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)

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 aim of this study was to explore the effects of diets containing saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and omega-3 and omega-6 polyunsaturated fatty acids (omega-3 and omega-6 PUFA, respectively) on the passive and active transport properties of rat jejunum using marker compounds. Rats were fed diets supplemented with 18.4% (w/w) lipid (4 groups) or standard rat chow (1 group) for a period of 30 days. At the end of the dietary period, mucosal scrapings were taken for the determination of membrane phospholipids, and the apparent jejunal permeability of radiolabelled marker compounds was determined using modified Ussing chambers. Changes in the phospholipid content of the brush border membrane reflected the different lipid content of the diets. The passive paracellular permeability of mannitol was not significantly affected by the fatty acid composition of the diet, although there was a trend toward decreased mannitol permeability in the rats fed both the omega-3 and omega-6 PUFA diets. In comparison, the transcellular diffusion of diazepam was reduced by 20% (P < 0.05) in rats fed diets supplemented with omega-3 and omega-6 PUFA. In the lipid-fed rats, the serosal to mucosal flux of digoxin, an intestinal P-glycoprotein substrate, was reduced by 20% (P < 0.05) relative to the chow-fed group, however there were no significant differences between the different lipid groups. The active absorption of D-glucose via the Na+-dependent transport pathway was highest in the SFA, MUFA and PUFA omega-3 dietary groups, intermediate in the low-fat chow group and lowest in the PUFA omega-6 group, and was positively correlated with short-circuit current. These studies indicate that dietary fatty acid changes can result in moderate changes to the active and passive transport properties of excised rat jejunum.
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PMID:Effect of dietary fatty acids on the intestinal permeability of marker drug compounds in excised rat jejunum. 1207 97

The objectives of this study were (1) to investigate the transporter inhibition activity of three nonionic surfactants on P-glycoprotein, the human intestinal peptide transporter, and the monocarboxylic acid transporter in Caco-2 cell monolayers, and (2) to evaluate the role of membrane fluidity and protein kinase C in surfactant-induced transporter inhibition. All three surfactants inhibited P-glycoprotein (P-gp). Over a range from 0 to 1 mM, Tween 80 and Cremophor EL increased apical-to-basolateral permeability (AP-BL) and decreased basolateral-to-apical (BL-AP) permeability of the P-gp substrate rhodamine 123. Vitamin E TPGS's effect was equally large, but essentially only reduced the BL-AP permeability of rhodamine 123, and did so at a vitamin E TPGS concentration of only 0.025 mM. These P-gp inhibition effects would appear to be related to these excipients' modulation of membrane fluidity, where Tween 80 and Cremophor EL fluidized cell lipid bilayers, while vitamin E TPGS rigidized lipid bilayers. However, among the three surfactants, only Tween 80 inhibited the peptide transporter, as measured by glycyl sarcosine permeability. Likewise, only Cremophor EL inhibited the monocarboxylic acid transporter, as measured by benzoic acid permeability. Nevertheless, at least one of these three surfactants inhibited each P-gp, the human intestinal peptide transporter, and the monocarboxylic acid transporter. A common functional feature of these three surfactants was their ability to modulate fluidity, although results indicate that even strong membrane fluidity modulation alone was not sufficient to reduce transporter activity. N-octyl glucoside, a nonionic surfactant that did not modulate membrane fluidity, did not affect transporter functioning. Protein kinase C inhibitors failed to affect rhodamine 123 and glycyl sarcosine permeability, suggesting protein kinase C inhibition was not the mechanism of transporter inhibition. These results suggest that surfactants can inhibit multiple transporters but that changes in membrane fluidity may not be a generalized mechanism to reduce transporter activity.
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PMID:Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers. 1220 53

