RESUMEN
OBJECTIVE: To identify factors secreted by the human embryo and correlate levels with embryo morphology and pregnancy outcome. DESIGN: A laboratory-based study of human embryo protein synthesis and secretion and a retrospective analysis of spent embryo culture media as it relates to pregnancy outcome. SETTING: University-based academic IVF program. PATIENT(S): IVF patients who had donated cryopreserved human pronuclear-stage embryos. Patients undergoing fresh IVF cycles resulting in a blastocyst transfer who donated spent media drops. INTERVENTION(S): In vitro embryo culture and collection of spent media. MAIN OUTCOME MEASURE(S): Protein analysis and identification by two-dimensional gel electrophoresis and mass spectrometry, ApoA1 quantification by ELISA, and mRNA analysis by quantitative reverse transcriptase-polymerase chain reaction. RESULT(S): By protein gel electrophoresis, apolipoprotein A1 (ApoA1) was increased in the culture media from good-quality blastocysts (n = 6 embryos) compared to either cleavage-arrested embryos (n = 6 embryos) or poor-quality blastocysts (n = 6 embryos) using spent media from culture days 4 and 5, respectively. Apolipoprotein A1 concentrations were 23.1% greater in day 5 spent culture media from good-grade blastocysts (n = 30) when compared to poor-grade embryos (n = 30). However, in a group of patients (n = 20) with transfer of two good-quality blastocysts, ApoA1 levels from day 5 spent media did not correlate with embryo implantation and pregnancy. Quantitative reverse transcriptase-polymerase chain reaction confirmed the presence of ApoA1 mRNA transcripts in human blastocysts. CONCLUSION(S): Apolipoprotein A1 is produced by human preimplantation embryos, and increased levels are present in spent culture media containing blastocysts of higher morphologic grade. These results suggest a role for lipoproteins in early embryologic development.
Asunto(s)
Apolipoproteína A-I/metabolismo , Blastocisto/metabolismo , Apolipoproteína A-I/genética , Medios de Cultivo/metabolismo , Electroforesis en Gel Bidimensional , Técnicas de Cultivo de Embriones , Implantación del Embrión , Transferencia de Embrión , Ensayo de Inmunoadsorción Enzimática , Femenino , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica , Humanos , Embarazo , Índice de Embarazo , Proteómica/métodos , ARN Mensajero/metabolismo , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Cardiolipinas/fisiología , Lesión Pulmonar/etiología , Neumonía Bacteriana/complicaciones , Animales , Sitios de Unión , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Pulmón/metabolismo , Pulmón/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas de Transferencia de Fosfolípidos , Neumonía Bacteriana/metabolismo , Surfactantes Pulmonares/metabolismoRESUMEN
HDL cholesterol levels are decreased in Crohn's disease, a tumor necrosis factor-alpha (TNF-alpha)-driven chronic inflammatory condition involving the gastrointestinal tract. ATP-binding cassette transporter A1 (ABCA1), one of several liver X receptor (LXR) target genes, is a cell surface transporter that mediates the rate-controlling step in HDL synthesis. The regulation of ABCA1 and HDL cholesterol efflux by TNF-alpha was investigated in the human intestinal cell line Caco-2. In response to cholesterol micelles or T0901317, an LXR nonsterol agonist, TNF-alpha decreased the basolateral efflux of cholesterol to apolipoprotein A1 (apoA1). TNF-alpha, by attenuating ABCA1 promoter activity, markedly decreased ABCA1 gene expression without attenuating the expression of LXR-alpha, LXR-beta, and most other LXR target genes, such as ABCG1, FAS, ABCG8, scavenger receptor-B1 (SR-B1), and apoC1. TNF-alpha also decreased ABCA1 mass by markedly enhancing the rate of ABCA1 degradation and modestly inhibiting its rate of synthesis. Inhibitors of the nuclear factor-kappaB (NF-kappaB) pathway, which is activated by TNF-alpha, partially reverse the effect of TNF-alpha on ABCA1 protein expression. The results suggest that TNF-alpha, the major cytokine implicated in the inflammation of Crohn's disease, decreases HDL cholesterol levels by attenuating the expression of intestinal ABCA1 and cholesterol efflux to apoA1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , HDL-Colesterol/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Intestinos/citología , Factor de Necrosis Tumoral alfa/farmacología , Transportador 1 de Casete de Unión a ATP , Animales , Células CACO-2 , Humanos , Mucosa Intestinal/metabolismo , Receptores X del Hígado , FN-kappa B/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , HDL-Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Transportador 1 de Casete de Unión a ATP , Células CACO-2 , Proteínas Portadoras/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Proteína Niemann-Pick C1RESUMEN
Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.
