(2) receptor, Gα(i), and Gα(o) proteins were determined in hippocampal synaptosomes by Western blotting. Hippocampal anandamide and 2-arachidonoyl glycerol levels were determined by LC-MS/MS. The inhibitory effect of the muscarinic receptor agonist oxotremorine on acetylcholine release in hippocampal slices was increased by genetic CB(1) receptor ablation (mouse) and the CB(1) antagonist rimonabant (rat but not mouse) and decreased by a cannabinoid receptor agonist (mouse). In mouse tissues, CB(1) receptor ablation also increased the effect of a δ opioid receptor agonist on acetylcholine release in the hippocampus and the effect of oxotremorine on noradrenaline release in the vas deferens. CB(1) receptor ablation, to a very slight extent, increased Gα(o) protein levels without affecting either Gα(i) and M(2) receptor protein or the levels of anandamide and 2-arachidonoyl glycerol in the hippocampus. In conclusion, the CB(1) receptor shows an inhibitory interaction with the muscarinic and δ opioid receptor on cholinergic neurones in the rodent hippocampus and with the muscarinic receptor on noradrenergic neurones in the mouse vas deferens.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Hipocampo/fisiología , Receptor Cannabinoide CB1/fisiología , Receptores Muscarínicos/fisiología , Receptores Opioides delta/fisiología , Conducto Deferente/fisiología , Analgésicos Opioides/farmacología , Animales , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/fisiología , Endocannabinoides , Encefalina D-Penicilamina (2,5)/farmacología , Glicéridos/metabolismo , Hipocampo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Muscarínicos/farmacología , Oxotremorina/farmacología , Piperidinas/farmacología , Alcamidas Poliinsaturadas/metabolismo , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores Opioides delta/agonistas , Rimonabant , Sinaptosomas/efectos de los fármacos , Sinaptosomas/fisiología , Conducto Deferente/efectos de los fármacosRESUMEN
We studied whether noradrenaline release is affected by interleukin-1ß and the neuropeptides urotensin II, melanin-concentrating hormone (MCH), neuropeptide W (NPW) and neuropeptide FF (NPFF). Rodent tissues preincubated with [3H]noradrenaline were superfused, and the effect of peptides on the electrically-evoked tritium overflow ("noradrenaline release") was studied. In mouse brain cortex, interleukin-1ß at 0.3 nM and the prostaglandin E2 analogue sulprostone at 3 nM inhibited noradrenaline release by about 40% the effect of interleukin-1ß developed gradually, whereas the effect of sulprostone occurred promptly. Urotensin II at 0.001-1 µM did not affect noradrenaline release in rat kidney cortex, whereas 0.01 µM angiotensin II increased it (positive control). MCH at 0.01-1 µM did not alter noradrenaline release in the rat brain cortex, and NPW 1 µM did not affect noradrenaline release in the mouse hypothalamus or hippocampus. In each model, 0.1 µM sulprostone inhibited noradrenaline release (positive control). NPFF and the NPFF2 receptor agonist dNPA (1 µM) did not affect noradrenaline release in the mouse atria; the inhibitory effect of the δ opioid receptor agonist 1 µM DPDPE on noradrenaline release in this tissue was not altered by NPFF or dNPA at 0.32 µM but was counteracted by the δ opioid antagonist naltrindole at 0.001 µM. In conclusion, interleukin-1ß inhibits noradrenaline release in the mouse cortex; the effect develops gradually, suggesting that it affects protein biosynthesis. Noradrenergic neurons in various tissues from rodents are devoid of presynaptic receptors for urotensin II, MCH, NPW and NPFF. Finally, an interaction between a δ opioid agonist and NPFF could not be detected.
Asunto(s)
Interleucina-1beta/farmacología , Neuropéptidos/farmacología , Norepinefrina/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Interleucina-1beta/administración & dosificación , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Masculino , Ratones , Neuropéptidos/administración & dosificación , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Cardiomiopatía Dilatada/inmunología , Cardiomiopatía Dilatada/metabolismo , Análisis por Matrices de Proteínas , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Cardiomiopatía Dilatada/diagnóstico , Humanos , Proteínas de Interacción con los Canales Kv/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.