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)

The pathogenesis of human immunodeficiency virus (HIV)-associated dementia has been linked to microglial responses after infection. We have recently confirmed expression of several ATP-dependent efflux transporters in microglia, namely, multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-gp). In the present study, we investigated whether cultured rat microglia express two additional MRP family members, rMRP4 and rMRP5. Using reverse transcriptase-polymerase chain reaction, rMRP4 and rMRP5 mRNA was detected in primary cultures of microglia and in a rat microglia cell line, MLS-9. Western blot analysis further confirmed protein expression of the two MRP isoforms in MLS-9 cells. Bis(pivaloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine [bis(POM)PMEA], a lipophilic ester prodrug of the well characterized MRP4 and 5 substrate 9-(2-phosphonylmethoxyethyl)adenine (PMEA), was chosen to examine transport characteristics in MLS-9. Using thin layer chromatography, we verified that more than 90% of radioactivity recovered in MLS-9 loaded with 1 microM [(3)H]bis(POM)PMEA for 1 h under ATP-depleting conditions was converted to PMEA. Efflux of PMEA by MLS-9 cell monolayers was ATP-dependent, glutathione-independent, and significantly inhibited by several MRP inhibitors (i.e., sulfinpyrazone, genistein, indomethacin, and probenecid) as well as the antiretroviral drug azidothymidine-monophosphate. Similar results were not observed in MRP1- or P-gp-overexpressing cell lines, suggesting that PMEA is not a substrate for either P-gp or MRP1. These studies provide further evidence that microglia express multiple subfamilies of ATP-binding cassette transporters (i.e., P-gp, MRP1, MRP4, and MRP5) that could restrict permeation of several different classes of antiretroviral drugs in a brain cellular target of HIV-1 infection.
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PMID:Multidrug resistance protein (MRP) 4- and MRP 5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine by microglia. 1476 2

The overexpression of multidrug resistance protein 1 (MDR1) and multidrug resistance protein 1 (MRP1) gene products is a major cause of multidrug resistance in cancer cells. A recent study suggested that disulfiram, a drug used to treat alcoholism, might act as a modulator of P-glycoprotein. In this study, we investigated the molecular and chemical basis of disulfiram as a multidrug resistance modulator. We demonstrate that in intact cells, disulfiram reverses either MDR1- or MRP1-mediated efflux of fluorescent drug substrates. Disulfiram inhibits ATP hydrolysis and the binding of [alpha-32P]8-azidoATP to P-glycoprotein and MRP1, with inhibition curves comparable with those of N-ethylmaleimide, a cysteine-modifying agent. However, if the ATP sites are protected with excess ATP, disulfiram stimulates ATP hydrolysis by both transporters in a concentration-dependent manner. Thus, in addition to modifying cysteines at the ATP sites, disulfiram may interact with the drug-substrate binding site. We demonstrate that disulfiram, but not N-ethylmaleimide, inhibits in a concentration-dependent manner the photoaffinity labeling of the multidrug transporter with 125I-iodoarylazidoprazosin and [3H]azidopine. This suggests that the interaction of disulfiram with the drug-binding site is independent of its role as a cysteine-modifying agent. Finally, we have exploited MRP4 (ABCC4) to demonstrate that disulfiram can inhibit ATP binding by forming disulfide bonds between cysteines located in the vicinity of, although not in, the active site. Taken together, our results suggest that disulfiram has unique molecular interactions with both the ATP and/or drug-substrate binding sites of multiple ATP binding cassette transporters, which are associated with drug resistance, and it is potentially an attractive agent to combat multidrug resistance.
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PMID:The molecular basis of the action of disulfiram as a modulator of the multidrug resistance-linked ATP binding cassette transporters MDR1 (ABCB1) and MRP1 (ABCC1). 1497 46

P-glycoprotein (P-gp) and the multidrug resistance-associated proteins (MRP), whose expression is associated with multidrug resistance, have been recently located in the brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB), without taking into account a possible influence or contribution of glial cells and pericytes. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the present study analysed the transcriptional expression of P-gp and the seven homologues of MRP transporters in BCECs in solo culture or in an in vitro model of the BBB consisting of a co-culture of BCECs and glial cells. Pericytes, glial cells, isolated brain capillaries and bovine grey matter extracts were also tested. P-gp mRNA, absent in glial cells, was found in brain capillaries and in co-cultured BCECs with an increased signal compared to the in solo culture. No amplification was observed in pericytes or grey matter. While MRP2, MRP3 and MRP7 remained undetected, MRP1, absent in capillaries or grey matter, was amplified in BCECs, glial cells and pericytes. MRP4 gave a low signal in most cultures. MRP5 was ubiquitously expressed, displaying a potent signal in all conditions. In spite of its presence in cultured glial cells, MRP6 mRNA expression appeared to be restricted to BCECs, with the same upregulation in the co-cultured condition as observed with P-gp. Moreover, MRP6 was the only transporter whose endothelial mRNA expression was influenced by the presence of pericytes. The tissue distribution of the expression of these transporters and the contribution of the different cell populations are discussed.
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PMID:Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier. 1526 98

