RESUMEN
Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.
Asunto(s)
Proteínas Portadoras , Glicosilfosfatidilinositoles/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN , Receptores de Lipoproteína/metabolismo , Anticuerpos/farmacología , Sitios de Unión , Humanos , Proteínas de la Membrana/inmunología , Fosfatidilcolinas/metabolismo , Fosfolipasas/antagonistas & inhibidores , Receptores de Lipoproteína/inmunología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
In [3H]phosphatidylcholine (PC) prelabelled HepG2 cells, HDL3 stimulates a biphasic increase in 1.2-diacylglycerol (DAG). The early phase is mediated in part by a phospholipase C which is inhibited by 10 microM D 609, RHC-80267 or U-73122 and less by 100 microM propranolol. A phospholipase D is more likely involved in the late phase, as the DAG peak lags behind phosphatidic acid rise and is blocked by 100 microM propranolol. Cellular preincubation with 200 microg/ml antibodies against the inositolphosphoglycan (IPG) moiety of the GPI-anchor (Ab(IPG)), or depletion in GPI-anchored proteins by cellular pretreatment with 0.5 U/ml PI-PLC, 1 mM insulin and 2 HU/ml streptolysin-O, or depletion in membrane cholesterol content by filipin (5 microg/ml), digitonin (5 microg/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL3-signal, suggesting the involvement of a lipolytic cleavage of GPI-anchored proteins. Inhibition of proteases by 1 mM leupeptin/PMSF improves the response time to HDL3, with a DAG peak at 2-3 min. In the presence of protease-inhibitors, HDL3 releases in the culture medium several proteins with a residual IPG that binds Ab(IPG) after SDS-PAGE analysis and immunoblotting. HDL3-signalling pathways comprise tyrosine kinases, as preincubation with 100 microg/ml genistein or tyrphostin inhibits the HDL3-signal. HDL3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Anticuerpos/inmunología , Medios de Cultivo/análisis , Diglicéridos/metabolismo , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Fosfatidilcolinas/metabolismo , Inhibidores de Proteasas/farmacología , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
To elucidate further the binding of high-density-lipoprotein subfraction 3 (HDL3) to cells, the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-proteins) was studied. Treatment of cultured cells, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinositol-specific phospholipase C (PI-PLC) significantly decreases specific HDL3 binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These proteins have a soluble form and can also bind HDL3, as revealed by ligand blotting experiments with HDL3. In order to obtain enriched GPI-proteins, we used a detergent-free purification method to prepare a caveolar membrane fraction. In the caveolar fraction, we obtained, by ligand blotting experiments, the enrichment of two HDL3-binding proteins with molecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. Electron microscopy also showed the binding of Au-labelled HDL3 inside the caveolar membrane invaginations. In SK-MES-1 cells, HDL3 are internalized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstration has been made of the implication of GPI-proteins as well as caveolae in the binding of HDL3 to cells.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Endocitosis , Glicosilfosfatidilinositoles/metabolismo , Lipoproteínas HDL/metabolismo , Transporte Biológico , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Unión Proteica/efectos de los fármacos , Piel/citología , Piel/metabolismo , Fosfolipasas de Tipo C/farmacologíaRESUMEN
The conversion of phosphatidylethanolamine (PE) into phosphatidylcholine (PC) by a sequence of three transmethylation reactions is shown to be stimulated by the apolipoprotein E-free subclass of high-density lipoprotein (HDL3) in isolated bovine brain capillary (BBC) membranes, HDL3-induced stimulation of BBC membranes pulsed with [methyl-14C]methionine causes a transient increase in each methylated phospholipid, i.e. phosphatidyl-N-monomethylethanolamine (PMME), phosphatidyl-NN-dimethylethanolamine (PDME) and PC. PC substrate arising from the activation of PE N-methyltransferase (PEMT) is hydrolysed by a phospholipase A2 (PLA2), as demonstrated by the accumulation of lysophosphatidylcholine (lyso-PC). When PE containing [14C]arachidonic acid in the sn-2 position ([14C]PAPE) is incorporated into BBC membranes, HDL3 stimulation induces the formation of PMME, PDME, PC and lyso-PC and the release of [14C]arachidonic acid, which correlates with the previous production of lyso-PC, suggesting that HDL3 stimulates a PLA2 that can release polyunsaturated fatty acids (PUFA). Both PEMT and PLA2 activities depend on a HDL3 concentration in the range 0-50 micrograms/ml and are strictly dependent on HDL3 binding, because HDL3 modified by tetranitromethane is no longer able to bind to specific receptors and to trigger PEMT and PLA2 activation. Moreover, HDL3 prelabelled with [14C]PAPE can stimulate PDME and lyso-PC synthesis in BBC membranes in the presence of S-adenosylmethionine, suggesting that HDL3 can supply BBC membranes in polyunsaturated PE and can activate enzymes involved in PE N-methylation and PUFA release. The results support the hypothesis of a close relationship between HDL3 binding, PE methylation and PUFA release, and suggest that the PC pool arising from PE could be used as a pathway for the supply of PUFA to the brain.
