Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling-time of about 22-31 hr, but did not grow at 39 degrees C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma-glutamyltranspeptidase activity in TR-BBBs was approximately 13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 +/- 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.
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PMID:mRna expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting. 1132 62

Well-characterised cell lines represent important tools for the study of endogenous solute or xenobiotic transport. A brain microvascular cell line, b.End3, isolated from mice transformed with the Polyoma virus middle T-antigen is available commercially. Here we report the characterisation of some features of b.End3 of relevance to its use in blood-brain barrier transport investigations. The b.End3 cells displayed a distinctive spindle-like squamous morphology in culture. Clathrin coated pits and numerous uncoated intracellular vesicles were evident within the cells, as was the expression of the vesicle-associated proteins, clathrin, caveolin-1, flotillin and dynamin II. In the presence of C6 astroglial co-culture b.End3 monolayers achieved a maximal transendothelial electrical resistance of 130 Omega cm2, but lacked real discrimination with respect to the permeation of transcellular and paracellular probes, e.g. permeability coefficients (x 10(-6) cm s(-1)) for propranolol of approximately 23 vs. 16 for sucrose. RT-PCR analysis confirmed the presence within the b.End3 cells of mRNA transcripts for the following transporters: GLUT-1; MCT 1 and 2; OAT1; Oatp1; mdr 1a and 1b; MRP 1 and 5; beta-alanine, system L and system y+L amino acid carriers; the nucleoside transporters cNT1 and 2, eNT1 and 2, and the tight junctional elements, ZO-1, JAM, occludin, claudin-1 and -5. The b.End3 cells actively accumulated D-glucose in a sodium-independent manner with characteristics consistant with that of GLUT-1. Functionality for P-glycoprotein efflux was evident as assessed by a rhodamine-123 accumulation and retention assay. The system L LAT1/4F2hc amino acid transporter was examined through uptake of L-leucine and L-phenylalanine and provided Km and Vmax values of approximately 16 microM and 350-480 pmol/mg protein/10 min, respectively; the affinity of transport for these substrates being weaker, approximately threefold, when the b.End3 cells were grown in the presence of C6 astroglial factors. Although the b.End3 cells appear unsuitable for transendothelial permeability assessments they display characteristics that would allow their worthwhile use in studies addressing blood-brain barrier transport mechanisms.
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PMID:Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. 1456 34

We previously found that the production of adrenomedullin (AM) is one magnitude higher in cerebral endothelial cells (CECs) than in the peripheral endothelium and the AM concentration in the cerebral circulation is significantly higher than in other tested parts of the circulation. We also showed that CECs express AM receptors, and AM as an autocrine hormone is important to regulate the intracellular cAMP level in CECs. Further we reported that acute AM treatment has cAMP-like effects on specific BBB functions: AM decreased endothelial fluid phase endocytosis, activated the P-glycoprotein, increased transendothelial electrical resistance (TEER) and reduced endothelial permeability for sodium fluorescein, which suggests a tightening of intercellular junctions. In the present study, we found chronic AM exposure also increased TEER. In contrast, we could not detect significant effect of AM on the expression of tight junction proteins (claudin-1, occludin and zonula occludens-1). While not affecting expression of tight junction proteins, chronic AM treatment may influence the localization of these proteins which has been reported to correlate with functional changes of the BBB without a change in protein expression.
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PMID:Chronic adrenomedullin treatment improves blood-brain barrier function but has no effects on expression of tight junction proteins. 1475 7

Poly(MePEG2000cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to reach the rat central nervous system after intravenous injection. For insight into the transport of colloidal systems across the blood-brain barrier (BBB), we developed a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells (RBECs) and rat astrocytes. The RBECs used in our model displayed and retained structural characteristics of brain endothelial cells, such as expression of P-glycoprotein, occludin and ZO-1, and immunofluorescence studies showed the specific localization of occludin and ZO1. The high values of transendothelial electrical resistance and low permeability coefficients of marker molecules demonstrated the functionality of this model. The comparative passage of polyhexadecylcyanoacrylate and PEG-PHDCA nanoparticles through this model was investigated, showing a higher passage of PEGylated nanoparticles, presumably by endocytosis. This result was confirmed by confocal microscopy. Thanks to a good in vitro/in vivo correlation, this rat BBB model will help in understanding the mechanisms of nanoparticle translocation and in designing new types of colloidal carriers as brain delivery systems.
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PMID:A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles. 1590 57

Miltefosine (hexadecylphosphocholine, HePC) is the first effective oral agent for the treatment of visceral leishmaniasis. This study aimed to determine whether this oral administration alters the integrity and transport capacities of the intestinal barrier. The objectives of this study were: (i) to evaluate the cytotoxicity of HePC, (ii) to investigate the effects of HePC on paracellular and transcellular transport and (iii) to investigate the influence of HePC on three major transporters of the intestinal barrier, namely, P-glycoprotein, the human intestinal peptide transporter (PepT-1) and the monocarboxylic acid transporter (MCT-1) in Caco-2 cell monolayers, used as an in vitro model of the human intestinal barrier. We show that HePC reduced the transepithelial electrical resistance and increased D-[14C]mannitol permeability in a dose-dependent manner but had no effect on [3H]testosterone permeability, demonstrating that HePC treatment enhances paracellular permeability via an opening of the tight junction complex without affecting the transcellular route. Morphological studies using confocal fluorescence microscopy showed no perturbation of the normal distribution of ZO-1, occludin or E-cadherin but revealed a redistribution of the tight junction-associated protein claudin-1 and the perijunctional actin after incubation with HePC. Finally, HePC was found to inhibit the intestinal P-glycoprotein in the Caco-2 cell model after a single short exposure. These results suggest that HePC could modify the oral bioavailability of other therapeutic compounds absorbed via the paracellular route or which are substrates of the intestinal P-glycoprotein.
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PMID:Modulation of intestinal barrier properties by miltefosine. 1633 52

