RESUMEN
One of the key functions of mammalian pulmonary surfactant is the reduction of surface tension to minimal values. To fulfill this function it is expected to become enriched in dipalmitoylphosphatidylcholine either on its way from the alveolar type II pneumocytes to the air/water interface of the lung or within the surface film during compression and expansion of the alveoli during the breathing cycle. One protein that may play a major role in this enrichment process is the surfactant protein B. The aim of this study was to identify the lipidic interaction partner of this protein. Time-of-flight secondary ion mass spectrometry was used to analyze the lateral distribution of the components in two SP-B-containing model systems. Either native or partly isotopically labeled lipids were analyzed. The results of both setups give strong indications that, at least under the specific conditions of the chosen model systems (e.g., concerning pH and lipid composition), the lipid interacting with surfactant protein B is not phosphatidylglycerol as generally accepted, but dipalmitoylphosphatidylcholine instead.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Liposomas/química , Fosfatidilgliceroles/química , Proteína B Asociada a Surfactante Pulmonar/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , 1,2-Dipalmitoilfosfatidilcolina/análisis , Sitios de Unión , Membrana Dobles de Lípidos/análisis , Fosfatidilgliceroles/análisis , Unión Proteica , Proteína B Asociada a Surfactante Pulmonar/análisis , Propiedades de SuperficieRESUMEN
Cryptococcus neoformans is an opportunistic pathogen invading the immunocompromised host. Infection starts with the inhalation of acapsular or sparsely encapsulated cells, after which capsule synthesis is initiated. The capsule is the main virulence factor of this yeast-like fungus. Pulmonary surfactant protein D (SP-D) is an important component of the local innate defense system. In the present study, interactions of SP-D with intact C. neoformans cells and their isolated capsular components were investigated. Although encapsulated cryptococci were bound, SP-D showed the highest affinity for acapsular C. neoformans. Only acapsular cryptococci were aggregated by SP-D. Furthermore, the cryptococcal capsular components glucuronoxylomannan (GXM) and mannoprotein 1 (MP1) were bound with relatively high affinity, in contrast to GalXM and MP2. Binding as well as aggregation of acapsular C. neoformans by SP-D could be inhibited by GXM in concentrations that are likely to be present in the lung after infection, suggesting that not only the capsule hampers SP-D function within the innate defense system of the lung but also the secreted capsular component GXM.
Asunto(s)
Adhesión Celular , Cryptococcus neoformans/fisiología , Polisacáridos/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Criptococosis/microbiología , Cryptococcus neoformans/química , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/metabolismo , Humanos , Polisacáridos/aislamiento & purificación , Unión ProteicaRESUMEN
BACKGROUND: There is evidence that surfactant protein (SP)-D is important in the innate, as well as in the adaptive pulmonary immune response. Serum concentrations of SP-D have been proposed as parameter of the integrity of the blood-airspace barrier in interstitial lung diseases. We hypothesized that serum SP-D concentrations are affected in allergic patients and correlate with changes in allergic airway inflammation. OBJECTIVE: To determine levels of serum SP-D in allergic patients compared with non-allergic controls. Furthermore, to investigate associations between serum SP-D concentrations on the one hand and changes in commonly used markers of bronchial inflammation in allergic airways disease on the other hand. MATERIALS AND METHODS: Fifty allergic patients were studied and bronchial allergen challenge was used as a model to increase bronchial allergic inflammation in these patients. Serum SP-D concentrations, inflammatory parameters in induced sputum and bronchial hyper-responsiveness (BHR) were determined before and after allergen challenge. Twenty-five non-allergic volunteers served as controls. RESULTS: Baseline serum SP-D was significantly higher in allergic patients as compared with controls (mean serum SP-D concentration (95% confidence interval): 62.7 (55.5, 70.0) in allergic patients vs. 49.5 (36.7, 62.3) ng/mL in non-allergic controls, P=0.006). In addition, baseline serum SP-D appeared to be an independent predictor for the magnitude of the late asthmatic response after allergen challenge. Furthermore, serum SP-D was predictive for the sputum eosinophil cationic protein concentration after allergen challenge. CONCLUSION: We propose that serum SP-D concentrations are associated with allergic bronchial inflammation and may give additional information, beside BHR and sputum eosinophils, about the degree of bronchial inflammation in allergic patients.
