RESUMEN
Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with beta,gamma-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 microM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36.
Asunto(s)
Plaquetas/metabolismo , Antígenos CD36/metabolismo , Palmitatos/metabolismo , Proteínas Quinasas/metabolismo , Humanos , FosforilaciónRESUMEN
Vitamin E supplementation exhibits anti-inflammatory properties. In the lung, the beneficial effects of vitamin E supplementation on inflammation and infections are well documented, but potential consequences of alimentary vitamin E deficiency to the immunological status of lung cells are not known. It is unclear if temporary vitamin E deficiency exhibits deleterious consequences or can be compensated for by other cellular antioxidants. To address this question, the alimentary vitamin E supply to rats was modified. We then investigated the effects on major histocompatibility molecule (MHC) class II, cell adhesion molecules, interleukin (IL)10, tumor necrosis factor (TNF)alpha in various lung cells. The constitutive expression of MHC class II, intercellular adhesion molecule (ICAM)-1, L-selectin, alpha5-integrin, and CD 166, was demonstrated by flow cytometry on type II pneumocytes, alveolar macrophages, and on co-isolated lymphocytes. Vitamin E depletion increased ICAM-1 and CD166 on type II cells and macrophages, whereas the expression of L-selectin increased only on macrophages. Furthermore, the vitamin E depletion increased the cellular content and secretion of IL10 in type II cells, but decreased the content and secretion of TNFalpha. Vitamin E depletion decreased the cellular vitamin E content, but did not change the activity of antioxidant enzymes (catalase, superoxide dismutase) and the glutathion (GSH)/oxidized glutathion (GSSG) ratio in alveolar type II cells. The shift of protein kinase C (PKC) from the cytosol to membranes indicates that a PKC-dependent signaling pathway may be involved in the change of the immunological status of type II cells. All these effects were reversed by vitamin E repletion. In summary, these results are clearly compatible with the view that a temporary vitamin E deficiency induces a reversible immunological dysregulation in alveolar type II cells and lung macrophages. This deficiency might predispose the lung to develop acute or chronic inflammation.
Asunto(s)
Pulmón/inmunología , Pulmón/patología , Deficiencia de Vitamina E/inmunología , Animales , Antioxidantes/metabolismo , Catalasa/metabolismo , Moléculas de Adhesión Celular/metabolismo , Supervivencia Celular , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Glutatión/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoquímica , Interleucina-10/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/patología , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vitamina E/metabolismo , Vitamina E/farmacología , Deficiencia de Vitamina E/dietoterapia , Deficiencia de Vitamina E/patologíaRESUMEN
The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesterol and cholesteryl ester (CE) from high density lipoprotein (HDL) into cells. The high expression in liver and steroidogenic tissues is compatible with a role of SR-BI in reverse cholesterol transport and steroid hormone synthesis. Ways of regulation thus far described include induction by trophic hormones via cAMP-activated protein kinase A (PKA) and the effects of cellular and plasma cholesterol. Here we show that vitamin E (vitE) has a major effect on the expression of SR-BI in rat liver and in a human hepatoma-derived cell line, HepG2. Feeding rats a vitE-depleted diet resulted in an 11-fold increase in the SR-BI protein level in liver tissue. This effect was readily reversed by feeding a vitE-enriched chow. In HepG2 cells, the expression of the human SR-BI homolog was reduced when the vitE content was increased by incubating the cells with vitE-loaded HDL or with phosphatidylcholine/vitE vesicles. The downregulation of human SR-BI (hSR-BI) was accompanied by a reduced level of protein kinase C (PKC) in the particulate cell fraction, and PKC inhibition decreased the expression of hSR-BI and the uptake of vitE and cholesterol from HDL. Our results are consistent with the view that the cellular level of vitE exerts a tight control over the expression of SR-BI. Furthermore, the inhibitory effect of vitE on PKC seems to be involved in the signaling pathway.
Asunto(s)
Antígenos CD36/metabolismo , Hígado/efectos de los fármacos , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Vitamina E/farmacología , Animales , Regulación hacia Abajo , Humanos , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Masculino , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Receptores Depuradores , Receptores Depuradores de Clase B , Transducción de Señal , Células Tumorales Cultivadas , Deficiencia de Vitamina E/metabolismoRESUMEN
Vitamin E is the most important lipophilic antioxidant, and beneficial effects on oxidant-caused injuries have been reported. Neonates are at high risk of oxidative injury in the lung and other organs because of a low vitamin E concentration, but the optimal timing of the application, a safe application form, and the optimal dosage of vitamin E are not known at present. We recently showed that alveolar type II cells take up vitamin E preferentially from high-density lipoprotein (HDL), probably by means of the candidate HDL receptor, scavenger receptor class B type I (SR-BI; Kolleck et al. Free Rad Biol Med 27; 882-890, 1999). Therefore, both the HDL-bound vitamin E in plasma and the expression of SR-BI on alveolar type II cells may determine the supply of the cells with vitamin E. We show here that the plasma level of vitamin E, total and HDL cholesterol, and the ratio of vitamin E to polyunsaturated fatty acids and to total fatty acids decrease during fetal rat development, reaching the minimum at the postconceptual day 21 (day -1). These parameters increase thereafter to about the same levels as in adult rats. SR-BI is not detectable until day -1 on fetal lung cells, but the expression during the postnatal phase follows the same pattern as the plasma lipid constituents. We conclude that the ability of alveolar type II cells to take up vitamin E develops perinatally in mature neonates. This aspect also has to be considered when the optimal timing of supplementation for the protection of preterm neonates with vitamin E against oxidative lung injury is established.