Asunto(s)
Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Capilares/metabolismo , Endotelio Vascular/metabolismo , Ácidos Grasos Insaturados/metabolismo , Lipoproteínas HDL/metabolismo , Metiltransferasas/metabolismo , Fosfolipasas A/metabolismo , Animales , Ácido Araquidónico/metabolismo , Bovinos , Activación Enzimática , Lipoproteínas HDL/farmacología , Lipoproteínas HDL3 , Metionina/metabolismo , Metilación , Fosfatidiletanolamina N-Metiltransferasa , Fosfatidiletanolaminas/metabolismo , Fosfolipasas A2 , Fosfolípidos/aislamiento & purificación , Fosfolípidos/metabolismoRESUMEN
We investigated the relationship between assessment of fatty acid intake by a 3-day food record and by capillary gas chromatography of erythrocyte phospholipid fatty acid. The study was performed in a sample of 244 men aged 45 to 66 years from the general population who were participating in the Monitoring of Cardiovascular Disease (MONICA)-Lille survey. The relationship between each nutrient and food item and erythrocyte phospholipid fatty acid was investigated by a regression model on proportion including each food item and nutrient as a dependent variable and percentage of fatty acid and covariables (nonalcoholic energy intake, age, alcohol intake, and smoking) as independent variables. Polyunsaturated fat and linoleic acid intake were positively correlated with linoleic acid content of erythrocytes (beta = 0.641 and 0.604, respectively, P < .001). Monounsaturated and saturated fat intake were correlated with oleic acid (beta = 0.375 and 0.373, respectively, P < .01). Fish intake correlated positively with docosahexaenoic acid (DHA) (beta = 0.383, P < .001) and negatively with arachidonic acid (beta = -0.509, P < .01). These data confirm, on a group level, a good relationship between assessment of polyunsaturated fat intake by a 3-day record and linoleic acid content of erythrocyte membranes. These data suggest that erythrocyte oleic acid content is a marker of both saturated and monounsaturated fat intake.
Asunto(s)
Enfermedades Cardiovasculares , Registros de Dieta , Grasas de la Dieta/administración & dosificación , Eritrocitos/química , Ácidos Grasos/sangre , Fosfolípidos/sangre , Animales , Ácido Araquidónico/administración & dosificación , Grasas Insaturadas en la Dieta/administración & dosificación , Ácidos Docosahexaenoicos/administración & dosificación , Peces , Alimentos , Humanos , Ácido Linoleico , Ácidos Linoleicos/administración & dosificación , Ácidos Linoleicos/sangre , Masculino , Persona de Mediana Edad , Ácido Oléico , Ácidos Oléicos/sangreRESUMEN
Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene (CYP27). So far several mutations causing CTX have been identified and characterized. A new mutation creating an insertion of cytosine at position 6 in the cDNA, which is expected to result in a frameshift and a premature termination codon at codon 179, has been identified in a French family. The mutation creates a new site for the restriction endonuclease HaeIII.
