RESUMEN
Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.
Asunto(s)
Barrera Hematoencefálica , Receptores de LDL/metabolismo , Receptores de LDL/fisiología , Animales , Astrocitos , Transporte Biológico , Encéfalo/irrigación sanguínea , Capilares/metabolismo , Capilares/fisiología , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Humanos , Modelos Biológicos , RatasRESUMEN
The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.
Asunto(s)
Barrera Hematoencefálica/citología , Células Cultivadas/metabolismo , Endotelio Vascular/citología , Modelos Biológicos , Xenobióticos/farmacocinética , Animales , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Bovinos , Perros , Endotelio Vascular/metabolismo , Humanos , Ratones , Permeabilidad , Ratas , Reproducibilidad de los ResultadosRESUMEN
A major challenge in correcting disorders affecting the central nervous system is to induce blood-brain barrier (BBB) crossing of exogenous biological compounds such as proteins or specific nucleic acid sequences. Fatty acids, due to their high membrane affinity and low toxicity, are good potential candidates to promote this barrier crossing when covalently bound to proteins. In this paper, we report that regiospecific monoacylation of ribonuclease A (RNase A) enables its transport across an in vitro model of the BBB. Myristoylated, palmitoylated and stearoylated RNases A were prepared using reversed micelles as microreactors. All the purified acylated RNases A kept their original enzymatic activity. A single fatty acid moiety was linked to RNase A through the alpha-amino group of its N-terminal lysine as shown by powerful analytical techniques. The ability of monoacylated RNases A to cross an in vitro model of the BBB is strictly dependent on the acyl chain length, which must be at least 16 carbon atoms long.
Asunto(s)
Barrera Hematoencefálica/fisiología , Ribonucleasa Pancreática/farmacocinética , Acetilación , Animales , Transporte Biológico , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Cinética , Espectrometría de Masas/métodos , Micelas , Mapeo Peptídico , Ribonucleasa Pancreática/metabolismoRESUMEN
PURPOSE: The objective of the current study was to determine the ability of some antiemetic compounds to cross the blood-brain barrier (BBB) and thereby to determine possible side effects of compounds for the central nervous system (CNS). METHODS: We compared the brain penetration of some antiemetic compounds using an in vitro BBB model consisting in brain capillary endothelial cells co-cultured with primary rat glial cells. RESULTS: This study clearly demonstrated that the metopimazine metabolite, metopimazine acid, has a very low brain penetration, lower than metopimazine and even less than the other antiemetic compounds tested in this study. CONCLUSIONS: The poor brain penetration of metopimazine acid, metopimazine biodisponible form, seems very likely related to the clinically observed difference in therapeutic and safety profile.
Asunto(s)
Antieméticos/farmacocinética , Barrera Hematoencefálica/metabolismo , Clorpromazina/farmacocinética , Domperidona/farmacocinética , Ácidos Isonipecóticos/farmacocinética , Metoclopramida/farmacocinética , Animales , Animales Recién Nacidos , Antieméticos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Permeabilidad Capilar , Células Cultivadas , Clorpromazina/metabolismo , Claudina-1 , Técnicas de Cocultivo/métodos , Domperidona/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Ácidos Isonipecóticos/metabolismo , Proteínas de la Membrana/análisis , Metoclopramida/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Ocludina , Fosfoproteínas/análisis , Ratas , Sacarosa/metabolismo , Uniones Estrechas/química , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Cyclodextrins (CDs) can be envisaged to cure some diseases related to the brain, but the behavior of these compounds toward the blood-brain barrier (BBB) remains largely unexplored to envisage such clinical applications. To fulfill this gap, the toxicity and endothelial permeability for native, methylated, and