Asunto(s)
Anticolesterolemiantes/farmacología , Azetidinas/farmacología , Membrana Celular/metabolismo , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Membrana Celular/efectos de los fármacos , Colesterol Oxidasa/metabolismo , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Ezetimiba , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Factores de TiempoRESUMEN
Bacterial infection triggers an acute inflammatory response that might alter phospholipid metabolism. We have investigated the acute-phase response of murine lung epithelia to Pseudomonas aeruginosa infection. Ps. aeruginosa triggered secretion of the pro-inflammatory lipase, sPLA2 IB (phospholipase A2 IB), from lung epithelium. Ps. aeruginosa and sPLA2 IB each stimulated basolateral PtdCho (phosphatidylcholine) efflux in lung epithelial cells. Pre-treatment of cells with glyburide, an inhibitor of the lipid-export pump, ABCA1 (ATP-binding cassette transporter A1), attenuated Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux. Effects of Ps. aeruginosa and sPLA2 IB were completely abolished in human Tangier disease fibroblasts, cells that harbour an ABCA1 genetic defect. Ps. aeruginosa and sPLA2 IB induced the heterodimeric receptors, PPARa (peroxisome-proliferator-activated receptor-a) and RXR (retinoid X receptor), factors known to modulate ABCA1 gene expression. Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux was blocked with PD98059, a p44/42 kinase inhibitor. Transfection with MEK1 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase 1), a kinase upstream of p44/42, increased PPARa and RXR expression co-ordinately with increased ABCA1 protein. These results suggest that pro-inflammatory effects of Ps. aeruginosa involve release of an sPLA2 of epithelial origin that, in part, via distinct signalling molecules, transactivates the ABCA1 gene, leading to export of phospholipid.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , PPAR alfa/fisiología , Fosfatidilcolinas/metabolismo , Fosfolipasas A/fisiología , Infecciones por Pseudomonas/fisiopatología , Receptores X Retinoide/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Flavonoides/farmacología , Gliburida/farmacología , Fosfolipasas A2 Grupo IB , Humanos , Pulmón/metabolismo , MAP Quinasa Quinasa 1/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Pseudomonas aeruginosa , Enfermedad de Tangier/fisiopatología , Transfección , Regulación hacia ArribaRESUMEN
The glia maturation factor (GMF), which was discovered in our laboratory, is a highly conserved protein predominantly localized in astrocytes. GMF is an intracellular regulator of stress-related signal transduction. We now report that the overexpression of GMF in astrocytes leads to the destruction of primary oligodendrocytes by interactions between highly purified cultures of astrocytes, microglia, and oligodendrocytes. We infected astrocytes with a replication-defective adenovirus carrying the GMF cDNA. The overexpression of GMF caused the activation of p38 MAP kinase and transcription factor NF-kappaB, as well as the induction of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA and protein in astrocytes. Small interfering RNA-mediated GMF knockdown completely blocked the GMF-dependent activation of p38 mitogen-activated protein kinase (MAPK), NF-kappaB, and enhanced expression of GM-CSF by astrocytes. Inhibition of p38 MAPK or NF-kappaB by specific inhibitors prevented GM-CSF production. The cell-free conditioned medium from overexpressing GMF astrocytes contained 320 +/- 33 pg/mL of GM-CSF, which was responsible for enhanced production and secretion of TNF-alpha, IL-1beta, IL-6, and IP-10 by microglia. Presence of these inflammatory cytokines in the conditioned medium from microglia efficiently destroyed oligodendrocytes in culture. These results suggest that GMF-induced production of GM-CSF in astrocytes is depending on p38 MAPK and NF-kappaB activation. The GM-CSF-dependent expression and secretion of inflammatory cytokine/chemokine, TNF-alpha, IL-1beta, IL-6, and IP-10, is cytotoxic to oligodendrocytes, the myelin-forming cells in the central nervous system, and as well as neurons. Our results suggest a novel pathway of GMF-initiated cytotoxicity of brain cells, and implicate its involvement in inflammatory diseases such as multiple sclerosis.
Asunto(s)
Astrocitos/metabolismo , Citocinas/metabolismo , Encefalitis/metabolismo , Factor de Maduración de la Glia/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Microglía/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/fisiopatología , Muerte Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Citocinas/toxicidad , Encefalitis/inmunología , Encefalitis/fisiopatología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Factor de Maduración de la Glia/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Ratones , Microglía/inmunología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Oligodendroglía/inmunología , Oligodendroglía/metabolismo , ARN Interferente Pequeño , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor delta (PPARdelta) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARdelta. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARdelta. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.
