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)

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

Brain capillaries contain a great variety of membrane proteins involved in the transport of hydrophilic nutrients or in the reception of hormonal signals. The use of Triton X-114 fractionation to purify membrane proteins according to their degree of hydrophobicity was investigated. Analysis by polyacrylamide gel electrophoresis showed a distinct polypeptide composition for each fraction. Most of the proteins (68%) were solubilized by Triton X-114 and, of these proteins, the majority (74%) was found in the detergent-poor phase. Alkaline phosphatase which possesses a glycosyl-phosphatidylinositol anchor partitioned in the pellet of insoluble proteins where it was enriched 2.3-fold. In contrast, gamma-glutamyltranspeptidase, the GLUT1 glucose transporter and P-glycoprotein, three integral membrane proteins, and p21ras and a 42 kDa G protein alpha subunit, both covalently modified by lipids, were efficiently solubilized and fractionated in the detergent-rich fraction where they were enriched 3.5-, 4.8-, 4.4-, 4.5- and 4.7-fold, respectively. Triton X-114 fractionation could therefore be used as a first step in the purification of many blood-brain barrier membrane proteins.
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PMID:Extraction of brain capillary membrane proteins using Triton X-114. 769 79

The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3.
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PMID:Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3. 901 20

The hypothesis that P-glycoprotein plays a functional role at the brain capillary endothelium, which makes up the blood-brain barrier in vivo, is based largely on immunocytochemical studies showing immunoreactive P-glycoprotein localized to either isolated brain microvessels or microvessels within tissue sections. The present studies use the MRK16 monoclonal antibody to human P-glycoprotein to demonstrate that the pattern of immunolocalization of P-glycoprotein in microvessels of human or primate brain is similar to the pattern of immunolocalization of an astrocyte protein, glial fibrillary acidic protein. In contrast, the discontinuous staining pattern of MRK16 is not colocalized with the continuous immunostaining of the brain endothelial GLUT1 glucose transporter. The MRK16 antibody was radiolabeled with [125I]-iodine, and 125I-MRK16 avidly bound isolated human brain capillaries via a saturable mechanism. However, the 125I-MRK16 antibody was not taken up by primate brain capillaries in vivo following intravenous injection. In conclusion, these studies provide evidence that P-glycoprotein does not play a functional role at the luminal membrane of the brain capillary endothelium in vivo, and that a principal site of immunoreactive P-glycoprotein in brain microvasculature is localized to astrocyte foot processes.
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PMID:Brain microvascular and astrocyte localization of P-glycoprotein. 904 75

Luminal membranes of the vascular endothelium were isolated from brain, heart and lungs by modification of their density. The presence of P-glycoprotein (P-gp) was detected by Western blotting in luminal membranes from the endothelium of the three tissues. Strong enrichment in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400-500-fold), indicates that P-gp is mainly located on the luminal side of the brain endothelium. Western blotting was also performed with antibodies directed against GLUT1, glial fibrillary acidic protein, adaptin, IP3R-3, integrins alphav and collagen IV as controls to determine whether the preparations were contaminated by other membranes. Strong enrichment of GLUT1 in brain capillary luminal membranes (9.9-fold) showed that the preparation consisted mainly of endothelial cell plasma membranes. Poor enrichment of glial fibrillary acidic protein (1.4-fold) and adaptin (2.4-fold) and a decreased level of IP3R-3, integrins alphav and collagen IV excludes the possibility of major contamination by astrocytes or internal and anti-luminal membranes. High levels of P-gp in the luminal membranes of brain capillary endothelial cells suggests that it may play an important role in limiting the access of anti-cancer drugs to the brain.
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PMID:P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. 929 Nov 29