Asunto(s)
Hipersensibilidad/sangre , Proteína D Asociada a Surfactante Pulmonar/sangre , Adulto , Alérgenos , Biomarcadores/sangre , Pruebas de Provocación Bronquial , Estudios de Casos y Controles , Proteína Catiónica del Eosinófilo/análisis , Femenino , Humanos , Hipersensibilidad/inmunología , Masculino , Esputo/inmunología , Factores de TiempoRESUMEN
Infection with gram-positive bacteria is a major cause of pneumonia. Surfactant proteins A (SP-A) and D (SP-D) are thought to play an important role in the innate immunity of the lung. Both proteins can bind to gram-positive bacteria. Until now, it was not known with which surface component(s) of gram-positive bacteria SP-A and SP-D interact. Lipoteichoic acid (LTA) and peptidoglycan (PepG) are components of the cell wall of gram-positive bacteria. By use of a solid phase-based binding assay, LTA of Bacillus subtilis was shown to be bound by SP-D but not by SP-A. Unmodified PepG of Staphylococcus aureus was bound by SP-D. SP-D binding to both LTA and PepG was calcium dependent and carbohydrate inhibitable. These results indicate that SP-D interacts with gram-positive bacteria via binding to the cell wall components LTA and PepG and that the carbohydrate recognition domain is responsible for this binding.
Asunto(s)
Bacillus subtilis/metabolismo , Glicoproteínas/metabolismo , Lipopolisacáridos/metabolismo , Peptidoglicano/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/metabolismo , Animales , Unión Competitiva , Calcio/metabolismo , Metabolismo de los Hidratos de Carbono , Pared Celular/química , Humanos , Proteína A Asociada a Surfactante Pulmonar , Proteína D Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante PulmonarRESUMEN
Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cysteines 5 and 6 before mature SP-C is formed by several proteolytic steps. To study the structural requirements for the palmitoylation of proSP-C, his-tagged human proSP-C (his-proSP-C) and his-proSP-C mutants were expressed in Chinese hamster ovary cells and analyzed by metabolic labeling with [(3)H]palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by serines showed that these were the only two cysteine residues palmitoylated in his-proSP-C. Substitution of the juxtamembrane basic residues lysine and arginine by uncharged glutamines led to a large decrease in palmitoylation level of proSP-C. The addition of brefeldin A nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutant increased also, but not to wild-type (WT) levels. Fluorescence immunocytochemistry showed that WT proSP-C was localized in punctate vesicles throughout the cell, whereas the mutant lacking the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). This indicates that the two basic juxtamembrane residues influence palmitoylation of proSP-C by preventing the transport of proSP-C out of the ER, implying that proSP-C becomes palmitoylated normally in a compartment distal to the ER.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arginina/química , Secuencia de Bases , Transporte Biológico Activo , Brefeldino A/farmacología , Células CHO , Cricetinae , Cisteína/química , Cartilla de ADN/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , Lisina/química , Datos de Secuencia Molecular , Mutación , Ácido Palmítico/química , Péptidos/genética , Proteína C Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.