Asunto(s)
Grasas Insaturadas en la Dieta/farmacología , Ácidos Docosahexaenoicos/farmacología , Aceites de Pescado/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Anexina A2/metabolismo , Células CACO-2 , Caveolina 1/metabolismo , Diferenciación Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetinae , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Enfermedades de Niemann-Pick/metabolismo , PPAR delta/metabolismo , Esterol O-Aciltransferasa/metabolismo , Esterol O-Aciltransferasa 2RESUMEN
We earlier reported that overexpression of glia maturation factor (GMF) in cultured astrocytes enhances the production of brain-derived neurotrophic factor (BDNF). The current study was conducted to find out whether BDNF production is impaired in animals devoid of GMF. To this end GMF-knockout (KO) mice were subjected to exercise and the neurotrophin mRNAs were determined by real-time RT-PCR. Compared to wild-type (WT) mice, there is a decrease in exercise-induced BDNF in the KO mice. The observation was correlated with the finding that, in WT mice, exercise increases GMF expression. The results are consistent with the hypothesis that GMF is necessary for exercise-induction of BDNF, and that GMF may promote neuroprotection through BDNF production.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor de Maduración de la Glia/metabolismo , Condicionamiento Físico Animal , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Femenino , Factor de Maduración de la Glia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Fármacos Neuroprotectores/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , ARN Mensajero/metabolismoRESUMEN
Possible mechanisms for the cholesterol-lowering effects of plant stanol esters were addressed by feeding hamsters diets containing stanol esters, cholesterol, or cholestyramine/lovastatin. ABCA1, ATP binding cassette G1 (ABCG1), ABCG5, ABCG8, and Niemann-Pick C1-like 1 (NPC1L1) mRNA levels were then estimated in duodenum, jejunum, and ileum. Plasma cholesterol was decreased by 36% and 94% in animals fed stanol esters and cholestyramine/lovastatin, respectively. Cholesterol feeding increased plasma cholesterol by 2.5-fold. Plasma plant sterols were unchanged by stanol ester feeding but became undetectable by feeding cholestyramine/lovastatin. Cholesterol and stanols accumulated in enterocytes of animals fed cholesterol and stanol esters, respectively. ABCG5 and ABCG8 mRNA levels were decreased by stanol esters and cholestyramine/lovastatin. Cholesterol feeding markedly increased ABCA1 and ABCG1 expression and modestly increased ABCG5/ABCG8. NPC1L1 mRNA was not significantly altered by any of the diets. ABCG1, ABCG5, ABCG8, and NPC1L1 mRNAs were highest in cells of the upper villus, whereas ABCA1 mRNA was highest in cells of the lower villus. The results suggest that cholesterol lowering effect of stanol esters is unrelated to changes in mRNA levels of intestinal ABC sterol transporters or NPC1L1. Cholesterol flux regulates ABC expression but not NPC1L1. The different localization of ABCA1 suggests a different function for this protein than for ABCG1, ABCG5, ABCG8, and NPC1L1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Colesterol/sangre , Proteínas de Transporte de Membrana/genética , Sitoesteroles/metabolismo , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Anticolesterolemiantes/farmacología , Resina de Colestiramina/farmacología , Cricetinae , Lovastatina/farmacología , Masculino , Proteínas de Transporte de Membrana/biosíntesis , ARN Mensajero/sangreRESUMEN
Diminished activity of peroxisome proliferator-activated receptor-gamma (PPARgamma) may play a role in the pathogenesis of hypertension and vascular dysfunction. To better understand what genes are regulated by PPARgamma, an experimental data set was generated by microarray analysis, in duplicate, of pooled aortic mRNA isolated from mice treated for 21 days with a PPARgamma agonist (rosiglitazone) or vehicle. Of the 12,488 probe sets present on the array (Affymetrix MG-U74Av2), 181 were differentially expressed between groups according to a statistical metric generated using Affymetrix software. A significant correlation was observed between the microarray results and real-time RT-PCR analysis of 39 of these genes. Cluster analysis revealed 3 expression patterns, 29 transcripts of moderate abundance that were decreased (-93%) to very low levels, 106 transcripts that were downregulated (-42%), and 46 transcripts that were upregulated (+70%). Functional groups that were decreased included inflammatory response (-93%, n = 6), immune response (-86%, n = 7), and cytokines (-82%, n = 7). There was an overall upregulation in the oxidoreductase activity group (+47%, n = 9). Individually, six transcripts in this group were increased (+72%), and three were decreased (-34%). Fourteen of the genes map to regions in the rat genome that have been linked to increased blood pressure, and of 142 upstream regions analyzed, sequences resembling the DNA binding site for PPARgamma were identified in 101 of the differentially expressed genes.