This study was undertaken to examine the secretory transport of arbekacin, an aminoglycoside antibiotic, in the rat small intestine and to compare it with those in Caco-2 and LLC-PK1 cells. In vitro permeation of arbekacin was examined using an Ussing chamber technique. Serosal-to-mucosal (secretory)/mucosal-to-serosal (absorptive) permeation ratios of 0.5 mM arbekacin were 2.8 in the jejunum and 7.0 in the ileum, respectively, indicating that arbekacin permeation was highly secretory-oriented. In the ileum, the ratios became smaller with increase in arbekacin concentration applied. When D-glucose was replaced with 3-o-methyl-D-glucose in the experimental medium, the directionality of the arbekacin permeation disappeared almost completely. Absorptive permeation of arbekacin was not significantly influenced by verapamil, cyclosporin A, or probenecid. On the other hand, when gentamicin sulfate was added to the serosal medium, secretory transport of arbekacin was significantly inhibited. The results of this study strongly suggest that a specialized efflux system other than P-glycoprotein and multidrug resistance proteins was involved in the secretory transport of arbekacin in the rat intestine. There was no directionality in arbekacin permeation across Caco-2 cell monolayers, suggesting the absence or very slight expression of the secretory system for arbekacin in this cell line.
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PMID:Arbekacin is actively secreted in the rat intestine via a different efflux system from P-glycoprotein. 1279 16

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

Effects of various surfactants on the transport of rhodamine123, a P-glycoprotein (P-gp) substrate, across the isolated rat intestinal membranes were examined by an in vitro diffusion chamber system. The jejunal serosal-to-mucosal transport (Jsm) of rhodamine123 was more than threefold greater than its mucosal-to-serosal transport (Jms), suggesting that the net movement of rhodamine123 across the rat jejunum was preferentially secretory direction. There exists a regional difference in the intestinal transport of rhodamine123 and the secretory directed transport was remarkably observed in the jejunum. The Jsm/Jms ratio of rhodamine123 decreased in the presence of 0.3 mM verapamil and 10 mM sodium azide (NaN3) + 1 mM sodium fluoride (NaF), confirming that rhodamine123 might be secreted from the intestinal tissue into the lumen by a P-gp-mediated efflux system. Nonionic surfactants [0.1% Cremophor EL, Tween 80 and n-dodecyl-beta-D-maltopyranoside (LM)] reduced the Jsm/Jms ratio of rhodamine123, whereas its ratio was not influenced in the presence of 0.1% cationic surfactant (hexadecyltrimethylammonium bromide, C16TAB) and anionic surfactant (sodium dodecyl sulfate, SDS). Therefore, these findings suggested that charge of surfactants was possibly related to the action of these surfactants on the intestinal absorption of P-gp substrates. On the other hand, the transfer of rhodamine123 was not affected by the addition of Cremophor EL to the serosal side. Because the c.m.c. of Cremophor EL is 0.0095 w/v%, interactions between rhodamine123 and the micellar form of Cremophor EL may decrease the P-gp-mediated efflux of rhodamine123 at higher concentrations. In the kinetic analysis, the Vmax value (nmol/min/g wet tissue) of rhodamine123 decreased, although the Km value (mM) was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the efflux transport of rhodamine123 in a noncompetitive manner. Cremophor EL did not affect the transport of [14C]Gly-Sar and [3H]3-O-methyl-D-glucose, suggesting that the action of Cremophor EL might be P-gp specific. These findings indicated that nonionic surfactants including Cremophor EL and Tween 80 may be useful pharmaceutical excipients for inhibiting the function of P-gp, thereby increasing the intestinal absorption of various drugs, which are secreted by a P-gp-mediated efflux system in the intestine.
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PMID:Modulation of intestinal P-glycoprotein function by cremophor EL and other surfactants by an in vitro diffusion chamber method using the isolated rat intestinal membranes. 1499 25