Asunto(s)
Antígenos CD36/metabolismo , Lipoproteínas HDL/sangre , Pulmón/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína/metabolismo , Vitamina E/sangre , Animales , Animales Recién Nacidos , Feto , Inmunohistoquímica , Hígado/metabolismo , Macrófagos Alveolares/metabolismo , Ratas , Receptores Depuradores , Receptores Depuradores de Clase B , Factores de TiempoRESUMEN
Numerous communications have indicated that specific binding proteins for high density lipoprotein (HDL) exist in addition to the well characterized candidate HDL receptor SR-BI, but structural information was presented only in a few cases, and most of the work was aimed at the liver and steroidogenic glands. In this study, we purified two HDL-binding proteins by standard procedures from rat lung tissue. One of these membrane glycoproteins was identified by N-terminal sequencing and with specific antibodies as HB2, a previously described HDL-binding protein, whereas the other one was identified as a glycosyl phosphatidylinositol-anchored membrane dipeptidase (MDP). The apparent dissociation constant of the HDL binding was determined by solid phase assay to be 2.1 microg/ml (HB2) and 25 microg/ml (MDP). MDP also exerts affinity to low density lipoprotein (LDL) on ligand blots, and competition between HDL and LDL was observed, but analysis by solid phase assay showed that very high concentrations of LDL are required. The physiologic relevance of this effect is therefore questionable. The level in type II pneumocyte membranes of both binding proteins, MDP and HB2, increased when the plasma lipoprotein concentration was reduced by treatment of rats with 4-aminopyrazolo[3,4-d]-pyrimidine, consistent with a function to facilitate lipid uptake in vivo. The binding proteins were also dramatically upregulated by feeding rats a vitamin E-depleted diet. Vitamin E uptake requires interaction between HDL and type II cells, suggesting a role of HB2 and MDP also in this process.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Dipeptidasas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Pulmón/citología , Pulmón/enzimología , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , HDL-Colesterol/análisis , HDL-Colesterol/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , Dieta , Electroforesis , Lectinas , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Deficiencia de Vitamina E/metabolismoRESUMEN
The alveolar surfactant is a prime target of reactive oxygen species present in air. Alveolar surfactant is supplemented with vitamin E during its assembly in type II pneumocytes. However, it is unknown which of the lipoproteins supply type II pneumocytes with vitamin E. The measurement of the uptake kinetics indicates that HDL might be the primary source of the vitamin E uptake by type II pneumocytes. Vitamin E depletion of rats caused an increase of vitamin E uptake by isolated type II pneumocytes from HDL but not from LDL or VLDL. We demonstrated that type II pneumocytes express the scavenger receptor class B type 1 (SR-B1), an HDL-specific receptor. Vitamin E depletion caused an increased expression of SR-B1 by a post-transcriptional mechanism. The increased vitamin E uptake from HDL and the increased expression of the SR-B1 were reversed by refeeding the vitamin. We propose that HDL is the primary source of vitamin E for type II pneumocytes. The rate of uptake of vitamin E by this cell type might be regulated by the expression of SR-B1.
Asunto(s)
Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Vitamina E/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Dieta , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Cinética , Lipoproteínas/metabolismo , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores Depuradores , Receptores Depuradores de Clase B , Deficiencia de Vitamina E/metabolismoRESUMEN
The alkenyl-acyl subclass of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (plasmalogens) are minor components of alveolar surfactant. Plasmalogens promote and stabilize hexagonal structures of phospholipids. In another study (W.R. Perkins, R.B. Dause, R.A. Parente, S.R. Michey, K.C. Neuman, S.M. Gruner, T.F. Taraschi, and A.S. Janoff. Science 273: 330-332, 1996), it was shown that polymorphic phase behavior may have an important role in the effective functioning of pulmonary surfactant. Therefore, we hypothesized that surface properties of phospholipid mixtures that contain plasmalogens are superior to plasmalogen-free mixtures. The effect of plasmalogens on surface tension of surfactant-like phospholipid mixtures (70 mol% dipalmitoyl phosphatidylcholine, 10 mol% phosphatidylglycerol, and 20 mol% PtdEtn) was measured. Using the pulsating bubble surfactometer, we show that an increasing amount of ethanolamine plasmalogens [plasmenylethanolamine (PlsEtn) results in reduction of surface tension (0 mol% PlsEtn 44.7 +/- 1.7, 2 mol% 33.5 +/- 1.7, 4 mol% 36 +/- 3.1, 6 mol% 26.2 +/- 2.9, and 8 mol% 22.2 +/- 0.3 mN/m). By means of the captive bubble surfactometer, minimal surface tension reached with 8 mol% PlsEtn was even lower (3.8 +/- 0.7 mN/m). With regard to morphological studies (B. Fringes, K. Gorgas, and A. Reith. Eur. J. Cell Biol. 46: 136-143, 1988), clofibrate treatment of rats might increase the plasmalogen content of alveolar surfactant. However, in the present study, we could not show that synthesis and secretion of plasmalogens are affected by clofibrate treatment.