Asunto(s)
Aorta/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Tiazolidinedionas/farmacología , Factores de Transcripción/fisiología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión/genética , Inflamación/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Ratas , Receptores Citoplasmáticos y Nucleares/agonistas , Rosiglitazona , Factores de Transcripción/agonistasRESUMEN
Many of the actions of angiotensin II (Ang II) are mediated by angiotensin type 1 receptors (AT1), of which there are 2 pharmacologically indistinguishable subtypes (AT1A and AT1B). The purpose of this study was to evaluate the effect of an AT1A homozygous deletion (AT1A-/-) on vascular reactivity. AT1A-/- mice and control littermates (AT1A+/+) were infused with vehicle (saline) or Ang II (1000 ng x kg(-1) x min(-1)) for 7 days by osmotic pumps. Systolic pressure was increased in AT1A+/+ mice (Delta45+/-8 mm Hg, P<0.0001) but unchanged in AT1A-/- mice (Delta5+/-3 mm Hg, P>0.13) on day 7. The carotid artery response to the vasodilators acetylcholine (ACh), nitroprusside, and papaverine and to the vasoconstrictors phenylephrine, U46619, 5-hydroxytryptamine (5-HT), and KCl were not different between vehicle-infused AT1A+/+ and AT1A-/- animals. Carotid relaxation to ACh was impaired and contraction to 5-HT was increased in Ang II-infused AT1A+/+ mice. Ang II did not affect carotid responses in AT1A-/- mice. Superoxide, measured by lucigenin (5 micromol/L), and hydroethidine staining were not different between AT1A+/+ and AT1A-/- mice after vehicle or Ang II infusion, suggesting that it was not contributing to the altered ACh and 5-HT responses. The Rho-kinase inhibitor Y-27632 (1 micromol/L) attenuated the 5-HT response in both vehicle- and Ang II-infused AT1A+/+ mice. Moreover, concentration-dependent relaxation to Y-27632 and RhoA protein expression were not different in vehicle- or Ang II-infused AT1A+/+. These data demonstrate that the AT1A receptor is required for Ang II-induced changes in carotid artery function.
Asunto(s)
Angiotensina II/toxicidad , Receptor de Angiotensina Tipo 1/fisiología , Vasoconstricción/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Acetilcolina/farmacología , Amidas/farmacología , Angiotensina II/administración & dosificación , Animales , Aorta/metabolismo , Arterias Carótidas/efectos de los fármacos , Bombas de Infusión Implantables , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/fisiología , Nitroprusiato/farmacología , Papaverina/farmacología , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , Piridinas/farmacología , Receptor de Angiotensina Tipo 1/biosíntesis , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 2/biosíntesis , Receptor de Angiotensina Tipo 2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Serotonina/farmacología , Superóxidos/metabolismo , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Proteína de Unión al GTP rhoARESUMEN
Surfactant is an apically-secreted surface-active material containing primarily disaturated phosphatidylcholine (DSPtdCho) that is released from alveolar epithelia into the alveolus. Surfactant deficiency is an important aspect of inflammatory lung disease and may result from extravasation of serum lipoproteins into the alveolus. We investigated whether one bioactive component of modified lipoproteins, oxysterols, might reduce surfactant PtdCho availability by altering its trafficking. The oxysterol, 22-hydroxycholesterol (22HC), in combination with its obligate partner, 9 cis-retinoic acid (RA), decreased surfactant PtdCho levels, in part, by stimulating basolateral phospholipid export in murine lung epithelia. 22HC/RA stimulated basolateral PtdCho efflux in cells via transcriptional activation of the ATP-binding cassette transporter 1 (ABCA1) gene. This effect was mediated by a DR-4 locus within the ABCA1 promoter. ABCA1 knockdown studies using ABCA1 siRNA or the ABCA1 inhibitor, glyburide, selectively attenuated 22HC/RA-driven basolateral PtdCho efflux. 22HC/RA significantly increased export of PtdCho molecular species containing saturated (16:0) fatty-acyl species typical of DSPtdCho. Overexpression of ABCA1 mimicked 22HC/RA effects by increasing cellular PtdCho efflux, whereas mutagenesis of ABCA1 at Trp590 attenuated PtdCho release. The results indicate the existence of an oxysterol-activated basolateral exit pathway for surfactant that might impact the availability of phospholipid destined for apical secretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Surfactantes Pulmonares/metabolismo , Esteroles/farmacología , Transportador 1 de Casete de Unión a ATP , Secuencia de Bases , Línea Celular , Cartilla de ADN , Transporte de ProteínasRESUMEN
