RESUMEN
Presenilin 1 (PS1) is linked with Alzheimer's disease but exhibits functional roles regulating growth and development. For instance, PS1 binds to beta-catenin and modulates beta-catenin signaling. In the current study, we observed that knockout of PS1 inhibited beta-catenin-mediated transcription by 35%, as shown by a luciferase reporter driven by the hTcf-4 promoter. Overexpressing wild-type PS1 increased beta-catenin-mediated transcription by 37.5%, and overexpressing PS1 with mutations associated with Alzheimer's disease decreased beta-catenin-mediated transcription by 66%. To examine whether regulation of beta-catenin by PS1 requires phosphorylation by glycogen synthase kinase 3beta (GSK 3beta), we examined whether inhibiting GSK 3beta activity overcomes the inhibition of beta-catenin transcription induced by mutant PS1 constructs. Cells expressing wild-type or mutant PS1 were treated with LiCl, which inhibits GSK 3beta, or transfected with beta-catenin constructs that lack the GSK 3beta phosphorylation sites. Neither treatment overcame PS1-mediated inhibition of beta-catenin signaling, suggesting that regulation of beta-catenin by PS1 was not affected by the activity of GSK 3beta. To investigate how PS1 might regulate beta-catenin signaling, we determined whether PS1 interacts with other elements of the beta-catenin signaling cascade, such as the Tcf-4 transcription factor. Coimmunoprecipitation studies showed binding of PS1 and hTcf-4, and examining nuclear isolates indicated that nuclear hTcf-4 was decreased in cells expressing mutant PS1. These data show that PS1 interacts with multiple components of the beta-catenin signaling cascade and suggest that PS1 regulates beta-catenin in a manner independent of GSK 3beta activity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Transactivadores , Transcripción Genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Humanos , Immunoblotting , Inmunohistoquímica , Cloruro de Litio/farmacología , Luciferasas/metabolismo , Ratones , Ratones Noqueados , Mutación , Plásmidos/metabolismo , Pruebas de Precipitina , Presenilina-1 , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/metabolismo , beta CateninaRESUMEN
The presenilin 1 (PS1) and PS2 proteins are thought to play roles in processing of amyloid precursor protein (APP), but the nature of this role is not fully understood. Recent studies have shown that PS1 is necessary for cleavage of APP at the gamma-secretase site. We now show that PS1 and PS2 participate in other aspects of APP processing. Fibroblasts generated from PS1 knockout mice have increased levels of the APP cleavage products, secreted APP (APPs), and APP C-terminal fragments, but lower secretion of APPs and Abeta. We have also observed that loss of PS1 prevents protein kinase C or extracellular regulated kinase from increasing production of the APP cleavage products, APPs, and APP C-terminal fragments. Transfection of PS1 -/- cells with PS1 restores the responsiveness of APP processing to protein kinase C and extracellular regulated kinase. This suggests that the changes in APP processing in PS1 -/- cells result strictly from the absence of PS1. Transfection of PS1 -/- cells with PS2 is also able to correct the deficits in APP secretion, which suggests that the PS2 also has the ability to regulate APP processing. Finally, transfection of the truncated PS2 construct, Alg3, into cells lacking PS1 increases APP C-terminal fragments. This suggests that Alg3 can interfere with the processing of APP by PS2. These data point to roles for both PS1 and PS2 in regulating APP processing and suggest that the role of these proteins also includes coupling APP to signal transduction pathways.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Transformación Celular Viral , Endopeptidasas/metabolismo , Fibroblastos , Homocigoto , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/metabolismo , Fenotipo , Presenilina-1 , Presenilina-2 , Proteína Quinasa C/metabolismo , Virus 40 de los Simios , TransfecciónRESUMEN