Sites of immunoreactive P-glycoprotein associated with human brain microvasculature were identified by labeling of unfixed isolated human brain capillaries, allowing visualization of the three-dimensional capillary structure by confocal microscopy. Capillaries isolated from human autopsy brain were dual-labeled with the MRK16 mouse monoclonal antibody (against human P-glycoprotein) and rabbit polyclonal antisera against the human brain microvascular glucose transporter (GLUT1), or glial fibrillary acidic protein (GFAP) on astrocyte foot processes. MRK16 and GLUT1 dual-labeling showed no signal overlap, identical to the staining pattern observed for dual-labeling with anti-GFAP and anti-GLUT1 antibodies: both GFAP and MRK16 labeling were discrete, discontinuous, and not co-localized with continuous GLUT1 labeling of capillary endothelium. In contrast, complete overlap of MRK16 and GFAP labeling demonstrated P-glycoprotein localization on astrocyte foot process remnants at the abluminal face of the brain microvasculature.
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PMID:P-Glycoprotein on astrocyte foot processes of unfixed isolated human brain capillaries. 1008 69

1. P-Glycoprotein is a 170-kDa transmembrane glycoprotein active efflux system that confers multidrug resistance in tumors, as well as normal tissues including brain. 2. The classical model of multidrug resistance in brain places the expression of P-glycoprotein at the luminal membrane of the brain microvascular endothelial cell. However, recent studies have been performed with human brain microvessels and double-labeling confocal microscopy using (a) the MRK16 antibody to human P-glycoprotein, (b) an antiserum to glial fibrillary acidic protein (GFAP), an astrocyte foot process marker, or (c) an antiserum to the GLUT1 glucose transporter, a brain endothelial plasma membrane marker. These results provide evidence for a revised model of P-glycoprotein function at the brain microvasculature. In human brain capillaries, there is colocalization of immunoreactive P-glycoprotein with astrocytic GFAP but not with endothelial GLUT1 glucose transporter. 3. In the revised model of multidrug resistance in brain, P-glycoprotein is hypothesized to function at the plasma membrane of astrocyte foot processes. These astrocyte foot processes invest the brain microvascular endothelium but are located behind the blood-brain barrier in vivo, which is formed by the brain capillary endothelial plasma membrane. 4. In the classical model, an inhibition of endothelial P-glycoprotein would result in both an increase in the blood-brain barrier permeability to a given drug substrate of P-glycoprotein and an increase in the brain volume of distribution (VD) of the drug. However, in the revised model of P-glycoprotein function in brain, which positions this protein transporter at the astrocyte foot process, an inhibition of P-glycoprotein would result in no increase in blood-brain barrier permeability, per se, but only an increase in the VD in brain of P-glycoprotein substrates.
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PMID:Brain microvascular P-glycoprotein and a revised model of multidrug resistance in brain. 1069 8

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.
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PMID:Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions. 1121 75

Analyses of specific interactions between solutes and a membrane protein can serve to characterize the protein. Frontal affinity chromatography of an interactant on a column containing the membrane protein immobilized in a lipid environment is a simple and robust approach for series of experiments with particular protein molecules. Regression analysis of the retention volumes at a series of interactant concentrations shows the affinity of the protein for the interactant and the amount of active binding sites. The higher the affinity, the fewer sites are required to give sufficient retention. Competition experiments provide the affinities of even weakly binding solutes and the non-specific retention of the primary interactant. Hummel and Dreyer size-exclusion chromatography allows complementary analyses of non-immobilized membrane materials. Analyses of the human facilitative glucose transporter GLUT1 by use of the inhibitor cytochalasin B (radioactively labeled) and the competitive substrate D-glucose (non-labeled) showed that GLUT1 interconverted between two states, exhibiting one or two cytochalasin B-binding sites per two GLUTI monomers, dependent on the membrane composition and environment. Similar analyses of a nucleoside transporter, a photosynthetic reaction center, nicotinic acetylcholine receptors and a P-glycoprotein, alternative techniques, and immobilized-liposome chromatographic approaches are presented briefly.
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PMID:Immobilized-biomembrane affinity chromatography for binding studies of membrane proteins. 1193 56

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


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