RESUMEN
Acetyl-L-carnitine (ALCAR) is an endogenous metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane. Exogenously administered ALCAR has been used as a nutritional supplement and also as an experimental drug with reported neuroprotective properties and effects on brain metabolism. The aim of this study was to determine oxidative metabolism of ALCAR in the immature rat forebrain. Metabolism was studied in 21-22 day-old rat brain at 15, 60 and 120 min after an intraperitoneal injection of [2-(13)C]acetyl-L-carnitine. The amount, pattern, and fractional enrichment of (13)C-labeled metabolites were determined by ex vivo(13)C-NMR spectroscopy. Metabolism of the acetyl moiety from [2-(13)C]ALCAR via the tricarboxylic acid cycle led to incorporation of label into the C4, C3 and C2 positions of glutamate (GLU), glutamine (GLN) and GABA. Labeling patterns indicated that [2-(13)C]ALCAR was metabolized by both neurons and glia; however, the percent enrichment was higher in GLN and GABA than in GLU, demonstrating high metabolism in astrocytes and GABAergic neurons. Incorporation of label into the C3 position of alanine, both C3 and C2 positions of lactate, and the C1 and C5 positions of glutamate and glutamine demonstrated that [2-(13)C]ALCAR was actively metabolized via the pyruvate recycling pathway. The enrichment of metabolites with (13)C from metabolism of ALCAR was highest in alanine C3 (11%) and lactate C3 (10%), with considerable enrichment in GABA C4 (8%), GLN C3 (approximately 4%) and GLN C5 (5%). Overall, our (13)C-NMR studies reveal that the acetyl moiety of ALCAR is metabolized for energy in both astrocytes and neurons and the label incorporated into the neurotransmitters glutamate and GABA. Cycling ratios showed prolonged cycling of carbon from the acetyl moiety of ALCAR in the tricarboxylic acid cycle. Labeling of compounds formed from metabolism of [2-(13)C]ALCAR via the pyruvate recycling pathway was higher than values reported for other precursors and may reflect high activity of this pathway in the developing brain. This is, to our knowledge, the first study to determine the extent and pathways of ALCAR metabolism for energy and neurotransmitter biosynthesis in the brain.
Asunto(s)
Acetilcarnitina/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Metabolismo Energético/fisiología , Neurotransmisores/biosíntesis , Acetilcarnitina/química , Animales , Encéfalo/citología , Ciclo del Ácido Cítrico/fisiología , Espectroscopía de Resonancia Magnética , Masculino , Neuronas/metabolismo , Fosforilación Oxidativa , Ratas , Ratas Sprague-DawleyRESUMEN
The enzyme aspartate aminotransferase (AAT) has a number of key roles in astrocytes and neurons in brain. An understanding of the regulation of AAT is important since AAT is involved in many aspects of glutamate metabolism including the synthesis of neurotransmitter glutamate. Mitochondrial AAT binds to a protein and lipids on the inner mitochondrial membrane and also forms a number of transient hetero-enzyme complexes with other enzymes. These complexes serve to facilitate metabolism by essentially channeling substrates and cofactors to other enzymes within the complex. The association and dissociation of transiently formed hetero-enzyme complexes may modulate enzyme activity in "real time" since these complexes are dynamically influenced by changes in the concentration of a number of key metabolites. The influence of several effectors that modulate AAT activity, either directly, or by altering the binding of AAT to mitochondrial lipids, or the association/dissociation into transient hetero-enzyme complexes was determined. The addition of palmitate, malate, citrate, glutamate, bovine serum albumin and Mg(2+) modulated AAT activity differently in synaptic and nonsynaptic mitochondria from brain. These findings suggest that AAT activity and also glutamate metabolism, may be regulated in part, by metabolites that influence binding of the enzyme to lipids or proteins in the inner mitochondrial membrane and/or the association/dissociation of transient hetero-enzyme complexes. This may have a role in the compartmentation of glutamate metabolism in brain.
Asunto(s)
Aspartato Aminotransferasas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Complejos Multienzimáticos/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/enzimología , Animales , Corteza Cerebral/enzimología , Corteza Cerebral/ultraestructura , Ácido Cítrico/farmacología , Ácido Glutámico/farmacología , Técnicas In Vitro , Cinética , Malatos/farmacología , Manganeso/farmacología , Membranas Mitocondriales/metabolismo , Ácido Palmítico/farmacología , Unión Proteica , Ratas , Albúmina Sérica Bovina/farmacología , Sinapsis/ultraestructuraRESUMEN
Nasal potential difference (NPD) has served as a non-invasive diagnostic method for cystic fibrosis (CF) a disease of chloride channel expression and function in secretory epithelia. Investigators have also used NPD to demonstrate ion transport abnormalities in newborns with respiratory distress. Standard perfusates for diagnostic NPD studies include the use of amiloride, replacement of chloride with gluconate, cAMP agonists, and nucleotides such as ATP. The pH of these perfusates may also be relevant to NPD studies as we have previously shown that the respiratory epithelia in mammals express CLC-2, which is a pH sensitive chloride channel. We hypothesized that acidic pH might activate chloride secretion in vivo if CLC-2 is present in human respiratory epithelia. Our objective was to determine the effect of acidic pH on NPD measurements and the frequency of expression of CLC-2 in normal subjects. Healthy adults were recruited and CLC-2 protein expression was detected in 20 of 29 primary nasal epithelial cell cultures. Acidic pH stimulated NPD responses in 33% of subjects. These findings suggest that pH sensitive alternative pathways are available for modulation in human respiratory epithelia and that NPD protocols should standardize pH of perfusates.