OBJECTIVE: To determine whether tolazoline reduces pulmonary vascular resistance (PVR) by means of endogenous nitric oxide production. DESIGN: Thirty newborn lambs (2 to 7 days of age) were anesthetized with pentobarbital, and their lungs were ventilated through an endotracheal tube. Intravascular catheters were placed in the left ventricle, descending aorta, right atrium, and pulmonary artery for continuous monitoring of intravascular pressures. Cardiac output was measured with radiolabeled microspheres. Arterial carbon dioxide pressure and pH were maintained in a normal range throughout the experiments. Animals were randomly assigned to the following groups: group 1, lungs ventilated with a hypoxic gas mixture and administered tolazoline; group 2, given N omega-nitro-L-arginine (L-NA) (5 mg/min intravenously for 60 minutes) and tolazoline; group 3, given L-NA with hypoxia and tolazoline. Acetylcholine (0.5 microgram/kg) was injected into the right atrium to assess pulmonary nitric oxide synthase activity before and after the L-NA infusion. Data were analyzed by analysis of variance. RESULTS: L-NA inhibited the acetylcholine-induced reduction in mean pulmonary artery pressure (MPAP) by more than 75%. Hypoxia and L-NA increased both MPAP and PVR. Tolazoline produced immediate reductions in both MPAP and PVR in all three groups (group 1, 27% +/- 3% and 50% +/- 5%; group 2, 34% +/- 5% and 50% +/- 6%; and group 3, 31% +/- 4% and 46% +/- 5%, respectively). CONCLUSIONS: These results suggest that tolazoline produces vasodilation independent of nitric oxide production. Understanding the mechanism by which tolazoline produces pulmonary vasodilation may provide insight into the clinical use of this drug and information regarding other potential endogenous mediators of pulmonary vasomotor tone in the neonate.
Asunto(s)
Óxido Nítrico/biosíntesis , Circulación Pulmonar/efectos de los fármacos , Tolazolina/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Animales Recién Nacidos , Músculo Liso Vascular/efectos de los fármacos , Ovinos , Resistencia Vascular/efectos de los fármacosRESUMEN
The effect of hypercholesterolemia for 10 wk on endothelium-dependent relaxations to acetylcholine was studied in isolated rings of rabbit carotid artery and abdominal aorta contracted with phenylephrine or elevated potassium. In these arteries obtained from hypercholesterolemic rabbits, endothelium-dependent relaxations to acetylcholine were not significantly different from those of normal rabbits. In normal and hypercholesterolemic arteries, partial relaxation persisted in the presence of NG-nitro-L-arginine methyl ester (L-NAME), which blocked acetylcholine-induced increases in arterial guanosine 3',5'-cyclic monophosphate (cGMP). Combined treatment with L-NAME and the calcium-dependent potassium-channel inhibitor, charybdotoxin, blocked relaxations in both groups, suggesting that L-NAME-resistant relaxations are mediated by an endothelium-derived hyperpolarizing factor. Charybdotoxin alone or depolarizing potassium had no significant effect on normal carotid artery or normal and hypercholesterolemic abdominal aorta but significantly inhibited relaxations of the carotid artery from cholesterol-fed rabbits. The enhanced role of calcium-dependent potassium channels and the hyperpolarizing factor in relaxation of the hypercholesterolemic carotid artery suggested by these results was likely related to the fact that acetylcholine failed to stimulate cGMP only in that artery. These data suggest that endothelium-dependent relaxation in these rabbit arteries is mediated by nitric oxide-cGMP-dependent and -independent mechanisms. In hypercholesterolemia, the contribution of nitric oxide-cGMP in the carotid artery is reduced, but a hyperpolarizing factor and calcium-dependent potassium channels maintain normal acetylcholine-induced relaxation.