hydroxypropylated alpha-, beta-, and gamma-CDs have been studied on an in vitro model of BBB. As shown by the endothelial permeability for sucrose and immunofluorescence stainings, the native CDs are the most toxic CDs (alpha- > beta- > gamma-CD). Whereas the chemical modification of beta-CD did not affect the toxicity of this CD, differences are observed for the alpha- and gamma-CD. To determine the origin of toxicity, lipid effluxes on the brain capillary endothelial cells were performed in the presence of native CDs. It was found that alpha-CD removed phospholipids and that beta-CD extracted phospholipids and cholesterol. gamma-CD was less lipid-selective than the other CDs. Finally, the endothelial permeability of each CD has been determined. Surprisingly, no structure/permeability relationship has been observed according to the nature and chemical modifications of CDs.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , alfa-Ciclodextrinas/farmacología , beta-Ciclodextrinas/farmacología , gamma-Ciclodextrinas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Permeabilidad/efectos de los fármacos , RatasRESUMEN
A cell culture model of the blood-brain barrier (BBB) consisting of a coculture of bovine brain capillary endothelial cells and rat astrocytes has been used to examine the ability of 60-nm nanoparticles with different physicochemical characteristics to cross the BBB. Neutral, anionic, and cationic nanoparticles were made from crosslinked malto-dextrins derivatized or not (neutral) with phosphates (anionic), quaternary ammoniums (cationic) ligands. Then, these particles were coated or not with a lipid bilayer made of dipalmitoyl phosphatidyl choline and cholesterol. Lipid coating of ionically charged nanoparticles was able to increase BBB crossing 3- or 4-fold compared with uncoated particles, whereas coating of neutral particles did not significantly alter their permeation characteristics across the endothelial cell monolayer. Lipid-coated nanoparticles were nontoxic toward BBB integrity, and crossed the BBB by transcytosis without any degradation. Furthermore, a 27-fold increase in albumin transport was observed when albumin had previously been loaded in the cationic lipid-coated nanoparticles. The influence of red blood cells was studied; a marked inhibition of the transport was observed, probably due to strong interaction between nanoparticles and red blood cells.
Asunto(s)
Barrera Hematoencefálica/fisiología , Microesferas , Animales , Astrocitos/metabolismo , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Endotelio/citología , Endotelio/metabolismo , Liposomas , Microscopía Fluorescente , Fosfolípidos/metabolismo , Polisacáridos , RatasRESUMEN
PURPOSE: To investigate the mechanism underlying the entry of the analgesic peptide dalargin into brain using biodegradable polybutylcyanoacrylate (PBCA) nanoparticles (NP) overcoated with polysorbate 80. METHODS: The investigations were carried out with PBCA NP and with non biodegradable polystyrene (PS) NP (200 nm diameter). Dalargin adsorption was assessed by HPLC. Its entry into the CNS in mice was evaluated using the tail-flick procedure. Locomotor activity measurements were performed to compare NP toxicities. BBB permeabilization by PBCA NP was studied in vitro using a coculture of bovine brain capillary endothelial cells and rat astrocytes. RESULTS: Dalargin loading was 11.7 microg/mg on PBCA NP and 16.5 microg/ mg on PS NP. Adding polysorbate 80 to NP led to a complete desorption. Nevertheless, dalargin associated with PBCA NP and polysorbate 80 induced a potent and prolonged analgesia, which could not be obtained using PS NP in place of PBCA NP. Locomotor activity dramatically decreased in mice dosed with PBCA NP, but not with PS NP. PBCA NP also caused occasional mortality. In vitro, PBCA NP (10 microg/ml) induced a permeabilization of the BBB model. CONCLUSIONS: A non specific permeabilization of the BBB, probably related to the toxicity of the carrier, may account for the CNS penetration of dalargin associated with PBCA NP and polysorbate 80.