Asunto(s)
Acidosis/metabolismo , Mucosa Nasal/fisiología , Adulto , Western Blotting , Canales de Cloruro CLC-2 , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Canales de Cloruro/biosíntesis , Fibrosis Quística/diagnóstico , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Potenciales de la Membrana/fisiología , Persona de Mediana Edad , Mucosa Nasal/citología , Valores de ReferenciaRESUMEN
BACKGROUND: Cystic fibrosis (CF) lung disease manifest by impaired chloride secretion leads to eventual respiratory failure. Candidate genes that may modify CF lung disease severity include alternative chloride channels. The objectives of this study are to identify single nucleotide polymorphisms (SNPs) in the airway epithelial chloride channel, CLC-2, and correlate these polymorphisms with CF lung disease. METHODS: The CLC-2 promoter, intron 1 and exon 20 were examined for SNPs in adult CF dF508/dF508 homozygotes with mild and severe lung disease (forced expiratory volume at one second (FEV1) > 70% and < 40%). RESULTS: PCR amplification of genomic CLC-2 and sequence analysis revealed 1 polymorphism in the hClC -2 promoter, 4 in intron 1, and none in exon 20. Fisher's analysis within this data set, did not demonstrate a significant relationship between the severity of lung disease and SNPs in the CLC-2 gene. CONCLUSIONS: CLC-2 is not a key modifier gene of CF lung phenotype. Further studies evaluating other phenotypes associated with CF may be useful in the future to assess the ability of CLC-2 to modify CF disease severity.
Asunto(s)
Canales de Cloruro/genética , Fibrosis Quística/genética , Enfermedades Pulmonares/genética , Polimorfismo de Nucleótido Simple , Adulto , Secuencia de Bases , Canales de Cloruro CLC-2 , Línea Celular , Canales de Cloruro/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/química , ADN/genética , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Exones/genética , Femenino , Genotipo , Humanos , Intrones/genética , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Masculino , Datos de Secuencia Molecular , Mutación , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Fenotipo , Regiones Promotoras Genéticas/genética , Homología de Secuencia de Ácido Nucleico , Índice de Severidad de la EnfermedadRESUMEN
Epithelial Cl(-) channels mediate Cl(-) and fluid secretion in the lung. In cystic fibrosis, aberrant Cl(-) secretion is one of the major causes for lung fluid imbalance. Regulation of Cl(-) channels is therefore an important issue in the lung. IFN-gamma regulates Na(+) and Cl(-) channels and fluid transport in the lung, but the mechanisms involved in these regulations are not clear. In expression studies, we found that IFN-gamma increased ClC-2 transcripts in Calu-3 cells. Studies of the promoter identified a minimal promoter which interacts with transcription factors Sp1 and Sp3. However, reporter gene assays showed that IFN-gamma did not activate the promoter. Instead, IFN-gamma significantly increased ClC-2 transcript stability. Using Ussing chamber experiments, we demonstrate that IFN-gamma activates a pH-regulated and Cd(2+)-sensitive short circuit current, characteristic properties of the ClC-2 Cl(-) channel. These data suggest that IFN-gamma activates ClC-2 channel activity in lung epithelial cells via mRNA stabilization.
Asunto(s)
Canales de Cloruro/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interferón gamma/farmacología , Pulmón/citología , Animales , Canales de Cloruro CLC-2 , Cadmio/metabolismo , Línea Celular Tumoral , Quimiocina CXCL9 , Quimiocinas CXC/farmacología , Canales de Cloruro/genética , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular/farmacología , Interleucina-10/farmacología , Pulmón/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Mensajero/metabolismoRESUMEN
Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology (26). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage (V(t)) of lung explants transfected with antisense CLC-2 was inhibited with V(t) = -1.5 +/- 0.2 mV (means + SE) compared with -3.7 +/- 0.3 mV (means + SE) for mock-transfected controls and -3.3 +/- 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.
Asunto(s)
Canales de Cloruro/genética , Regulación del Desarrollo de la Expresión Génica , Pulmón/embriología , Pulmón/fisiología , Animales , Líquidos Corporales/metabolismo , Canales de Cloruro CLC-2 , Células Cultivadas , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Femenino , Pulmón/citología , Oligonucleótidos Antisentido , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Coloración y Etiquetado , TransfecciónRESUMEN
BACKGROUND & AIMS: Enteric infections have been implicated in the pathogenesis of both food intolerance and autoimmune diseases secondary to the impairment of the intestinal barrier. On the basis of our recent discovery of zonulin, a modulator of small-intestinal tight junctions, we asked whether microorganisms might induce zonulin secretion and increased small-intestinal permeability. METHODS: Both ex vivo mammalian small intestines and intestinal cell monolayers were exposed to either pathogenic or nonpathogenic enterobacteria. Zonulin production and changes in paracellular permeability were monitored in Ussing chambers and micro-snapwells. Zonula occludens 1 protein redistribution after bacteria colonization was evaluated on cell monolayers. RESULTS: Small intestines exposed to enteric bacteria secreted zonulin. This secretion was independent of either the species of the small intestines or the virulence of the microorganisms tested, occurred only on the luminal aspect of the bacteria-exposed small-intestinal mucosa, and was followed by a decrease in small-intestinal tissue resistance (transepithelial electrical resistance). The transepithelial electrical resistance decrement was secondary to the zonulin-induced tight junction disassembly, as also shown by the disengagement of the protein zonula occludens 1 protein from the tight junctional complex. CONCLUSIONS: This zonulin-driven opening of the paracellular pathway may represent a defensive mechanism, which flushes out microorganisms and contributes to the host response against bacterial colonization of the small intestine.