RESUMO
OBJECTIVE: To evaluate the impact of different types of hypertension on the development of intimal hyperplasia (IH). METHOD: Genetic, surgical, and pharmacological models of hypertension were used to compare IH formation in a murine model of carotid artery ligation (CAL). CAL was performed in normotensive WT male mice and in three mouse models of hypertension: (1) L-NAME (Nω-nitro-l-arginine-methyl-ester) treatment for 2 weeks prior to CAL to instate renin-independent hypertension; (2) 2K1C (two kidneys, one clip) surgery 1 week prior to CAL to induce renin-dependent hypertension; (3) Cx40-/- mice, a genetic model of renin-dependent hypertension. Mice were sacrificed prior to CAL or 3, 14, or 28 days post CAL. Data collection included tail blood pressure measurements, and morphometric and histological assessment of the ligated carotids. RESULTS: CAL triggered the formation of a VSMC-rich neointima layer after 14-28 days, which was increased in all hypertensive mice. Despite similarly increased blood pressure, L-NAME treated mice displayed more IH than all other hypertensive groups. In addition, L-NAME induced hypertension triggered more cell proliferation and recruitment of CD45 positive inflammatory cells to the ligated vessel wall compared with Cx40-/- or normotensive WT mice. CONCLUSIONS: NO deficiency is a major aspect of vascular inflammation, VSMC proliferation, and IH in hypertensive conditions.
Assuntos
Artérias Carótidas/patologia , Hiperplasia/etiologia , Hipertensão/complicações , Óxido Nítrico/deficiência , Animais , Modelos Animais de Doenças , Hiperplasia/patologia , Hipertensão/induzido quimicamente , Hipertensão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/patologia , NG-Nitroarginina Metil Éster/farmacologiaRESUMO
AIMS/HYPOTHESIS: Pro-atherogenic and pro-oxidant, oxidised LDL trigger adverse effects on pancreatic beta cells, possibly contributing to diabetes progression. Because oxidised LDL diminish the expression of genes regulated by the inducible cAMP early repressor (ICER), we investigated the involvement of this transcription factor and of oxidative stress in beta cell failure elicited by oxidised LDL. METHODS: Isolated human and rat islets, and insulin-secreting cells were cultured with human native or oxidised LDL or with hydrogen peroxide. The expression of genes was determined by quantitative real-time PCR and western blotting. Insulin secretion was monitored by EIA kit. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Exposure of beta cell lines and islets to oxidised LDL, but not to native LDL raised the abundance of ICER. Induction of this repressor by the modified LDL compromised the expression of important beta cell genes, including insulin and anti-apoptotic islet brain 1, as well as of genes coding for key components of the secretory machinery. This led to hampering of insulin production and secretion, and of cell survival. Silencing of this transcription factor by RNA interference restored the expression of its target genes and alleviated beta cell dysfunction and death triggered by oxidised LDL. Induction of ICER was stimulated by oxidative stress, whereas antioxidant treatment with N-acetylcysteine or HDL prevented the rise of ICER elicited by oxidised LDL and restored beta cell functions. CONCLUSIONS/INTERPRETATION: Induction of ICER links oxidative stress to beta cell failure caused by oxidised LDL and can be effectively abrogated by antioxidant treatment.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/fisiologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/fisiopatologia , Estresse Oxidativo/fisiologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Modulador de Elemento de Resposta do AMP Cíclico/efeitos dos fármacos , Modulador de Elemento de Resposta do AMP Cíclico/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Masculino , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Left ventricular hypertrophy (LVH) is due to pressure overload or mechanical stretch and is thought to be associated with remodeling of gap-junctions. We investigated whether the expression of connexin 43 (Cx43) is altered in humans in response to different degrees of LVH. The expression of Cx43 was analyzed by quantitative polymerase chain reaction, Western blot analysis and immunohistochemistry on left ventricular biopsies from patients undergoing aortic or mitral valve replacement. Three groups were analyzed: patients with aortic stenosis with severe LVH (n=9) versus only mild LVH (n=7), and patients with LVH caused by mitral regurgitation (n=5). Cx43 mRNA expression and protein expression were similar in the three groups studied. Furthermore, immunohistochemistry revealed no change in Cx43 distribution. We can conclude that when compared with mild LVH or with LVH due to volume overload, severe LVH due to chronic pressure overload is not accompanied by detectable changes of Cx43 expression or spatial distribution.