In vitro studies demonstrated that the accumulation of 2-deoxy-D-glucose was reduced in multidrug resistant cell lines. In animal study, it has been suggested that 2-[18F]fluoro-2-deoxy-D-glucose (FDG) may be a marker for multidrug resistance (MDR). The aim of this clinical study was to compare MDR characteristics by immunohistochemical assay with FDG uptake and investigate whether FDG is a marker for MDR in patients with untreated lung cancer. Forty-seven patients with 49 untreated lung cancers, who had undergone both preoperative FDG PET imaging and thoracotomy, were enrolled in this study. Before surgery, FDG PET was performed 40 min after injection, and standardized uptake values (SUVs) were obtained. Patients were classified into low-SUV (< or = 5) and high-SUV (> 5) groups. After surgery, the expression of P-glycoprotein (Pgp) was investigated by immunohistochemistry, and the lung cancer FDG uptake was analysed for possible association with Pgp expression. The strong intensity of Pgp immunoreactivity was seen only in the low-SUV group. The percentage of the Pgp positive area was significantly lower in the high-SUV group (21.7 +/- 13.4%) than in the low-SUV group (44.1 +/- 29.7%) (P = 0.015). In the high-SUV group, the percentage of Pgp positive area did not exceed 50%. In lung adenocarcinoma, the intensity of Pgp immunoreactivity and the percentage of Pgp positive area increased with degree of cell differentiation, while FDG uptake decreased with degree of cell differentiation. Bronchioloalveolar carcinoma, in particular, showed overexpression of Pgp and modest uptake of FDG. In conclusion, Pgp expression was found to be inversely related to FDG uptake in untreated lung cancer. Pgp expression correlated with the degree of cell differentiation in adenocarcinomas, whilst FDG uptake was inversely related to cell differentiation. FDG may be an in vivo marker for MDR in patients with untreated lung cancer.
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PMID:P-glycoprotein expression is associated with FDG uptake and cell differentiation in patients with untreated lung cancer. 1506 Dec 61

Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [(3)H]genistein than K562 cells. [(3)H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [(3)H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.
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PMID:Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance. 1520 50

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3-4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.
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PMID:Effects of miltefosine on membrane permeability and accumulation of [99mTc]-hexakis-2-methoxyisobutyl isonitrile, 2-[18F]fluoro-2-deoxy-D-glucose, daunorubucin and rhodamine123 in multidrug-resistant and sensitive cells. 1578 39

Membrane vesicles from the multidrug-resistant KB-V1 and KB-C1 cell lines overexpressing P-glycoprotein (Pgp), responsible for pleiotropic chemotherapeutic agents resistance, were solubilized with octyl-glucoside (OG-EX) and further fractionated on DEAE-sepharose column with increased concentrations of NaCl. The fraction containing Pgp (F3) was reconstituted into proteoliposomes (F3-PLP). Comparisons of the phosphorylation levels of Pgp achieved throughout the purification and reconstitution steps were addressed in this study. The [delta32 P] ATP-driven phosphorylation of Pgp was strongly increased in OG-EX, decreased in F3 and not detected in F3-PLP, when compared to Pgp phosphorylation in native plasma membrane vesicles. [delta32 P]ATP-phosphorylation of Pgp in F3-PLP could be restored by exogenously added PKC or by the catalytic sub-unit of PKA. The vanadate-induced hyperphosphorylation effect on Pgp by [delta32 P]ATP observed with plasma membrane vesicles was maintained in OG-EX, but was lost in F3 and did not enable labelling in F3-PLP. Enhancement of [delta32 P]-labelling of native Pgp via [delta32 P]ATP combined with GTP was maintained and also triggered phosphorylation of purified/reconstituted Pgp in F3-PLP as well. Altogether, our data suggest differential phosphorylation patterns of the transporter linked to environmental molecular composition (lipids, presence of detergent) and structure (unfolded versus embedded). In addition, restoration by GTP of Pgp phosphorylation by [delta32 P]ATP in the frame of F3-PLP suggests intra-molecular modulations and hints that other phosphorylation sites and processes, different from the classic ones involving PKC and/or PKA, may participate in the transporter's mechanism.
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PMID:Differential phosphorylation patterns of P-glycoprotein reconstituted into a proteoliposome system: insight into additional unconventional phosphorylation sites. 1630 79


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