Asunto(s)
Liposomas/química , Proteolípidos/química , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/química , Elasticidad , Fosfatidilgliceroles/química , Tensión SuperficialRESUMEN
A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observed. Spread phospholipid films containing 50% DPPC over a subphase containing 50% DPPC vesicles did not form stable surface films with a low minimum surface tension. Addition of surfactant protein B (SP-B) to the surface film led to a progressive decrease in minimum surface tension toward 1 mN/m upon cycling, indicating an enrichment in DPPC. Surfactant protein C (SP-C) had no such detectable refining effect on the film. Surfactant protein A (SP-A) had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SP-A was combined with SP-B and incubated with subphase vesicles before addition to the air bubble containing sample chamber. Comparison of spread films with adsorbed films indicated that refinement induced by SP-B occurs by selective removal of non-DPPC lipids upon cycling. SP-A, combined with SP-B, induces a selective adsorption of DPPC from subphase vesicles into the surface film. This is achieved by formation of large lipid structures which might resemble tubular myelin.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Liposomas/química , Proteolípidos/química , Surfactantes Pulmonares/química , Animales , Glicoproteínas/química , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Propiedades de Superficie , PorcinosRESUMEN
Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.
Asunto(s)
Fragmentos de Péptidos/química , Proteolípidos/química , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/química , Secuencia de Aminoácidos , Calcio/química , Dimerización , Fluorescencia , Humanos , Lípidos/química , Liposomas/química , Membranas Artificiales , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Solubilidad , Propiedades de Superficie , Tensión SuperficialRESUMEN
Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high alpha-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.
Asunto(s)
Baculoviridae/genética , Proteolípidos/biosíntesis , Surfactantes Pulmonares/biosíntesis , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Expresión Génica , Insectos , Espectrometría de Masas , Ácido Palmítico/química , Pliegue de Proteína , Proteolípidos/química , Proteolípidos/genética , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genética , Espectroscopía Infrarroja por Transformada de FourierAsunto(s)
Productos Biológicos , Proteínas , Surfactantes Pulmonares/fisiología , Transporte Biológico/fisiología , Hormonas/fisiología , Humanos , Metabolismo de los Lípidos , Vaina de Mielina , Fosfatidilcolinas/biosíntesis , Proteolípidos/metabolismo , Surfactantes Pulmonares/biosíntesis , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismoRESUMEN
To gain more insight into the regulation of the expression of insulin-like growth factor (IGF) binding proteins (IGFBPs) in the lung, the developmental patterns of the abundance of the mRNAs encoding IGFBPs were measured in the perinatal rat lung and in explant cultures of fetal rat lung. In hormone-free explant cultures, the levels of the mRNAs encoding IGFBP-2 through -5 changed with a pattern similar to that occurring in vivo (although in the case of IGFBP-3 to -5 at a faster rate), indicating that the developmental regulation of the expression of these IGFBPs in perinatal lung is mimicked in the explants. For the IGFBP-6 mRNA level, the pattern in vitro differed from that in vivo. In the explant cultures, dexamethasone decreased the production of IGFBP-3 and -4 and decreased the abundance of the mRNAs encoding IGFBP-2 to -5 but increased the abundance of IGFBP-6 mRNA. These observations indicate that glucocorticoids may be involved in the developmental regulation of the expression of these components of the IGF system and that the IGF system may be involved in the physiological effects of glucocorticoids on lung development. No appreciable effects of 3,3',5-triiodothyronine on the expression of the IGFBPs were observed.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Pulmón/metabolismo , Transcripción Genética , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Dexametasona/farmacología , Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Edad Gestacional , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Técnicas de Cultivo de Órganos , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacosRESUMEN
The complete sequence of the nonspecific lipid-transfer protein (nsL-TP; sterol carrier protein 2) including the presequence is present at the C-terminus (residues 405-547) of a 58-kDa protein. To be able to study this 58-kDa protein without the interference of nsL-TP, antibodies were raised against predicted epitope regions in the N-terminal part (peptide I, residues 23-43; peptide II, residues 130-149). Using these antibodies, rat tissues were analyzed by immunoblotting. In rat liver, in addition to the 58-kDa protein the antibody against peptide I (alpha-58K23) as well as the antibody against peptide II (alpha-58K130) detected a 46-kDa protein. This suggests that both peptide sequences are present in this 46-kDa protein. Both the 46- and the 58-kDa-proteins were abundantly present in liver and adrenals, but could also be detected in brain, kidney, heart, lung, testes, and ovary. This distribution was observed in tissues from both male and female rats. Immunogold labeling of cryosections of liver showed that alpha-58K23 labels the peroxisomes. From double-labeling experiments using alpha-nsL-TP and alpha-58K23 we conclude that the 46-kDa protein is peroxisomal. We propose that in the peroxisomes the protease that processes pre-nsL-TP also cleaves the 58-kDa protein giving rise to the 46-kDa protein and nsL-TP. In addition to the 58- and 46-kDa proteins, an immunoreactive 44-kDa protein was prominently present in rat heart and at low levels also in small intestine and brain. Immunogold labeling of cryosections of heart and Western blotting of purified mitochondria showed that the 44-kDa protein is localized in the mitochondria. The 44-kDa protein was shown to be identical to mitochondrial sarcomeric creatine kinase, which has a peptide segment of five amino acid residues in common with peptide I.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Proteínas Portadoras/metabolismo , Microcuerpos/metabolismo , Acetil-CoA C-Acetiltransferasa/análisis , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/análisis , Catalasa/análisis , Epítopos/análisis , Femenino , Immunoblotting , Hígado/metabolismo , Hígado/ultraestructura , Masculino , Microcuerpos/ultraestructura , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Ratas , Ratas Wistar , Esteroles/metabolismoRESUMEN
In fetal lung the amounts of mRNAs encoding fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and ATP citrate lyase (ACL) increase in late gestation and drop around birth. To study the mechanism of the perinatal decrease, pregnancy was prolonged from 22 (term) to 25 days in rats with daily injections of progesterone. Progesterone did not affect the levels of lipogenic enzyme mRNAs in fetal lung prior to term, but significantly delayed the perinatal decrease in the levels of lung FAS and ACC mRNA. Although for ACL mRNA abundance the differences were not statistically significant, its pattern in the control and progesterone groups were similar to those of FAS and ACC mRNA. Malic enzyme mRNA did not change between 20 and 25 days after conception in either group. These results suggest that the decrease in FAS and ACC mRNA at term can be partially explained by labor, delivery, air-breathing or switch from carbohydrate to fat metabolism.
Asunto(s)
Enzimas/genética , Ácidos Grasos/biosíntesis , Feto/metabolismo , Expresión Génica , Pulmón/enzimología , Embarazo Prolongado/genética , ATP Citrato (pro-S)-Liasa/genética , Acetil-CoA Carboxilasa/genética , Animales , Ácido Graso Sintasas/genética , Femenino , Pulmón/embriología , Malato Deshidrogenasa/genética , Embarazo , Progesterona/farmacología , ARN Mensajero/metabolismo , RatasRESUMEN
In view of the proposed role of the non-specific lipid-transfer protein (nsL-TP; sterol carrier protein 2) in the metabolism of pulmonary surfactant lipids (Batenburg et al. (1994) Biochem. J. 298, 223-229), its subcellular localization was studied in the surfactant producing alveolar type II cells. It was shown by immuno-electron microscopy that nsL-TP colocalizes with the peroxisomal marker catalase. The peroxisomal localization of nsL-TP was confirmed by gradient fractionation of type II cell homogenates. As a peroxisomal marker acyl-CoA:dihydroxyacetone-phosphate acyltransferase was assayed. Given this subcellular localization, it is very unlikely that nsL-TP plays a role in the transfer of surfactant lipids from the endoplasmic reticulum to the lamellar bodies. These results strengthen the opinion that peroxisomes are involved in surfactant synthesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Microcuerpos/metabolismo , Proteínas de Plantas , Alveolos Pulmonares/metabolismo , Esteroles/metabolismo , Aciltransferasas/metabolismo , Animales , Biomarcadores , Masculino , Microscopía Inmunoelectrónica , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismo , Ratas , Ratas Wistar , Fracciones Subcelulares/metabolismoRESUMEN
Fetal rat lung epithelial cells were isolated on gestational day 17 (term is 22), separated from fibroblasts, and cultured up to 6 days in a serum-free medium on a basement membrane matrix. Surfactant protein (SP) A, barely detectable by immunostaining at the beginning of the culture, considerably increased in cells and subsequently in the lumen of the epithelial cell clusters. SP-A mRNA, already detectable at culture initiation, progressively increased. By contrast, SP-B and its mRNA appeared after 2-3 days. SP-C mRNA appeared only after 4 days of culture. Cells cultured 6 days had a phospholipid composition similar to that of freshly isolated adult rat type II cells. The enhancement of lipid synthesis between the first and the sixth culture days, reported earlier to occur in these cells, was found to be accompanied by a two- to fivefold increase in amount of mRNAs of lipogenic enzymes and choline phosphate cytidylyltransferase. In conclusion, alveolar epithelial type II cells appear to be capable of full differentiation in vitro, and components of the surfactant system are all regulated developmentally at a pretranslational level.