Assuntos
Estenose da Valva Aórtica/complicações , Conexina 43/análise , Hipertrofia Ventricular Esquerda/mortalidade , Insuficiência da Valva Mitral/complicações , Miocárdio/química , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/fisiopatologia , Biópsia , Pressão Sanguínea , Western Blotting , Conexina 43/genética , Feminino , Regulação da Expressão Gênica , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/metabolismo , Insuficiência da Valva Mitral/patologia , Insuficiência da Valva Mitral/fisiopatologia , Miocárdio/patologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Função Ventricular EsquerdaRESUMO
Vessel wall trauma induces vascular remodeling processes including the development of intimal hyperplasia (IH). To assess the development of IH in human veins, we have used an ex vivo vein support system (EVVSS) allowing the perfusion of freshly isolated segments of saphenous veins in the presence of a pulsatile flow which reproduced arterial conditions regarding shear stress, flow rate and pressure during a period of 7 and 14 days. Compared to the corresponding freshly harvested human veins, histomorphometric analysis showed a significant increase in the intimal thickness which was already maximal after 7 days of perfusion. Expression of the endothelial marker CD31 demonstrated the presence of endothelium up to 14 days of perfusion. In our EVVSS model, the activity as well as the mRNA and protein expression levels of plasminogen activator inhibitor 1, the inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), were increased after 7 days of perfusion, whereas the expression levels of tPA and uPA were not altered. No major change was observed between 7 and 14 days of perfusion. These data show that our newly developed EVVSS is a valuable setting to study ex vivo remodeling of human veins submitted to a pulsatile flow.
Assuntos
Veia Safena/fisiologia , Idoso , Velocidade do Fluxo Sanguíneo , Técnicas de Cultura de Células/métodos , Endotélio Vascular/fisiologia , Feminino , Humanos , Masculino , Perfusão/métodos , Inibidor 1 de Ativador de Plasminogênio/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Pulso Arterial , Veia Safena/citologia , Veia Safena/imunologia , Veia Safena/patologia , Ativador de Plasminogênio Tecidual/genética , Coleta de Tecidos e Órgãos/métodos , Túnica Média/patologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Procedimentos Cirúrgicos VascularesRESUMO
We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca(++) for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10-100 nM), cyclosporin A (1 microM),and BMS605401 (100 nM), a specific inhibitor of Ca(2+)-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100-200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was >or=2.2 mM, while Cx43 was independent from extracellular [Ca(++)]. In cultured cells, 4 mM Ca(++)-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca(2+)-induced changes. Moreover, cyclosporin A completely abolished the Ca(2+)-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.
Assuntos
Cálcio/fisiologia , Junções Comunicantes/fisiologia , Animais , Fibrilação Atrial/metabolismo , Células Cultivadas , Conexina 43/análise , Conexina 43/fisiologia , Conexinas/análise , Conexinas/fisiologia , Ciclosporina/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Transdução de Sinais , Verapamil/farmacologia , Proteína alfa-5 de Junções ComunicantesRESUMO
Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Regulação Enzimológica da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Sequência de Bases , Northern Blotting , Proteínas de Transporte/metabolismo , DNA Complementar/metabolismo , Inibidores Enzimáticos/farmacologia , Biblioteca Gênica , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Células PC12 , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
The cellular distribution of connexin40 (Cx40), a newly cloned gap junction structural protein, was examined by immunofluorescence microscopy using two different specific anti-peptide antibodies. Cx40 was detected in the endothelium of muscular as well as elastic arteries in a punctate pattern consistent with the known distribution of gap junctions. However, it was not detected in other cells of the vascular wall. By contrast, Cx43, another connexin present in the cardiovascular system, was not detected in endothelial cells of muscular arteries but was abundant in the myocardium and aortic smooth muscle. We have tested the ability of these connexins to interact functionally. Cx40 was functionally expressed in pairs of Xenopus oocytes and induced the formation of intercellular channels with unique voltage dependence. Unexpectedly, communication did not occur when oocytes expressing Cx40 were paired with those expressing Cx43, although each could interact with a different connexin, Cx37, to form gap junction channels in paired oocytes. These findings indicate that establishment of intercellular communication can be spatially regulated by the selective expression of different connexins and suggest a mechanism that may operate to control the extent of communication between cells.