To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and beta-sitosterol absorption, ABCA1-facilitated beta-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of beta-sitosterol without altering apical efflux. LXR-mediated enhanced beta-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in beta-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented beta-sitosterol efflux in response to the LXR ligand activation. Influx of beta-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of beta-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and beta-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in beta-sitosterol absorption.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores X Retinoide/metabolismo , Sitoesteroles/metabolismo , Transportador 1 de Casete de Unión a ATP , Células CACO-2 , Proteínas de Unión al ADN , Activación Enzimática , Humanos , Ligandos , Receptores X del Hígado , Receptores Nucleares HuérfanosRESUMEN
The peroxisome proliferator activated receptor (PPARgamma) agonist rosiglitazone has been reported to yield cardiovascular benefits in patients by a mechanism that is not completely understood. We tested whether oral rosiglitazone (25 mg/kg per day, 21 days) treatment improves blood pressure and vascular function in a transgenic mouse expressing both human renin and human angiotensinogen transgenes (R(+)A(+)). Rosiglitazone decreased systolic (138+/-5 versus 128+/-5 mm Hg) and mean blood pressure (145+/-5 versus 126+/-7 mm Hg) of R(+)A(+) mice as measured by tail-cuff and indwelling carotid catheters, respectively. Relaxation of carotid arteries to acetylcholine and authentic nitric oxide, but not papaverine, was impaired in R(+)A(+) mice when compared with littermate controls (RA(-)). There were no effects of rosiglitazone on RA(-) mice; however, relaxation to acetylcholine (49+/-10 versus 82+/-9% at 100 micromol/L) and nitric oxide (51+/-11 versus 72+/-6% at 10 micromol/L) was significantly improved in treated R(+)A(+) mice. Rosiglitazone treatment of R(+)A(+) mice did not alter the expression of genes, including endothelial nitric oxide synthase (eNOS), angiotensin 1 receptors, and preproendothelin-1, nor did it alter the levels of eNOS or soluble guanylyl cyclase protein. In separate studies, carotid arteries from R(+)A(+) and RA(-) mice relaxed in a concentration-dependent manner to rosiglitazone, suggesting possible PPARgamma-independent effects in the vasculature. This response was not inhibited with the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (200 micromol/L) or the PPARgamma antagonist bisphenol A diglycidyl ether; 4,4'-isopropylidenediphenol diglycidyl ether (100 micromol/L). These data suggest that in addition to potential genomic regulation caused by PPARgamma activation, the direct effect of rosiglitazone in blood vessels may contribute to the improved blood pressure and vessel function.
Asunto(s)
Antihipertensivos/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazolidinedionas/uso terapéutico , Factores de Transcripción/agonistas , Angiotensinógeno/genética , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Endotelio Vascular/fisiopatología , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Ratones , Ratones Transgénicos , Renina/genética , Rosiglitazona , Vasodilatación/efectos de los fármacosRESUMEN
The effect of fatty acids on LXR (liver X receptors)-mediated enhancement of ABCA1 (ATP-binding cassette transporter A1) expression and cholesterol efflux was investigated in human intestinal cells CaCo-2. LXR activation by T0901317 increased basolateral cholesterol efflux to lipoprotein particles isolated at a density of 1.21 g/ml or higher. Oleic and arachidonic acids attenuated the amount of cholesterol isolated from these particles. Stearic, linoleic and docosahexaenoic acids also decreased cholesterol efflux from basolateral membranes, with the polyunsaturated fatty acids being the most potent. Although oleic, arachidonic and docosahexaenoic acids modestly decreased ABCA1 mRNA levels in response to LXR activation, stearic and linoleic acids did not. Except for oleic acid, all fatty acids substantially attenuated an increase in ABCA1 mass secondary to LXR activation. Inhibiting acyl-CoA:cholesterol acyltransferase activity prevented the decrease in cholesterol efflux caused by oleic acid. Thus, in response to LXR activation, all fatty acids decreased the efflux of cholesterol from the basolateral membrane of CaCo-2 cells. Although modest suppression of ABCA1 gene expression by oleic, arachidonic and docosahexaenoic acids cannot be completely excluded as a mechanism, the predominant effect of fatty acids on ABCA1 expression and cholesterol efflux is at a post-transcriptional level.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Células CACO-2/química , Colesterol/metabolismo , Ácidos Grasos/farmacología , Lipoproteínas HDL/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Ácidos Araquidónicos/farmacología , Células CACO-2/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , HDL-Colesterol/química , HDL-Colesterol/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Receptores X del Hígado , Ácido Oléico/farmacología , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/fisiologíaRESUMEN
