Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.
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PMID:A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs. 167 25

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.
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PMID:SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. 898 3

Vascular endothelial cells (EC) exhibit organ-to-organ heterogeneity in their functions and morphologies. In particular, brain capillary EC have unique characteristics exemplified by the blood-brain barrier (BBB). The formation and the maintenance of BBB have been ascribed to EC responses to inductive signal(s) or factor(s) from astrocytes that encircle microvessels in the central nervous system. These EC responses were demonstrated in numerous in vivo studies, exemplified by those of Janzer and Raff (Nature 325:253, 1987) and Tout et al. (Neuroscience 55:291, 1993) showing that transplanted astrocytes induced BBB properties in non-neural vascular EC. In this study, we constructed a heterologous co-culture system, in which rat fetal brain astrocytes were cultivated on one surface of a porous membrane and human umbilical vein EC on the opposite surface. Electron microscopic examination revealed that astrocytes passed their endfeet through the pores, making contact with EC. In this system, gamma-glutamyltranspeptidase (gamma-GTP) activity in EC was found to be significantly increased by contacting astrocytes in a density- and time-dependent manner, but not when the astrocyte feeder layer was apart from EC or replaced by COS cells; astrocyte-derived extracellular matrix partially activated gamma-GTP. mRNAs for some of the representative BBB markers, including transferrin receptor, P-glycoprotein, brain-type glucose transporter (GLUT-1), and gamma-GTP were also demonstrated by reverse transcription-polymerase chain reaction to be upregulated in EC co-cultured with astrocytes. Astrocyte inductions of close membrane apposition resembling a zonula occludens and of an increase in the content of mitochondria in EC were also noted in electron micrographs. Furthermore, an increased barrier activity against inulin was conferred on EC when they were lined with astrocytes. The results obtained with this heterologous co-culture system thus indicate that through contact with their feet, astrocytes are capable of transdifferentiating non-neural EC into the brain type, endowing them with the BBB properties.
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PMID:Induction of various blood-brain barrier properties in non-neural endothelial cells by close apposition to co-cultured astrocytes. 898 64

We have previously shown that the hypersensitivity of KB MDR cells to 2-deoxy-D-glucose is associated with diminished plasma membrane GLUT-1 levels compared with parental, non-MDR cells. Here we report that MDR cells are hypersensitive to the N-linked glycosylation inhibitor tunicamycin, which induces partial inhibition of GLUT-1 glycosylation and diminishes GLUT-1-mediated transport. The effect of tunicamycin, which also enhances the hypersensitivity of MDR cells to 2-deoxy-D-glucose, could not be attributed to alterations in P-glycoprotein activity. The use of agents that act synergistically to diminish the level and activity of GLUT-1 in MDR cells may be of clinical potential.
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PMID:The human KB multidrug-resistant cell line KB-C1 is hypersensitive to inhibitors of glycosylation. 914 28

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

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37 degrees C but not at 39 degrees C. However, growth was restored when the temperature of the culture was lowered to 33 degrees C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33 degrees C, there was less of this complex at 37 degrees C and 39 degrees C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. The alkaline phosphatase and gamma-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 +/- 1.16 mmol/l. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr1a and mdr1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 micromol/l verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.
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PMID:Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene. 1174 Dec 43

The blood-brain barrier (BBB) is formed by brain endothelial cells lining the cerebral microvasculature, and is an important mechanism for protecting the brain from fluctuations in plasma composition, and from circulating agents such as neurotransmitters and xenobiotics capable of disturbing neural function. The barrier also plays an important role in the homeostatic regulation of the brain microenvironment necessary for the stable and co-ordinated activity of neurones. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of more complex tight junctions than in other capillary endothelia, and a number of specific transport and enzyme systems which regulate molecular traffic across the endothelial cells. Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT-1 glucose carrier, L1 amino acid transporter) and efflux transporters (e.g. P-glycoprotein). In addition to a role in long-term barrier induction and maintenance, astrocytes and other cells can release chemical factors that modulate endothelial permeability over a time-scale of seconds to minutes. Cell culture models, both primary and cell lines, have been used to investigate aspects of barrier induction and modulation. Conditioned medium taken from growing glial cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors. However, for some features of endothelial differentiation and induction, the extracellular matrix plays an important role. Several candidate molecules have been identified, capable of mimicking aspects of glial-mediated barrier induction of brain endothelium; these include TGFbeta, GDNF, bFGF, IL-6 and steroids. In addition, factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) have been shown to induce astrocytic differentiation. Thus endothelium and astrocytes are involved in two-way induction. Short-term modulation of brain endothelial permeability has been shown for a number of small chemical mediators produced by astrocytes and other nearby cell types. It is clear that endothelial cells are involved in both long- and short-term chemical communication with neighbouring cells, with the perivascular end feet of astrocytes being of particular importance. The role of barrier induction and modulation in normal physiology and in pathology is discussed.
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PMID:Astrocyte-endothelial interactions and blood-brain barrier permeability. 1216 30

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.
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PMID:Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier. 1295 Apr 65

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

Increased expression of the multidrug transporter P-glycoprotein (Pgp; ABCB1) has previously been found in epileptogenic brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE) as well as in the hippocampus and other limbic brain regions in the rat kainate model of TLE. Approaches to the quantification of Pgp expression have mainly been based on subjective visual estimation of the level of Pgp immunoreactivity in brain sections. In the present study, computer-assisted image analysis based on optical density (OD) measurements was used to examine immunohistochemical expression of Pgp in the kindling model of TLE. Sections from kainate-treated rats were used for comparison. Using diaminobenzidine as chromogen, Pgp was exclusively located in brain capillary endothelial cells, which was confirmed by double-labeling with an antibody against the endothelial glucose transporter (GLUT-1). After kainate-induced seizures, the intensity of endothelial Pgp staining significantly increased by 70-80% in the dentate gyrus. A significant, albeit less marked increase in Pgp expression in this area was also seen after amygdala-kindled seizures. Furthermore, Pgp was upregulated after kindling in the hilus of the dentate gyrus, the CA1 and CA3 sectors of the hippocampus, and the piriform and cerebral cortex. In kindled rats, most Pgp alterations occurred ipsilateral to the electrode in the basolateral amygdala. The data demonstrate that computer-assisted image analysis using OD is an accurate and rapid method to determine the relative amount of Pgp protein in brain sections and the effects of seizures on this multidrug transporter. The fact that Pgp overexpression in brain capillary endothelial cells occurs in two established models of difficult-to-treat TLE substantiates the notion that seizure-induced upregulation of Pgp contributes to multidrug resistance (MDR) in epilepsy.
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PMID:Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats. 1506 76


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