Assuntos
Endotélio Vascular/metabolismo , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Cardiovascular/metabolismo , Comunicação Celular/fisiologia , Conexinas , DNA/genética , Eletroquímica , Endotélio Vascular/ultraestrutura , Imunofluorescência , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Oócitos/metabolismo , Oócitos/ultraestrutura , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ratos , XenopusRESUMO
Previous studies have provided evidence for the transcripts of Cx43 and Cx45 within pancreatic islets. As of yet, however, it has proven difficult to unambiguously demonstrate the expression of these proteins by islet cells. We have investigated whether Cx36, a new connexin species recently identified in mammalian brain and retina, may also be expressed in pancreatic islets. Using probes that permitted the original identification of Cx36 in the central nervous system, we show that a transcript for Cx36 is clearly detectable in rat pancreatic islets. Using novel and affinity-purified polyclonal antibodies, we have found that Cx36 is actually expressed in pancreatic islets. Both in situ hybridization and immunolabeling indicated that this connexin is abundant in the centrally located insulin-producing beta-cells and is expressed much less, if at all, by the other endocrine cell types. This differential expression was further confirmed on fluorescence-activated cell sorter-purified preparations enriched in either beta- or non-beta-cells. The finding of a differential distribution of Cx36 within distinct regions of pancreatic islets creates the possibility that this connexin may provide the establishment of selective pathways of communication between the different types of endocrine cells comprising the pancreatic islet.
Assuntos
Conexinas/metabolismo , Proteínas do Olho/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Conexinas/genética , Proteínas do Olho/genética , Técnicas Imunológicas , Hibridização In Situ , Insulina/biossíntese , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Proteína delta-2 de Junções ComunicantesRESUMO
OBJECTIVES: To elucidate the structural basis for the electrophysiologic remodeling induced by chronic atrial fibrillation (AF), we investigated connexin40 and connexin43 (Cx40 and Cx43) expression and distribution in atria of patients with and without chronic AF and in an animal model of AF with additional electrophysiologic investigation of anisotropy (ratio of longitudinal and transverse velocities). BACKGROUND: Atrial fibrillation is a common arrhythmia that has a tendency to become persistent. Since gap junctions provide the syncytial properties of the atrium, changes in expression and distribution of intercellular connections may accompany the chronification of AF. METHODS: Atrial tissues isolated from 12 patients in normal sinus rhythm at the time of cardiac surgery and from 12 patients with chronic AF were processed for immunohistology and immunoblotting for the detection of the gap junction proteins. The functional study of the cardiac tissue anisotropy was performed in rat atria in which AF was induced by 24 h of rapid pacing (10 Hz). RESULTS: Immunoblotting revealed that AF did not induce any significant change in Cx43 content in human atria. In contrast, a 2.7-fold increase in expression of Cx40 was observed in AF. Immunohistologic analysis indicated that AF resulted in an increase in the immunostaining of both connexins at the lateral membrane of human atrial cells. A similar spatial redistribution of the Cx43 signal was seen in isolated rat atria with experimentally-induced AF. In addition, AF in rat atria resulted in decreased anisotropy with slightly enhanced transverse conduction velocity. CONCLUSIONS: This experimental study showed that AF is accompanied by spatial remodeling of gap junctions that might induce changes in the biophysical properties of the tissue.
Assuntos
Fibrilação Atrial/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Átrios do Coração/metabolismo , Idoso , Animais , Anisotropia , Western Blotting , Doença Crônica , Técnicas Eletrofisiológicas Cardíacas , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Pessoa de Meia-Idade , Modelos Animais , Ratos , Distribuição Tecidual , Proteína alfa-5 de Junções ComunicantesRESUMO
Human complement component C8 exhibits an unusual structure in that it contains three chains, two of which, alpha and beta, display high sequence homology to other complement and CTL pore-forming proteins. The third chain, C8 gamma, is covalently linked to C8 alpha by a disulfide linkage; it is demonstrated that Cys40 of C8 gamma is linked to Cys164 of C8 alpha, a unique cysteine located in a loop located between the cysteine-rich LDL-receptor class A module and the membrane-inserting region of C8 alpha. C8 gamma was recently identified as a member of the lipocalin protein family, in which all proteins were either shown to, or are believed to bind small hydrophobic ligands. The present results now demonstrate that C8 gamma incorporates retinol and retinoic acid in the presence of 2 M NaCl. Molecular modeling of C8 gamma, based on the crystal structure of the homologous beta-lactoglobulin, reveals a structure of eight antiparallel beta-strands, bearing a highly hydrophobic binding pocket. The residues participating in the pocket formation are highly conserved when compared with the structures of beta-lactoglobulin and retinol-binding protein, both of which are known to interact with retinol. It is therefore proposed that C8 gamma may act as a retinol transporting protein in plasma.