Asunto(s)
Encefalopatías/genética , Codón de Terminación/genética , Sistema Enzimático del Citocromo P-450/genética , Esteroide Hidroxilasas/genética , Xantomatosis/genética , Adulto , Secuencia de Bases , Niño , Colestanotriol 26-Monooxigenasa , Femenino , Francia , Humanos , Masculino , Datos de Secuencia Molecular , LinajeRESUMEN
In isolated human platelets, exposure of subfraction 3 high-density lipoprotein (HDL3) binding sites to high concentrations of HDL3 (1 mg/mL) causes rapid desensitization of HDL3 (50 micrograms/mL)-stimulated breakdown of phosphatidylcholine, as shown in approximately a 70% depression of the maximal 1,2-diacylglycerol release activity by phospholipase C. This desensitization is HDL3 dose dependent (IC50, 150 +/- 20 micrograms/mL, n = 6) and time dependent (t1/2, < 30 seconds). It requires the binding of HDL3, as pretreatment of HDL3 by tetranitromethane does not cause the desensitization of HDL3-induced phospholipase C activity. Permeabilization of human platelets with 10 micrograms/mL digitonin, used to permit access of charged inhibitors to the cytosol, does not interfere with the pattern of HDL3 (1 mg/mL)-induced desensitization of HDL3 (50 micrograms/mL)-stimulated phospholipase C. Inhibitors of protein kinase C (100 mumol/L H-7 and 10 mumol/L staurosporine) markedly inhibit desensitization of HDL3-induced phospholipase C activity, whereas cAMP-dependent protein kinase inhibitor (1 mumol/L), heparin (100 nmol/L), or concanavalin A (0.25 mg/mL) were ineffective. HDL3-induced desensitization is accompanied at least by the phosphorylation of the 94- and 110-kD proteins. Inhibition of HDL3-induced desensitization by 100 mumol/L H-7 or 10 mumol/L staurosporine is characterized by a marked reduction of the phosphorylation state of these proteins in permeabilized platelets. Whereas protein kinase C inhibitors fully inhibited the phosphorylation of the 94- and 110-kD proteins, inhibitors of protein kinase A were less effective. These data establish that phosphorylation by protein kinase C represent a step in the desensitization of HDL3 binding sites in human platelets.
Asunto(s)
Plaquetas/enzimología , Lipoproteínas HDL/farmacología , Proteína Quinasa C/fisiología , Fosfolipasas de Tipo C/sangre , Sitios de Unión , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Fosforilación , Proteína Quinasa C/farmacología , Transducción de SeñalRESUMEN
This study examined the relationship between the fatty acid composition of red blood cell phospholipids and lipid markers of atherosclerotic risk in an urban male population aged 45 to 66 years. There was a surprisingly significant positive association between the docosahexaenoic acid ([DHA] 22:6n-3) content of erythrocyte phospholipids and the following risk markers: plasma cholesterol (P < .01), low-density lipoprotein (LDL) cholesterol (P < .01), apolipoprotein (apo) B (P < .05), and apo B-containing lipoprotein particles (P < .05) recognized by a monoclonal antibody (LpBL3). On the other hand, phospholipid alpha-linolenate was positively correlated with apo A-I and high-density lipoprotein (HDL) cholesterol levels (P < .05), while arachidonate showed an inverse relationship with plasma cholesterol (P < .05). There was a negative association between palmitoleic acid and apo B (P < .01) and LpBL3 (P < .001); the latter showed a negative association with stearic acid (P < .001). These interesting findings emphasize the beneficial effect on atherosclerotic risk markers of dietary n-6 polyunsaturated and monounsaturated fatty acids, and suggest that long-chain n-3 polyunsaturated fatty acids (DHA) could have an adverse effect on some of the lipid risk markers.
Asunto(s)
Apolipoproteínas/sangre , Eritrocitos/metabolismo , Ácidos Grasos/sangre , Lípidos/sangre , Fosfolípidos/sangre , Humanos , Masculino , Persona de Mediana Edad , Análisis de RegresiónRESUMEN
Initially we established that, in human platelets, low concentrations of HDL3 stimulate phosphatidylcholine (PC) hydrolysis and a transient increase in 1,2-diacylglycerol (DAG). In (3H) PC prelabelled platelets, phosphocholine is released into the medium during HDL3 induced PC turnover with a 1.5 to 2 fold increment, indicating that HDL3 stimulated DAG generation in platelets is likely due to phospholipase C (PLC). GTP or GTP-gamma-S augments, and pertussis toxin inhibits HDL3 stimulated DAG production. Treatment of platelet membranes with HDL3 or with proteoliposome containing apo A-I or A-II substantially prevents 41 kDa protein ADP-ribosylation that was induced by pertussis toxin, with apo A-II having an inhibitory potency greater than apo A-I. These data provide strong evidence that the pertussis sensible G protein (Go or Gi) is directly involved in coupling PLC to HDL3 receptor in platelets.