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mm), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[alpha-32P]ATP binds to MRP4 (concentration for half-maximal binding approximately 3 microm) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 microm). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4.8-azido[alpha-32P]ADP.Vi and MRP4.8-azido[alpha-32P]ADP.beryllium fluoride can be generated, nucleotide trapping is approximately 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co2+ increases 8-azido[alpha-32P]ATP binding and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mm, respectively) and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping, whereas Pi has no effect up to 20 mm. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.
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PMID:Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state. 1536 14

Efflux transporters such as P-glycoprotein and multidrug resistance-associated proteins (MRPs) in the intestinal wall restrict intestinal drug transport. To overcome this limitation for enteral drug absorption, galenical targeting approaches have been proposed for site-specific luminal drug release in segments of the gut, where expression of the respective absorption-limiting transporter is minimal. Therefore, expression of multidrug resistance gene 1 (MDR1) and MRP1-5 was systematically investigated in 10 healthy subjects. Biopsies were taken from different segments of the gastrointestinal tract (from duodenum and terminal ileum, as well as ascending, transverse, descending, and sigmoid colon). Gene expression was investigated by quantitative real-time PCR (TaqMan). MRP3 appeared to be the most abundantly expressed transporter in investigated parts of the human intestine, except for the terminal ileum, where MDR1 showed the highest expression. The ranking of transporter gene expression in the duodenum was MRP3 >> MDR1 > MRP2 > MRP5 > MRP4 > MRP1. In the terminal ileum, the ranking order was as follows: MDR1 > MRP3 >> MRP1 approximately MRP5 approximately MRP4 > MRP2. In all segments of the colon (ascending, transverse, descending, and sigmoid colon), the transporter gene expression showed the following order: MRP3 >> MDR1 > MRP4 approximately MRP5 > MRP1 >> MRP2. We have shown, for the first time, systematic site-specific expression of MDR1 and MRP mRNA along the gastrointestinal tract in humans. All transporters showed alterations in their expression levels from the duodenum to sigmoid colon. The most pronounced changes were observed for MRP2, with high levels in the small intestine and hardly any expression in colonic segments. This knowledge may be useful to develop new targeting strategies for enteral drug delivery.
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PMID:Mapping of multidrug resistance gene 1 and multidrug resistance-associated protein isoform 1 to 5 mRNA expression along the human intestinal tract. 1552 49

Pancreatic ductal adenocarcinoma is among the top 10 causes of death from cancer in industrialized countries. In comparison with other gastrointestinal malignancies, pancreatic cancer is one of the tumors most resistant to chemotherapy. An important mechanism of tumor multidrug resistance is increased drug efflux mediated by several transporters of the ABC superfamily. Especially BCRP (ABCG2), MDR1 P-glycoprotein (ABCB1) and members of the MRP (ABCC) family are important in mediating drug resistance. The MRP family consists of 9 members (MRP1-MRP9) with MRP1-MRP6 being best characterized with respect to protein localization and substrate selectivity. Here, we quantified the mRNA expression of BCRP and of all MRP family members in normal human pancreas and pancreatic carcinoma and analyzed the mRNA level of the transporters most abundantly expressed in pancreatic tissue, BCRP, MRP1, MRP3, MRP4 and MRP5, in 37 tissue samples. In addition, we determined the localization of the 4 MRP proteins in normal human pancreas and in pancreatic carcinoma. The expression of BCRP, MRP1 and MRP4 mRNA did not correlate with tumor stage or grading. On the other hand, the expression of MRP3 mRNA was upregulated in pancreatic carcinoma samples and was correlated with tumor grading. The MRP5 mRNA level was significantly higher in pancreatic carcinoma tissue compared to normal pancreatic tissue. These data suggest that MRP3 and MRP5 are involved in drug resistance of pancreatic tumors and that quantitative analysis of their expression may contribute to predict the benefit of chemotherapy in patients with pancreatic cancer.
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PMID:Expression and localization of human multidrug resistance protein (ABCC) family members in pancreatic carcinoma. 1568 70