Assuntos
Complemento C8/química , alfa-Globulinas/química , Sequência de Aminoácidos , Complemento C8/metabolismo , Brometo de Cianogênio/química , Dissulfetos/química , Humanos , Lactoglobulinas/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/química , Proteínas de Ligação ao Retinol/química , Proteínas Plasmáticas de Ligação ao Retinol , Solventes , Tretinoína/metabolismo , Vitamina A/metabolismoRESUMO
The aim of this investigation was to examine the interrelation between renal mRNA levels of renin and angiotensin II receptor type 1 (AT1) in a renin-dependent form of experimental hypertension. Rats were studied 4 weeks after unilateral renal artery clipping. Mean blood pressure and plasma renin activity were significantly higher in the hypertensive rats (n = 10 206 +/- mm Hg and 72.4 +/- 20.9 ng/mL-1/h-1, respectively) than in sham-operated controls (n = 10, 136 +/- 3 mm Hg and 3.3 +/- 0.5 ng/mL-1/h, respectively). Northern blot analysis of polyA+ RNA obtained from the kidneys of renal hypertensive rats showed increased levels of renin mRNA in the clipped kidney, whereas a decrease was observed in the unclipped kidney. Plasma renin activity was directly correlated with renin mRNA expression of the poststenotic kidney (r = .94, P < .01). AT1 mRNA expression was lower in both kidneys of the hypertensive rats. This downregulation was specific for the AT1A subtype since the renal expression of the AT1B subtype remained normal in hypertensive rats. The downregulation of the renal AT1A receptor may be due to high circulating angiotensin II levels. This is supported by the significant inverse correlation (r = .71, P < .01) between plasma renin activity and AT1A mRNA expression measured in the clipped kidney of the hypertensive rats.
Assuntos
Hipertensão Renovascular/metabolismo , Rim/metabolismo , RNA Mensageiro/análise , Receptores de Angiotensina/biossíntese , Renina/biossíntese , Animais , Regulação para Baixo , Hipertensão Renovascular/genética , Masculino , Ratos , Ratos Wistar , Receptores de Angiotensina/genética , Renina/sangue , Renina/genéticaRESUMO
Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Conexinas/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Urotélio/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Comunicação Celular/fisiologia , Conexina 26 , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Proteínas de Ligação a DNA/metabolismo , Junções Comunicantes/metabolismo , Técnicas de Transferência de Genes , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Transdução de Sinais/fisiologia , Estresse Mecânico , Transativadores/genética , Fator de Transcrição AP-1/metabolismo , Obstrução UretralRESUMO
The secretory, duct, connective and vascular cells of pancreas are connected by gap junctions, made of different connexins. The insulin-producing beta-cells, which form the bulk of endocrine pancreatic islets, express predominantly Cx36. To assess the function of this connexin, we have first studied its expression in rats, during sequential changes of pancreatic function which were induced by the implantation of a secreting insulinoma. We observed that changes in beta-cell function were paralleled by changes in Cx36 expression. We have also begun to investigate mutant mice lacking Cx36. The absence of this protein did not affect the development and differentiation of beta-cells but appeared to alter their secretion. We have studied this effect in MIN6 cells which spontaneously express Cx36. After stable transfection of a construct that markedly reduced the expression of this connexin, we observed that MIN6 cells were no more able to secrete insulin, in contrast to wild type controls, and differentially displayed a series of still unknown genes. The data provide evidence that Cx36-dependent signaling contributes to regulate the function of native and tumoral insulin-producing cells.
Assuntos
Conexinas/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Conexinas/genética , Junções Comunicantes/metabolismo , Insulinoma , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Neoplasias Pancreáticas , Ratos , Células Tumorais Cultivadas , Proteína delta-2 de Junções ComunicantesRESUMO
The high Km glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic beta-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic beta-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.