Asunto(s)
Desarrollo Embrionario y Fetal , Enzimas/metabolismo , Feto/metabolismo , Metabolismo de los Lípidos , Pulmón/embriología , Surfactantes Pulmonares/metabolismo , Animales , Células Cultivadas , Citidililtransferasa de Colina-Fosfato , ADN/metabolismo , Enzimas/genética , Feto/citología , Técnicas Inmunológicas , Queratinas/metabolismo , Pulmón/citología , Nucleotidiltransferasas/genética , Fosfolípidos/metabolismo , ARN Mensajero/metabolismo , Ratas/embriología , Ratas Wistar , Tubulina (Proteína)/metabolismo , Vimentina/metabolismoRESUMEN
Gene expression of non-specific lipid-transfer protein (nsL-TP; identical with sterol carrier protein 2) and phosphatidylinositol-transfer protein (PI-TP) was investigated in developing rat lung. During the late prenatal period (between days 17 and 22) there is a 7-fold increase in the level of nsL-TP and a 2-fold rise in that of PI-TP. The prenatal increases in the levels of nsL-TP and PI-TP are accompanied by parallel increases in the levels of their mRNAs, indicating pretranslational regulation. Compared with whole lung, isolated alveolar type-II cells are enriched in nsL-TP and its mRNA, but not in PI-TP and its mRNA. The observation that the levels of nsL-TP and its mRNA in rat lung show a pronounced increase in the period of accelerated surfactant formation, together with the observation that the surfactant-producing type-II cells are enriched in nsL-TP and its mRNA, suggest that nsL-TP plays a role in the metabolism of pulmonary surfactant.
Asunto(s)
Proteínas Portadoras/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Alveolos Pulmonares/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas Portadoras/genética , Femenino , Pulmón/embriología , Proteínas de la Membrana/genética , Embarazo , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Ratas , Ratas WistarRESUMEN
In hormone-free explant cultures of 18-day fetal rat lung the levels of the mRNAs for fatty acid synthase, ATP citrate lyase and surfactant proteins A, B, and C, increased as they do in vivo. Also CTP:phosphocholine cytidylyltransferase mRNA increased spontaneously. Unlike in vivo, malic enzyme mRNA increased drastically in cultured explants. Culture with dexamethasone increased the abundance of fatty acid synthase and surfactant protein mRNAs, but considerably depressed that of malic enzyme mRNA. Dexamethasone had little effect on CTP:phosphocholine cytidylyltransferase mRNA, supporting the concept that the increased activity of this enzyme caused by glucocorticoid is due to increased fatty acid synthesis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Lípidos/biosíntesis , Pulmón/enzimología , Biosíntesis de Proteínas , Tensoactivos/metabolismo , ATP Citrato (pro-S)-Liasa/genética , ATP Citrato (pro-S)-Liasa/metabolismo , Animales , Northern Blotting , Citidililtransferasa de Colina-Fosfato , Técnicas de Cultivo , Dexametasona/farmacología , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Femenino , Feto , Pulmón/embriología , Pulmón/metabolismo , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Embarazo , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Regulation of CTP:choline-phosphate cytidylyltransferase activity was studied in regenerating rat liver. The formation of phosphatidylcholine from [14C]choline in hepatocytes isolated from regenerating liver at 22 h after surgery was increased 1.9-fold when compared with hepatocytes from sham-operated animals. This effect was accompanied by a 1.4-fold increase in cytosolic cytidylyltransferase activity as well as by a 1.5-fold increase in the amount of immunoreactive