Asunto(s)
Encéfalo/metabolismo , Enbucrilato/toxicidad , Excipientes/toxicidad , Polisorbatos/toxicidad , Analgésicos/administración & dosificación , Analgésicos/toxicidad , Animales , Barrera Hematoencefálica/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Enbucrilato/administración & dosificación , Endotelio/citología , Endotelio/metabolismo , Leucina Encefalina-2-Alanina/administración & dosificación , Leucina Encefalina-2-Alanina/análogos & derivados , Leucina Encefalina-2-Alanina/toxicidad , Excipientes/administración & dosificación , Masculino , Ratones , Microesferas , Actividad Motora/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Polisorbatos/administración & dosificaciónRESUMEN
PURPOSE: The objective of the current study was to investigate whether blood-brain barrier (BBB) permeability studies in vitro could be accelerated by running several compounds together in the same experiment. METHODS: To address this question, we compared the transport of six compounds run separately with the results of the same compounds run together (cocktails). RESULTS: The study clearly demonstrated that the outcome of the experiments were totally different depending on the strategy used. Furthermore, the study highlights the importance of having the resistance to drug transport offered by filters without cells under control, as the filter membrane itself can be the rate-limiting step for some compounds; in addition, there is always a potential risk of interactions between molecules in cocktails as well as drug-drug interaction at the level of BBB transporters. In this study, the presence of several P-glycoprotein substrates in the drug cocktail was found to cause breakdown of the BBB. CONCLUSIONS: The results demonstrate that unless a strategy that involves running several compounds in the same experiment is properly validated, the results are of little predictive value.
Asunto(s)
Barrera Hematoencefálica/fisiología , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos/métodos , Preparaciones Farmacéuticas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico Activo , Células Cultivadas , Diseño de Fármacos , Endotelio Vascular/metabolismo , Microscopía Fluorescente , Neuroglía/metabolismo , Ratas , SacarosaRESUMEN
Tetanus neurotoxin reaches the CNS by axonal retrograde transport and thus becomes inaccessible to current treatments. A possible strategy to improve current therapy for tetanus disease would be the vectorization of Fab'2 fragments, allowing their delivery into the CNS. The purpose of this study was to investigate whether after cationization anti-tetanus Fab'2 fragments are able to cross the blood-brain barrier, the first obstacle to CNS delivery. We used primary cocultures of bovine brain capillary endothelial cells and newborn rat astrocytes as an in vitro model to study the binding and transport of cationized Fab'2 (cFab'2) fragments across the brain endothelium. We first show that cationization does not alter Fab'2 affinity for tetanus toxin. Then we demonstrate that after cationization Fab'2 fragments are able to bind to the negative charges on the surface of endothelial cells and subsequently to be transported across the endothelial cell monolayer without any modification of affinity. Finally, using fluorescence microscopy, we show that cFab'2 fragments are transported through endocytotic vesicles. The present study demonstrates that cationization allows Fab'2 directed against tetanus toxin to be transported through brain endothelium by adsorptive-mediated transcytosis. We suggest that this vectorization way could be a promising delivery strategy for carrying anti-tetanic immunoglobulin fragments across the blood-brain barrier to improve tetanus treatment.
Asunto(s)
Barrera Hematoencefálica/fisiología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Biológicos , Tétanos/inmunología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Transporte Biológico , Encéfalo/irrigación sanguínea , Cationes , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Endotelio Vascular/metabolismo , Radioisótopos de Yodo , Microscopía Fluorescente , RatasRESUMEN
Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.
Asunto(s)
Endocitosis , Lactoferrina/metabolismo , Animales , Membrana Basal/metabolismo , Barrera Hematoencefálica , Bovinos , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Unión ProteicaRESUMEN
PURPOSE: The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. METHODS: An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. RESULTS: We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. CONCLUSIONS: Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antibióticos Antineoplásicos/farmacocinética , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos/farmacología , Barrera Hematoencefálica/fisiología , Encéfalo/irrigación sanguínea , Ciclosporina/farmacocinética , Doxorrubicina/farmacocinética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inmunosupresores/farmacocinética , Piperidinas/farmacología , Triazinas/farmacología , Vincristina/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Astrocitos/citología , Astrocitos/metabolismo , Transporte Biológico/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Bovinos , Técnicas de Cocultivo , Endotelio Vascular/citología , Valor Predictivo de las Pruebas , Ratas , Verapamilo/farmacologíaRESUMEN
Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.