In vitro models of the blood-brain barrier (BBB) play a major role in the study of BBB permeability of drug candidates. However, most established in vitro models use cells of non-human origin, which is not optimal for the prediction of brain permeability in humans. The aim of this study was to assess the human brain capillary endothelial cell line BB19 for its usefulness as an in vitro model of the human BBB. Restrictive tight junctions are a prerequisite for drug transport studies. Sucrose permeability of BB19 cells on different filters was compared to porcine brain capillary endothelial cells (BCEC). Tightness of BB19 cell monolayers still needs further optimization. Hardly any discrimination between Sucrose and Propranolol (P(app) = 1.30 x 10(-5) vs. 2.18 x 10(-5) cm/s) was seen. Cells showed an improvement towards a more primary BCEC morphology with C6 conditioned medium, dexamethasone, and 1,25-dihydroxyvitamin D3. The presence of P-glycoprotein (P-gp), MRP4 and BCRP, ABC-transporters located in the BBB, has been shown on mRNA level, by immunostaining, and western blot. MRP1, MRP2, MRP5, OAT3, and OAT4 were also detected by RT-PCR. Functional properties of the BBB were shown with uptake of propranolol, morphine, and sucrose. Uptake studies with daunomycin and the P-gp inhibitor verapamil showed functional activity of P-gp. We conclude that BB19 cells might be feasible as a human in vitro model of the BBB for drug uptake studies. However, for the assessment of transport studies, further improvements of this model are necessary.
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PMID:Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood-brain barrier. 1628 83

Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both dose-limiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated K(m) of 1.66 microM and V(max) of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 microM) for 24 hr, celecoxib (50 microM), or MK-571 (100 microM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POM-PMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.
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PMID:Topotecan is a substrate for multidrug resistance associated protein 4. 1645 95

Rifampicin (RIF) and ursodeoxycholic acid (UDCA) therapies have beneficial effects in chronic cholestatic diseases. These may result in part from the induction of multidrug-resistance protein 2 (MRP2/ABCC2) expression in the liver and kidney. However, the precise mechanisms by which RIF and UDCA act in cholestasis remain unclear. In the present study, we report the effects of chronic administration of both drugs in a patient with Dubin-Johnson syndrome (DJS), an inherited autosomal recessive disorder characterized by the absence of functional MRP2 protein at the canalicular hepatocyte membrane. A novel 974C-->G nonsense mutation was identified in the MRP2 gene sequence from this patient. RIF induced further increase in conjugated bilirubinemia, whereas concomitant administration of RIF and UDCA led to a dramatic rise in serum bile acid concentrations. These biochemical effects, which are in marked contrast to those observed in cholestatic settings, were concomitant with an increased MRP3, but not MRP4, expression on basolateral hepatocyte membrane. Such findings highlight the key role of MRP2 in the pharmacological properties of RIF and UDCA and suggest that both drugs should be used with caution in pathologic settings in which MRP2 expression may be downregulated, as in advanced stage of cholestatic diseases.
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PMID:Identification of a novel 974C-->G nonsense mutation of the MRP2/ABCC2 gene in a patient with Dubin-Johnson syndrome and analysis of the effects of rifampicin and ursodeoxycholic acid on serum bilirubin and bile acids. 1695 91

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor recently approved for the treatment of type 2 diabetes, is excreted into the urine via active tubular secretion and glomerular filtration in humans. In this report, we demonstrate that sitagliptin is transported by human organic anion transporter hOAT3 (Km=162 microM), organic anion transporting polypeptide OATP4C1, and multidrug resistance (MDR) P-glycoprotein (Pgp), but not by human organic cation transporter 2 hOCT2, hOAT1, oligopeptide transporter hPEPT1, OATP2B1, and the multidrug resistance proteins MRP2 and MRP4. Our studies suggested that hOAT3, OATP4C1, and MDR1 Pgp might play a role in transporting sitagliptin into and out of renal proximal tubule cells, respectively. Sitagliptin did not inhibit hOAT1-mediated cidofovir uptake, but it showed weak inhibition of hOAT3-mediated cimetidine uptake (IC50=160 microM). hOAT3-mediated sitagliptin uptake was inhibited by probenecid, ibuprofen, furosemide, fenofibric acid, quinapril, indapamide, and cimetidine with IC50 values of 5.6, 3.7, 1.7, 2.2, 6.2, 11, and 79 microM, respectively. Sitagliptin did not inhibit Pgp-mediated transport of digoxin, verapamil, ritonavir, quinidine, and vinblastine. Cyclosporine A significantly inhibited Pgp-mediated transport of sitagliptin (IC50=1 microM). Our data indicate that sitagliptin is unlikely to be a perpetrator of drug-drug interactions with Pgp, hOAT1, or hOAT3 substrates at clinically relevant concentrations. Renal secretion of sitagliptin could be inhibited if coadministered with OAT3 inhibitors such as probenecid. However, the magnitude of interactions should be low, and the effects may not be clinically meaningful, due to the high safety margin of sitagliptin.
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PMID:Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug resistance P-glycoprotein. 1731 1


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