Asunto(s)
Acetilcolina/farmacología , Arginina/análogos & derivados , Arterias Carótidas/fisiopatología , Hipercolesterolemia/fisiopatología , Relajación Muscular/fisiología , Canales de Potasio/fisiología , Análisis de Varianza , Animales , Arginina/farmacología , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Arterias Carótidas/fisiología , Caribdotoxina , Colesterol en la Dieta , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Hipercolesterolemia/metabolismo , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/fisiopatología , NG-Nitroarginina Metil Éster , Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Conejos , Valores de Referencia , Venenos de Escorpión/farmacologíaRESUMEN
To determine if endogenous local levels of nitric oxide (NO) modulate atherogenesis, we studied the effect of inhibiting NO with NG-nitro-L-arginine methyl ester (L-NAME) on early neointima formation in cholesterol-fed rabbits. Male rabbits were fed for 5 weeks with a 0.5% cholesterol diet alone or treated in addition during the last 4 weeks with L-NAME (12 mg/kg per day SC) via osmotic minipump. Endothelial cell function was assessed in isolated aortic rings by vascular reactivity and levels of cyclic GMP. In L-NAME-treated rabbits there was inhibition of endothelium-dependent relaxations to acetylcholine and the calcium ionophore A23187 as well as impaired cyclic GMP accumulation in response to acetylcholine. Neointima formation in the ascending thoracic aorta was assessed by determining media and intima cross-sectional areas with computerized image analysis. Compared with rabbits that consumed the cholesterol diet alone, L-NAME-treated rabbits had significant increases in lesion area (0.29 +/- 0.04 versus 0.15 +/- 0.03 mm2) and in lesion/media ratio (0.06 +/- 0.01 versus 0.03 +/- 0.01). Plasma levels of cholesterol and fluorescent lipid peroxide products were unchanged, suggesting no difference in cholesterol metabolism or oxidation. Because arterial blood pressure was not altered by L-NAME treatment, the increased atherogenesis could not be attributed to an increase in blood pressure. These results indicated that local inhibition of NO accelerates early neointima formation possibly because of modulating monocyte recruitment or foam cell lipid accumulation.
Asunto(s)
Arteriosclerosis/etiología , Endotelio Vascular/fisiología , Hipercolesterolemia/complicaciones , Óxido Nítrico/fisiología , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Aorta/patología , Arginina/análogos & derivados , Arginina/farmacología , GMP Cíclico/análisis , Hipercolesterolemia/patología , Hipercolesterolemia/fisiopatología , Peroxidación de Lípido , Masculino , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintasa , Conejos , VasodilataciónRESUMEN
Nitric oxide is the major endothelium-derived relaxing factor (EDRF), and it is thought to relax smooth muscle cells by stimulation of guanylate cyclase, accumulation of its product cyclic GMP, and cGMP-dependent modification of several intracellular processes, including activation of potassium channels through cGMP-dependent protein kinase. Here we present evidence that both exogenous nitric oxide and native EDRF can directly activate single Ca(2+)-dependent K+ channels (K+Ca) in cell-free membrane patches without requiring cGMP. Under conditions when guanylate cyclase was inhibited by methylene blue, considerable relaxation of rabbit aorta to nitric oxide persisted which was blocked by charybdotoxin, a specific inhibitor of K+Ca channels. These studies demonstrate a novel direct action of nitric oxide on K+Ca channels.
Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , Canales de Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Membrana Celular/metabolismo , Caribdotoxina , GMP Cíclico/metabolismo , Etilmaleimida/farmacología , Técnicas In Vitro , Magnesio/metabolismo , Masculino , Potenciales de la Membrana , Azul de Metileno/farmacología , Músculo Liso Vascular/efectos de los fármacos , Ácido Mirístico , Ácidos Mirísticos/farmacología , Canales de Potasio/efectos de los fármacos , Conejos , Venenos de Escorpión/farmacologíaRESUMEN
Endothelium-dependent relaxation is associated with smooth muscle hyperpolarization in many arteries which may account for relaxation that persists in the presence of nitric oxide inhibitors such as NG-nitro-L-arginine methyl ester (L-NAME). Acetylcholine (ACh)-induced relaxations of the rabbit thoracic and abdominal aorta and iliac and carotid arteries were studied for the relative contribution of nitric oxide-dependent and -independent mechanisms in rings suspended for measurement of isometric tension. Although relaxation of the thoracic aorta to ACh (10(-6) M) was almost blocked completely by L-NAME (3 x 10(-5) M), the maximal relaxation in the abdominal aorta, carotid and iliac arteries was only reduced by 28, 26 and 62%, respectively. In rings of abdominal aorta, L-NAME blocked the ACh-stimulated (10(-6) M) rise in cyclic GMP verifying that relaxation which persists in L-NAME-treated rings is not mediated by nitric oxide. The L-NAME resistant response was nearly abolished by elevated external K+ in rings of abdominal aorta and carotid artery, suggesting this relaxation may be mediated by a membrane potential sensitive mechanism. Furthermore, tetraethylammonium (10(-3) M) partially and charybdotoxin (5 x 10(-8) M) completely inhibited the remaining L-NAME-resistant relaxation in both abdominal aorta and carotid artery, suggesting a role for Ca(++)-activated K(+)-channels. Blockers of ATP-sensitive K+ channels also inhibited the L-NAME resistant relaxation in the abdominal aorta only.