Asunto(s)
Plaquetas/metabolismo , Proteínas Portadoras , Proteínas de Unión al GTP/fisiología , Lipoproteínas HDL , Toxina del Pertussis , Fosfatidilcolinas/metabolismo , Proteínas de Unión al ARN , Receptores de Superficie Celular/metabolismo , Receptores de Lipoproteína , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacología , Apolipoproteína A-I/farmacología , Apolipoproteína A-II/farmacología , Diglicéridos/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/farmacología , HumanosRESUMEN
In vitro and in vivo data have indicated that tumor cells actively internalize the low-density lipoprotein (LDL) from the circulation. A family of 2-(aminomethyl) acrylophenones (AMA) possesses an in vitro antileukemic activity but is devoid of any in vivo antineoplastic activity, because the compounds are actively captured by proteins in solution in the blood. In order to achieve a selective delivery of these drugs via the LDL pathway, we have incorporated an AMA drug, 2-morpholinomethyl-2',3',4'- trimethoxy acrylophenone hydrochloride (ILE) into LDL particles. ILE spontaneously associated with LDL to produce an LDL-ILE complex containing 200 +/- 100 molecules of drug per LDL particle. The LDL-ILE complex was highly electronegative as detected by electrophoresis. Further, this complex presented an immunologically detected over expression of the ligand-binding domain to the LDL receptor. In spite of these modifications, the LDL receptor processing bound, internalized, and degraded the LDL-ILE complex. Nevertheless, these biological properties were reduced by 32, 20, and 40%, respectively, in comparison to native LDL. Despite its high electronegativity, the LDL-ILE complex was not recognized by the macrophagic scavenger receptor. The LDL-ILE complex showed specific LDL receptor mediated in vitro cytotoxicity as judged from the growth inhibition of neoplastic A549 cells and of normal fibroblasts, but no activity on defective LDL receptor cells. Further, the pharmacological activity of the complex against A549 cells has been demonstrated to be equally potent as that of the free drug (median inhibitory dose, 5 microM). It is suggested that LDL drug targeting of AMA molecules could specifically deliver active molecules to cancer cells, avoiding their entrapment by other blood proteins and their rapid clearance by the reticuloendothelial system.
Asunto(s)
Antineoplásicos/administración & dosificación , Lipoproteínas LDL/administración & dosificación , Morfolinas/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antineoplásicos/farmacología , Portadores de Fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Morfolinas/farmacología , Receptores de LDL/metabolismoRESUMEN
Theromyzon tessulatum vitellin was identified as a lipoglycoprotein of 490 kDa. The insolubility of this molecule in low-ionic-strength media was used to extract it from the ovaries. Antiserum prepared against vitellin was shown to react with a coelomic fluid component of 520 kDa. This vitellin precursor, or vitellogenin, was purified by gel permeation and ion-exchange column chromatography. These two lipoglycoproteins were characterized by amino acid, carbohydrate and lipid analysis and subunit composition. In spite of differences in terms of native molecular mass, solubility and isoelectric point, the lipoglycoproteins isolated from the coelomic fluid and the ovary were similar in their subunit components (a single polypeptide of 165 kDa) and in their amino acid and carbohydrate compositions. However, vitellogenin was found to be more highly lipidated (31.8% by mass) than vitellin (24% by mass) and lipid analysis indicated a higher amount of sterols and phospholipids in vitellogenin. From these data, we conclude that vitellogenin and vitellin are probably dimers of two identical subunit polypeptides plus lipid and that, after vitellogenin is sequestered in the oocyte, part of its lipid component is stripped from the molecule to give vitellin. Furthermore, electrophoretic analysis seems to indicate that vitellogenin synthesis and secretion is initiated following the third and last blood meal of the animal but that vitellogenin significantly accumulates in the coelomic fluid before being incorporated in the oocytes suggesting a complex mode of vitellogenesis regulation.