Hamsters were fed a control diet or diets containing palm, olive, safflower, or fish oil for 2 weeks. In villus cell populations from duodenum, jejunum, and ileum, rates of intestinal fatty acid and cholesterol synthesis were estimated, as were sterol regulatory element-binding protein (SREBP)-1a, SREBP-1c, SREBP-2, HMG-CoA synthase, fatty acid synthase, ATP citrate lyase, acetyl-CoA carboxylase mRNA levels, and SREBP-1 and SREBP-2 mass. Plasma cholesterol and triacylglcerol levels were increased in animals ingesting palm oil and decreased in animals ingesting fish oil. Fatty acid synthesis and fatty acid synthase activity were decreased in the proximal intestine of animals ingesting all the fat-containing diets. Intestinal cholesterol synthesis was unaltered. In animals fed fat, SREBP-1c gene expression was modestly increased in the duodenum of hamsters fed palm oil or olive oil, and decreased in animals ingesting safflower oil or fish oil. Fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, SREBP-2, and HMG-CoA synthase mRNA levels were not altered, nor were SREBP-1 or SREBP-2 mass. In the intestine, dietary polyunsaturated fatty acids suppress SREBP-1c mRNA without altering expression of its target genes, fatty acid synthase, acetyl-CoA carboxylase, or ATP citrate lyase. Fatty acid influx decreases intestinal fatty acid synthesis by a posttranscriptional mechanism independent of the SREBP pathway.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Grasas Insaturadas en la Dieta/farmacología , Ácidos Grasos/metabolismo , Mucosa Intestinal/metabolismo , Factores de Transcripción/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Animales , Peso Corporal , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/genética , Colesterol/biosíntesis , Colesterol/sangre , Cricetinae , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Grasas Insaturadas en la Dieta/metabolismo , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos/sangre , Expresión Génica , Intestinos/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Triglicéridos/sangreRESUMEN
Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1 mM taurocholate with or without 250 microM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Ácido Graso Sintasas/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Expresión Génica , Factores de Transcripción , Triglicéridos/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Anticolesterolemiantes/farmacología , Apolipoproteínas B/metabolismo , Células CACO-2 , Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Eicosapentaenoico/farmacología , Ácidos Grasos/metabolismo , Humanos , Hidrocarburos Fluorados , Immunoblotting , Plásmidos/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , SulfonamidasRESUMEN
Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.
Asunto(s)
Colesterol/metabolismo , Regulación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Alitretinoína , Anticolesterolemiantes/farmacología , Células CACO-2 , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular , Colesterol/farmacología , Proteínas de Unión al ADN , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/biosíntesis , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Hidrocarburos Fluorados , Ligandos , Receptores X del Hígado , Micelas , Receptores Nucleares Huérfanos , Receptores X Retinoide , Sulfonamidas , Tretinoina/farmacologíaRESUMEN
We infected a mixed culture of primary rat astrocytes and microglia with a replication-defective adenovirus carrying the rat glia maturation factor (GMF) cDNA. Affymetrix microarray analysis showed a big increase in the expression of several major histocompatibility complex (MHC) class II proteins along with interleukin-1beta (IL-1beta). Subsequent study using reverse transcription-polymerase chain reaction (RT-PCR) yielded the same results with the mixed culture, but not with pure astrocytes or pure microglia. We also noticed that the GMF/virus construct infected only astrocytes but not microglia. This led us to suspect that overexpression of GMF in astrocytes resulted in the secretion of an active substance that stimulated the microglia to express MHC II and IL-1beta. We identified this substance as granulocyte-macrophage-colony stimulating factor (GM-CSF). MHC II are unique to antigen-presenting cells such as microglia and monocytes. The results suggest that GMF in astrocytes can initiate a series of events, leading to immune activation in the nervous system, and implicates its involvement in autoimmune diseases such as multiple sclerosis.