cytidylyltransferase protein, and a 1.7-fold increase in [35S]methionine incorporation into cytidylyltransferase protein. Northern blot analysis of cytidylyltransferase mRNA showed two signals at 1.5 and 5.0 kb. Partial hepatectomy caused a significant 2-3-fold increase in the 1.5-kb and 5.0-kb messengers at 12 h after surgery. During the next 10 h after partial hepatectomy cytidylyltransferase mRNA levels slightly decreased, although they were still elevated in comparison with sham-operated rats 20-22 h after surgery. In contrast to the elevated cytidylyltransferase mRNA levels, the amount of acetyl-CoA carboxylase mRNA did not increase between 12 and 22 h after surgery, which is in line with the unchanged activity of this enzyme. In conclusion, our data demonstrate that in regenerating liver phosphatidylcholine biosynthesis and cytidylyltransferase activity are regulated at a pretranslational level.
Asunto(s)
Regulación de la Expresión Génica , Regeneración Hepática/fisiología , Hígado/metabolismo , Nucleotidiltransferasas/biosíntesis , Fosfatidilcolinas/biosíntesis , ARN Mensajero/metabolismo , Animales , Citidililtransferasa de Colina-Fosfato , Citosol/enzimología , Hepatectomía , Isoenzimas/aislamiento & purificación , Hígado/enzimología , Masculino , Nucleotidiltransferasas/aislamiento & purificación , Ratas , Ratas WistarRESUMEN
Exposure to fibroblast-conditioned cortisol-containing medium increased fatty acid synthase activity and fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase mRNA abundance in fetal type II alveolar epithelial cells. Both fibroblast conditioning and cortisol in the medium were required for maximal effect on the mRNA levels, indicating involvement of mesenchymal-epithelial interaction in the cortisol effects. The observed effects provide evidence for an earlier hypothesis that increased activity of CTP:phosphocholine cytidylyltransferase in lung tissue caused by glucocorticoid is due to increased fatty acid synthesis. However, evidence suggesting pre-translational regulation of this enzyme by glucocorticoid was also found.
Asunto(s)
Ácidos Grasos/biosíntesis , Glucocorticoides/fisiología , Pulmón/metabolismo , Fosfatidilcolinas/biosíntesis , Biosíntesis de Proteínas , ATP Citrato (pro-S)-Liasa/genética , ATP Citrato (pro-S)-Liasa/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Animales , Células Cultivadas , Citidililtransferasa de Colina-Fosfato , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/genética , Pulmón/citología , Pulmón/embriología , Pulmón/enzimología , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Fosfatidilcolinas/genética , ARN Mensajero/genética , RatasRESUMEN
Pulmonary surfactant, a complex consisting of 90% lipids and 10% specific proteins, lines the alveoli of the lung and prevents alveolar collapse and transudation by lowering the surface tension at the air-liquid interface. Dipalmitoylphosphatidylcholine constitutes approximately 50% of the surfactant lipids and is primarily responsible for the surface tension-lowering property of the surfactant mixture. This phospholipid, together with the other surfactant phospholipids, is produced at the endoplasmic reticulum of the alveolar type II epithelial cells. The characteristic lamellar bodies in these cells serve as storage depot for the surfactant before this is secreted onto the alveolar surface. This article reviews the pathways via which the surfactant lipids are synthesized, our current knowledge of the regulation of these pathways, and what is known about intracellular traffic of phospholipids from their site of synthesis to the lamellar bodies.