Asunto(s)
Proteínas del Huevo/análisis , Sanguijuelas/química , Vitelogeninas/análisis , Aminoácidos/análisis , Animales , Carbohidratos/análisis , Proteínas del Huevo/química , Proteínas del Huevo/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Sanguijuelas/crecimiento & desarrollo , Sanguijuelas/metabolismo , Lípidos/análisis , Peso Molecular , Concentración Osmolar , Solubilidad , Vitelogeninas/química , Vitelogeninas/metabolismoRESUMEN
Apolipoprotein A-I (apo A-I)*/DMPC complexes have been previously shown to promote cholesterol efflux from cholesterol-preloaded adipose cells whereas apo A-II/DMPC complexes, which bind to the same cell surface binding sites, were ineffective. Addition of apo A-I/DMPC complexes led to a rapid and transient formation of diacylglycerol. However, in contrast to PGF2 alpha (Doglio et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 1148), no accumulation of inositol phosphates was observed. Apo A-II/DMPC complexes had no effect on diacylglycerol formation. Stimulation by apo A-I/DMPC complexes or native HDL3 of cells prelabelled with (2-palmitoyl 9,10[3H])phosphatidylcholine induced also the formation of labelled diacylglycerol whereas apo A-II/DMPC complexes and HDL3 treated with tetranitromethane showed no effect. Direct activation of protein kinase C(s) by PMA promoted cholesterol efflux providing that DMPC liposomes were present as cholesterol acceptor. It is proposed that lipoprotein particles have two separate effects, i.e. a ligand-induced effect leading to cholesterol translocation from intracellular stores to the cell surface and a bilayer-induced effect allowing cholesterol efflux from the cell surface to the acceptor.
Asunto(s)
Tejido Adiposo/metabolismo , Colesterol/metabolismo , Diglicéridos/metabolismo , Proteína Quinasa C/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/enzimología , Apolipoproteínas/sangre , Células Cultivadas , Dimiristoilfosfatidilcolina/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Cinética , Membrana Dobles de Lípidos/metabolismo , Ésteres del Forbol/farmacologíaRESUMEN
Low concentrations of HDL3 stimulate a transient biphasic increase in 1,2-diacylglycerol (DAG), with an early phase peaking at 30 seconds and a late phase at 60 seconds in (3H)-phosphatidylcholine prelabelled platelets. DAG generation is coupled to apolipoprotein AII or AI binding to specific surface receptors. Coincubations with HDL3 and 0.2 microM phorbol ester induced a significant rise in the second phase DAG indicating the involvement of protein kinase C in this late phase. The HDL3 induced production of DAG in platelets pretreated with 6 microM R 59022 is enhanced, while phosphatidic acid (PA) content was reduced, suggesting that DAG attenuation is derived at least in part from a pathway involving DAG-Kinase.
Asunto(s)
Plaquetas/metabolismo , Lipoproteínas HDL/fisiología , Fosfatidilcolinas/sangre , Plaquetas/efectos de los fármacos , Diacilglicerol Quinasa , Diglicéridos/biosíntesis , Humanos , Técnicas In Vitro , Lipoproteínas HDL/aislamiento & purificación , Lipoproteínas HDL3 , Ácidos Fosfatidicos/farmacología , Fosfotransferasas/metabolismo , Proteína Quinasa C/farmacología , Esfingosina/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [3H] palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [14C] palmitic acid, 2mM ATP, 50 microM CoA and 10 mM MgCl2 and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [3H] cerebrosides and [14C] palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37 degrees C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80 degrees C. Omission of ATP, CoA, MgCl2 or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.
Asunto(s)
Encéfalo/metabolismo , Cerebrósidos/metabolismo , Galactosilceramidas/metabolismo , Vaina de Mielina/metabolismo , Animales , Técnicas In Vitro , RatasRESUMEN
The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.
Asunto(s)
Encéfalo/irrigación sanguínea , Endotelio Vascular/ultraestructura , Uniones Intercelulares/ultraestructura , Monoaminooxidasa/metabolismo , Animales , Antígenos , Capilares , Bovinos , Células Cultivadas , Circulación Cerebrovascular , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Epoprostenol/biosíntesis , Factor VII/análisis , Factor VII/inmunología , Microscopía Electrónica , Peptidil-Dipeptidasa A/metabolismo , Factores de Tiempo , gamma-Glutamiltransferasa/metabolismo , Factor de von WillebrandRESUMEN
High-density lipoprotein 3 (HDL3) binds to capillary endothelial cells when their lumen surfaces are exposed to 125I-HDL3 by post-mortem perfusion of whole brain. Kinetic studies of binding of HDL3 to isolated membranes show that HDL3 binds only to endothelial membranes with high affinity (Kd = 7 micrograms/ml). Trypsin treatment of membranes abolishes HDL3 binding. High-affinity binding sites for HDL3 were recovered when endothelial cells from bovine brain capillaries were maintained in culture (Kd = 13 micrograms/ml HDL3 protein). The characteristics of the binding were preserved up to the 6th passage. Competition experiments using isolated luminal membranes or cultured endothelial cells indicate that only HDL3 and not LDL or methylated LDL, are able to compete binding of 125I-HDL3. Furthermore, the inhibition of 125I-HDL3 binding by lipoprotein A-I and lipoprotein A-I:A-II strongly suggests that apolipoprotein A-I is implicated in the formation of HDL3-receptor complexes. The binding is increased by loading cells with free cholesterol or LDL cholesterol. In addition, surface-bound 125I-HDL3 remains sensitive to mild trypsin treatment after subsequent incubation of BBCE at 37 degrees C. HDL3 bound to the cell surface is not endocytosed, but rather rapidly released into the medium after binding (t1/2 = 5 min).