Asunto(s)
Barrera Hematoencefálica/fisiología , Ciclofilinas , Endotelio Vascular/metabolismo , Inmunofilinas/metabolismo , Receptores de Péptidos/fisiología , Animales , Apéndice/irrigación sanguínea , Astrocitos/fisiología , Transporte Biológico , Encéfalo/irrigación sanguínea , Capilares , Bovinos , Línea Celular , Técnicas de Cocultivo , Ciclosporina/metabolismo , Humanos , Radioisótopos de Yodo , Cinética , Isomerasa de Peptidilprolil , Venas UmbilicalesRESUMEN
The passage of either unesterified docosahexaenoic acid (DHA) or lysophosphatidylcholine-containing DHA (lysoPC-DHA) through an in vitro model of the blood-brain barrier was investigated. The model was constituted by a brain capillary endothelial cell monolayer set over the medium of an astrocyte culture. Cells were incubated for 4 h with a medium devoid of serum, then the endothelial cell medium was replaced by the same medium containing labeled DHA or lysoPC-DHA and incubations were performed for 2 h. DHA uptake by cells and its transfer to the lower medium (astrocyte medium when they were present) were measured. When the lower medium from preincubation and astrocytes were maintained during incubation, the passage of lysoPC-DHA was higher than that of unesterified DHA. The passage of both forms decreased when astrocytes were removed. The preference for lysoPC-DHA was not seen when the lower medium from preincubation was replaced by fresh medium, and was reversed when albumin was added to the lower medium. A preferential lysoPC-DHA passage also occurred after 2 h with brain endothelial cells cultured without astrocytes but not with aortic endothelial cells cultured and incubated under the same conditions. Altogether, these results suggest that the blood-brain barrier cells released components favoring the DHA transfer and exhibit a preference for lysoPC-DHA.
Asunto(s)
Barrera Hematoencefálica/fisiología , Ácidos Docosahexaenoicos/farmacocinética , Lisofosfatidilcolinas/farmacocinética , Animales , Aorta/citología , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/irrigación sanguínea , Capilares/citología , Permeabilidad Capilar/fisiología , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Ratas , TritioRESUMEN
Association between doxorubicin (DOX) and gamma-cyclodextrin (gamma-CD) or hydroxypropyl-gamma-CD (HP-gamma-CD) has been examined to increase the delivery of this antitumoral agent to the brain. The stoichiometry and the stability constant of gamma-CD or HP-gamma-CD and DOX complexes were determined in physiological medium by UV-visible spectroscopy. By using an in vitro model of the blood-brain barrier (BBB), endothelial permeability and toxicity toward the brain capillary endothelial cells of DOX, gamma-CD, and HP-gamma-CD were performed. For each CD, endothelial permeability was relatively low and a disruption of the BBB occurred at 20 microM, 20 mM, and 50 mM DOX, gamma-CD, and HP-gamma-CD, respectively. Increasing amounts of CDs were added to a fixed DOX concentration. Addition of gamma-CD or HP-gamma-CD, up to 15 and 35 mM, respectively, decreased the DOX delivery, probably due to the low complex penetration across the BBB and the decrease in free DOX concentration. Higher CD concentrations increased the DOX delivery to the brain, but this effect is due to a loss of BBB integrity. In contrast to what was observed on Caco-2 cell model with various drugs, CDs are not able to increase the delivery of DOX across our in vitro model of BBB.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Doxorrubicina/metabolismo , gamma-Ciclodextrinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/farmacología , Transporte Biológico Activo/efectos de los fármacos , Capilares/citología , Capilares/efectos de los fármacos , Capilares/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Técnicas de Cocultivo , Conductividad Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Microscopía Fluorescente , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Piperidinas/farmacología , Ratas , Espectrofotometría Ultravioleta , Triazinas/farmacologíaRESUMEN
To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n-6 and 18:3n-3 into 20:4n-6 and 22:6n-3, respectively, a coculture of the two cell types mimicking the in vivo blood-brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n-6 and 18:3n-3 (10 microM each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n-6 and 22:6n-3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum. These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.
Asunto(s)
Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Encéfalo/irrigación sanguínea , Endotelio Vascular/metabolismo , Ácidos Grasos Insaturados/metabolismo , Animales , Radioisótopos de Carbono , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Ácidos Grasos/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Fosfolípidos/metabolismoRESUMEN
The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the 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 some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.