Asunto(s)
Fosfolípidos/química , Plasmalógenos/farmacología , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina , Animales , Clofibrato/farmacología , Cinética , Fosfatidiletanolaminas , Fosfatidilgliceroles , Alveolos Pulmonares , Surfactantes Pulmonares/efectos de los fármacos , Ratas , Ratas Wistar , Tensión Superficial/efectos de los fármacosRESUMEN
Experimental and clinical studies have provided evidence for the involvement of oxygen free radicals in development of acute and chronic lung diseases. Hyperoxia is very often an indispensable therapeutic intervention which seems to impose oxidative stress on lung tissue. We measured the effect of hyperoxia (80% O2 for 20 h) (1) on the lipid composition of pulmonary surfactant treated in vitro, (2) on surfactant lipid synthesis and secretion of type II pneumocytes in primary culture, (3) on the lipid composition and on the SP-A content of rat lung lavages and (4) on the turnover of phospholipids, cholesterol, plasmalogens and vitamin E in type II pneumocytes, lamellar bodies and lavages of adult rat lungs. (1) Hyperoxia of lung lavages in vitro reduces the vitamin E content significantly but does not change the relative proportion of PUFA or the content of plasmalogens. (2) Hyperoxia does not affect the biosynthesis or secretion of surfactant lipids and plasmalogens by type pneumocytes in primary culture. (3) Hyperoxic treatment of rats increases the SP-A content and reduces the vitamin E content significantly but does not change the concentration of other lipid components of lung lavage. (4) The vitamin E turnover, measured in type II pneumocytes, lamellar bodies and lung lavages, is increased 2-fold in these fractions. In contrast, the turnover of surfactant cholesterol and surfactant lipids does not change. (5) Hyperoxia caused an increase of the vitamin E uptake by type II pneumocytes resulting in a vitamin E enrichment of lamellar bodies. From these results we conclude that type II pneumocytes are able to regulate the turnover of lipophilic constituents of the alveolar surfactant independently of each other. Hyperoxia caused type II pneumocytes to increase the vitamin E content of lamellar bodies. The lipid and SP-A content of alveolar fluid can be regulated independently each other.
Asunto(s)
Hiperoxia/metabolismo , Metabolismo de los Lípidos , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Colesterol/metabolismo , Lípidos/química , Masculino , Estrés Oxidativo/fisiología , Fosfolípidos/metabolismo , Plasmalógenos/metabolismo , Alveolos Pulmonares/citología , Surfactantes Pulmonares/química , Ratas , Ratas Wistar , Vitamina E/metabolismoRESUMEN
Alveolar surfactant (exposed to air and therefore a prime target of air oxidants) is supplied with antioxidants during its intracellular formation on type-II pneumocytes [Rüstow, Haupt, Stevens and Kunze (1993) Am. J. Physiol. 265, L133-L139]. Plasmalogens can protect animal cells against lipid peroxidation caused by u.v. radiation. It has been suggested that plasmalogens play a direct role in protecting animal cell membranes against oxidative stress [Zoeller, Morand and Raetz (1988) J. Biol. Chem. 263, 11590-11596]. We investigated biosynthesis and secretion of plasmalogens and phospholipids by type-II cells of adult rat lungs. The plasmalogens of type-II cells consist of 93% ethanolamine plasmalogens (EthPlas) and 7% choline plasmalogens (ChoPlas). Plasmalogens isolated from alveolar surfactant, however, consist of 36.5% ChoPlas and 63.5% EthPlas. The different incorporation rates of [14C]hexadecanol into both types of plasmalogen by type-II pneumocytes are reflected in the relative proportions of their total cellular plasmalogen content. Type-II cells cultured in the presence of labelled hexadecanol or labelled hexadecylglycerol and of labelled palmitate secrete labelled ChoPlas and labelled phospholipids, both spontaneously and in response to isoprenaline. The spontaneous and stimulated secretion rates of labelled ChoPlas are 3-6 times higher than those of labelled EthPlas. This higher relative secretion rate of ChoPlas corresponds to its higher proportion in the total plasmalogen content of alveolar surfactant compared with type-II cells. Added extracellular surfactant-specific protein A inhibits the secretion of plasmalogens as well as that of phospholipids by type-II cells. The molecular species of EthPlas and ChoPlas isolated from type-II cells or lung lavage do not differ significantly and consist mainly of molecular species containing poly-unsaturated fatty acids. We conclude that ChoPlas are secreted partly as integral constituents of the alveolar surfactant. Type-II cells select between both types of plasmalogens for secretion as a constituent of surfactant. The intramolecular sorting signal presumably is the choline moiety.