Asunto(s)
Proteínas Portadoras , Circulación Cerebrovascular , Endotelio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Unión al ARN , Receptores de Superficie Celular/metabolismo , Receptores de Lipoproteína , Unión Competitiva , Capilares , Células Cultivadas , Cloroquina/farmacología , Humanos , Cinética , Lipoproteínas HDL3 , Receptores de Superficie Celular/fisiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The presence of lipoproteins, apolipoproteins, and their receptors in the brain could provide a system for cholesterol homeostasis, as they do in other tissues. This study was undertaken to determine whether plasma low-density lipoprotein, the major carrier of cholesterol, is involved in the delivery of lipids through the blood-brain barrier. 125I-Labeled low-density lipoprotein bound to a specific receptor on the endothelium of brain capillaries when it was injected immediately postmortem into bovine brain circulation. In contrast, no specific binding of 125I-low density lipoprotein was found when the incubations were performed with isolated capillaries. Incubations of endothelial or basement membranes of brain capillaries with 125I-low density lipoprotein demonstrated a high-affinity association of low-density lipoprotein with the membranes of bovine cerebral endothelial cells. The specificity of the low-density lipoprotein binding was determined in several ways using a dot blot assay. This receptor shows the same characteristics as the low-density lipoprotein receptor on human fibroblasts. The molecular weight of the bovine brain capillary low-density lipoprotein receptor (132,000) was determined by ligand blotting. These results demonstrated the occurrence of a low-density lipoprotein receptor on the endothelial cells of brain capillaries.
Asunto(s)
Encéfalo/metabolismo , Capilares/metabolismo , Endotelio Vascular/metabolismo , Receptores de LDL/metabolismo , Animales , Membrana Basal/metabolismo , Encéfalo/irrigación sanguínea , Bovinos , Circulación Cerebrovascular , Endotelio Vascular/citología , Fibroblastos/metabolismo , Técnicas In Vitro , Lipoproteínas LDL/metabolismoRESUMEN
Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their Rf values into two main fractions which comigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography-mass spectrometry analysis of both ester- and amide-linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.
Asunto(s)
Ceramidas/metabolismo , Ratones Mutantes Neurológicos/metabolismo , Vaina de Mielina/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Ratones , Ratones Mutantes Neurológicos/crecimiento & desarrollo , Vaina de Mielina/fisiología , Valores de Referencia , Nervio Ciático/crecimiento & desarrollo , Nervio Ciático/metabolismoRESUMEN
Electron microscopic examination of the interaction of gold labelled HDL with platelets indicates that the internalized lipoprotein becomes closely associated with surface connected canaliculae and endocytic vacuoles. At the same time granule centralization occurs. Using fluorescent derivatives of naturally occurring lipids we have further investigated lipid exchange between HDL and platelets. Analogs of phosphatidylcholine containing fluorescent fatty acids are rapidly transferred from the lipoproteins to the cells and remain at the plasma membrane as long as they are kept at 4 degrees C. However when the platelets are warmed to 37 degrees C, a rapid degradation of the fluorescent lipids occurs, generating fluorescent diacylglycerol as a consequence of the activation of platelet enzymes.
Asunto(s)
Plaquetas/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/análisis , Plaquetas/ultraestructura , Membrana Celular/metabolismo , Colchicina/farmacología , Colorantes Fluorescentes/metabolismo , Oro , Humanos , Cuerpos de Inclusión/metabolismo , Microscopía Electrónica , Fosfatidilcolinas/síntesis química , Fosfolípidos/metabolismo , Unión Proteica , TemperaturaRESUMEN
This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).