The isolation and culture of spinal cord microvascular endothelial cells (SCMEC), which form the blood-spinal cord barrier (BSCB), is described. Though morphologically similar to brain microvascular endothelial cells (BMEC) that form the blood-brain barrier (BBB), SCMEC express reduced amounts of several prominent BBB proteins, including tight junction-associated proteins ZO-1 and occludin, adherens junction-associated proteins beta-catenin and VE-cadherin, and the efflux transporter P-glycoprotein. These distinguishing features may reflect more widespread differences between the BBB and BSCB that impact physiological and pathophysiological processes.
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PMID:Isolation and culture of microvascular endothelial cells from murine spinal cord. 1680 99

The blood-brain barrier (BBB) impedes the influx of intravascular compounds from the blood to the brain. The elements composing the BBB are endothelial cells, pericytes and the end-feet of astrocytes. Among them, the endothelial cell barrier line is the most critical for preventing toxic substances from entering the brain. In this review, we focus on the ultrastructural distribution of important components in the intracellular junction and cytoplasm of brain endothelial cells. The ultrastructural distribution of tight junction-specific integral membrane proteins such as occludin, junctional adhesion molecules, claudin, peripheral zonula occludens protein-1 (ZO-1), adherens junction-specific transmembrane protein cadherin, and adherens junction-associated peripheral proteins alpha-catenin, beta-catenin, and p120 catenin is reviewed. P-glycoprotein and some other transporters recently discovered in endothelial cells prevent several compounds from entering the brain parenchyma. It is likely that the transient inhibition of P-glycoprotein by antidepressants enables other medicines to enter the brain. Vesicular transport with clathrin-mediated or adsorptive endocytosis through endothelial cells is also critical for transportation of blood-born substances from the bloodstream to the brain. How medicines pass the BBB to reach the brain parenchyma is discussed.
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PMID:Molecular anatomy of the brain endothelial barrier: an overview of the distributional features. 1750 40

The purpose of the study was to assess the suitability of the mouse endothelial cell line bEnd5 as a blood-brain barrier (BBB) model under normal or pathologic (stroke) conditions. In comparison to the well-established bovine brain endothelial cell (BBMEC) model, cultured bEnd5 monolayers reached a maximal transendothelial electrical resistance (TEER) of 121 Omega cm(2) on day 7, and possessed oval and spindle shape morphology. Structurally, confluent monolayers of bEnd5 cells and BBMECs exhibit peripheral band staining of the tight junction protein ZO-1 and occludin. Both bEnd5 and BBMECs express important tight junctional proteins, ZO-1, occludin and claudin-1, as well as the transporters P-glycoprotein (P-gp), NKCC, GLUT1, and most PKC isoforms. Marker permeability experiments suggest that bEnd5 cells form a tight barrier that compares to well-established in vitro BBB models, such as the BBMEC. After short durations of hypoxia/aglycemia (H/A), hyperpermeability was seen in the bEnd5 endothelial monolayer compared to later time periods for BBMECs, suggesting that bEnd5 cells are more sensitive to hypoxia/algycemia treatment than BBMECs. Taken together, bEnd5 cell culture model may provide a useful in vitro model of the BBB for drug delivery studies and modeling pathological states such as oxygen glucose deprivation associated with stroke.
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PMID:Evaluation of bEnd5 cell line as an in vitro model for the blood-brain barrier under normal and hypoxic/aglycemic conditions. 1782 43

In-vitro models that maintain complex transport mechanisms and structural properties associated with the blood-brain barrier in vivo would be useful in drug permeability and neurotoxicological studies. To evaluate the suitability of a human retinal pigment epithelial cell line for a blood-brain barrier model, we have compared the barrier properties of the human retinal pigment epithelial cell line ARPE-19, the human colonic adenocarcinoma cell line Caco-2, and primary porcine microvessel endothelial cells. The tight junction proteins occludin and ZO-1 were stained immunocytochemically. The paracellular ionic permeability was evaluated by measuring the trans-epithelial or trans-endothelial electric resistance. To evaluate the active transport mechanisms, the existence and the activity of the efflux transporters, P-glycoprotein and multidrug resistance-associated proteins, were studied. All the cell types in this study stained positively for occludin and ZO-1. However, the trans-endothelial electric resistance of ARPE-19 cells was low compared with that of primary porcine microvessel endothelial cell and Caco-2 cells. In addition, both the P-glycoprotein expression and its activity in ARPE-19 cells were low. In conclusion, the barrier properties of the human ARPE-19 cell line were not satisfactory for a blood-brain barrier model. For future studies, it is important to develop a human brain endothelial cell line with expression of the complex in-vivo properties of the blood-brain barrier.
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PMID:Evaluation of the selected barrier properties of retinal pigment epithelial cell line ARPE-19 for an in-vitro blood-brain barrier model. 1904 59

Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.
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PMID:A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. 1911 69


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