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acetilcolina/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Canales de Potasio/fisiología , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/fisiología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Arginina/análogos & derivados , Arginina/farmacología , GMP Cíclico/metabolismo , Endotelio Vascular/fisiología , Gliburida/farmacología , Técnicas In Vitro , Masculino , NG-Nitroarginina Metil Éster , Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Conejos , Estimulación Química , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Tolbutamida/farmacologíaRESUMEN
The effect of chronic inhibition of nitric oxide (NO) synthesis by NG-nitro-L-arginine methyl ester (L-NAME) on cutaneous ear blood flow (EBF) in the rabbit was examined in vivo with use of laser Doppler flowmetry. Additionally, the efficacy of inhibition of NO by L-NAME was studied ex vivo in isolated preparations of aortic rings from these rabbits. Before surgical implantation of osmotic pumps loaded with L-NAME, resting EBF was not significantly different in rabbits selected for control or L-NAME treatment. Although chronic L-NAME treatment had no significant effect on resting core temperature, resting EBF was reduced significantly in L-NAME-treated rabbits as compared with EBF in control rabbits. Short-term body warming for 10-20 min caused a significant increase in EBF, but not in body temperature, to levels that were not significantly different between groups at 0, 1 and 2 weeks. Prolonged body warming for a further 30-40 min produced a rise in body temperature and a further increase in EBF of 22% to levels that were not significantly different in control and L-NAME-treated groups. Acetylcholine-induced relaxations of aortic rings and levels of cyclic GMP were significantly reduced in L-NAME-treated rabbits as compared with control rabbits, whereas relaxations to sodium nitroprusside were enhanced significantly. These findings demonstrate that the synthesis of NO can be inhibited chronically in the rabbit and are consistent with the concept that NO participates in the control of skin blood flow by counteracting vasoconstrictor tone but not in the vasodilation induced by body warming.
Asunto(s)
Arginina/análogos & derivados , Oído Externo/irrigación sanguínea , Óxido Nítrico/biosíntesis , Vasoconstricción/efectos de los fármacos , Acetilcolina/metabolismo , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Arginina/farmacología , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , GMP Cíclico/biosíntesis , Oído Externo/efectos de los fármacos , Técnicas In Vitro , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa , Nitroprusiato/farmacología , Conejos , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguíneaRESUMEN
A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from alloxan-induced diabetic rabbits. Isolated aorta of diabetic rabbits, contracted submaximally with phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine or adenosine diphosphate compared to those from normal rabbits. Basal and acetylcholine-stimulated levels of cyclic GMP and the relaxations in response to an endothelium-independent vasodilator, sodium nitroprusside, were not significantly different between diabetic and normal rabbits, indicating that nitric oxide release and action on the vascular smooth muscle were unchanged. The release of thromboxane A2 from diabetic vessels was increased, as previously demonstrated. Treatment with an aldose reductase inhibitor, zopolrestat, normalized the elevated red blood cell sorbitol levels in diabetic rabbits. Zopolrestat also restored the abnormal acetylcholine- and adenosine diphosphate-induced relaxations of the aorta. The aldose reductase inhibitor had no effect on the levels of cyclic GMP or on the increased release of thromboxane A2 in diabetic aorta. These findings suggest that increased activity of the aldose reductase pathway in hyperglycemia is responsible for the abnormal endothelium-dependent relaxation in diabetic blood vessels. Significant alterations in endothelial production of neither nitric oxide nor vasoconstrictor prostanoids could be directly implicated in the improvement caused by the drug, suggesting another mechanism of action.