Assuntos
Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , Primers do DNA/genética , Expressão Gênica , Genes Reporter , Transportador de Glucose Tipo 2 , Imuno-Histoquímica , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Neuropeptide Y (NPY) is a key modulator of the autonomic nervous system playing pivotal roles in cardiovascular and neuronal functions. In this study, we assessed the cellular localization and gene expression of NPY in rat kidneys. We also examined the relationship between NPY gene expression and renin in two rat models of hypertension (two-kidney, one-clip renal hypertension (2K1C), and deoxycorticosterone-salt-induced hypertension (DOCA-salt)) characterized by a similar blood pressure elevation. In situ hybridization and immunohistochemistry, using anti-NPY or anti-C-flanking peptide of NPY (CPON) antibodies, showed that NPY transcript and protein were colocalized in the tubules of rat kidneys. During experimental hypertension, NPY mRNA was decreased in both kidneys of the 2K1C animals, but not in the kidney of DOCA-salt rats. In 2K1C rats, renal NPY content was also decreased. The difference in NPY gene expression between 2K1C rats (a high renin model of hypertension) and DOCA-salt rats (a low renin model of hypertension) suggests that circulating angiotensin II plays a role in local renal NPY gene expression and that the elevated blood pressure per se is not the primary factor responsible for the control of NPY gene expression in the kidney.
Assuntos
Hipertensão Renal/fisiopatologia , Rim/metabolismo , Neuropeptídeo Y/análise , Neuropeptídeo Y/genética , Animais , Peso Corporal , Expressão Gênica , Regulação da Expressão Gênica , Hemodinâmica , Hipertensão Renal/metabolismo , Imuno-Histoquímica , Rim/química , Rim/patologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Renina/sangue , Renina/genéticaRESUMO
The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Biomarcadores , Encéfalo/citologia , Mapeamento Encefálico , Junções Comunicantes/metabolismo , Imuno-Histoquímica , Masculino , Neurônios/citologia , Proteínas Nucleares/metabolismo , Parvalbuminas/metabolismo , Glândula Pineal/citologia , Glândula Pineal/metabolismo , Hipófise/citologia , Hipófise/metabolismo , RNA Mensageiro/metabolismo , Ratos , Proteína delta-2 de Junções ComunicantesRESUMO
Connexin43 (Cx43), the predominant gap junction protein of muscle cells in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension on the expression of Cx43 in aorta and heart in three different models of experimental hypertension. Rats were made hypertensive either by clipping one renal artery (two kidney, one-clip renal (2K,1C) model) by administration of deoxycorticosterone and salt (DOCA-salt model) or by inhibiting nitric oxide synthase with N G-nitro-L-arginine methyl ester (L-NAME model). After 4 weeks, rats of the three models showed a similar increase in intra-arterial mean blood pressure and in the thickness of the walls of both aorta and heart. Analysis of heart mRNA demonstrated no change in Cx43 expression in the three models compared to their respective controls. The same 2K,1C and DOCA-salt hypertensive animals expressed twice more Cx43 in aorta, and the 2K, 1C rats showed an increase in arterial distensibility. In contrast, the aortae of L-NAME hypertensive rats were characterized by a 50% decrease in Cx43 and the carotid arteries did not show increased distensibility. Western blot analysis indicated that Cx43 was more phosphorylated in the aortae of 2K,1C rats than in those of L-NAME or control rats, indicating a differential regulation of aortic Cx43 in different models of hypertension. The data suggest that localized mechanical forces induced by hypertension affect Cx43 expression and that the cell-to-cell communication mediated by Cx43 channels may contribute to regulating the elasticity of the vascular wall.
Assuntos
Conexina 43/biossíntese , Junções Comunicantes/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Aorta/metabolismo , Western Blotting , Comunicação Celular , Doença Crônica , Conexina 43/genética , Modelos Animais de Doenças , Expressão Gênica , Tono Muscular , Músculo Liso Vascular/patologia , RNA Mensageiro/metabolismo , RatosRESUMO
Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.
Assuntos
Aorta/metabolismo , Conexina 43/biossíntese , Hipertensão Renovascular/fisiopatologia , Hipertensão/fisiopatologia , Miocárdio/metabolismo , Animais , Aorta/patologia , Pressão Sanguínea , Desoxicorticosterona , Hipertensão/metabolismo , Hipertensão/patologia , Hipertensão Renovascular/metabolismo , Hipertensão Renovascular/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miocárdio/patologia , Ratos , Sódio na DietaRESUMO
Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic ß-cell function. We have recently demonstrated that Cx36 also supports ß-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1ß, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca(2+) stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca(2+) homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Conexinas/metabolismo , Citocinas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Conexinas/genética , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Interleucina-1beta/farmacologia , Metformina/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Ratos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Proteína delta-2 de Junções ComunicantesRESUMO
Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic ß-cells. Pro-inflammatory cytokines are early mediators of ß-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in ß-cells, but the role for CHOP overexpression in cytokine-induced ß-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating ß-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting ß-cell death: (1) CHOP directly contributes to cytokine-induced ß-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.