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 investigated the time kinetics of P-glycoprotein (P-gp), a membrane bound drug efflux pump for many anti-cancer drugs in multidrug resistant cells, using a rat ischemic brain model. Frozen sections of the brain were studied immunohistochemically with anti-Factor VIII antibody for endothelial cells, with anti-glial fibrillary acidic protein (GFAP) antibody for reactive astrocytes, and with MC6-4 monoclonal antibody for P-gp. A putative blood-brain barrier (BBB) marker, gamma-glutamyl transpeptidase (gamma-GTP), and the progression of the brain edema were also studied. P-gp positive endothelial cells disappeared in the ischemic lesion by post-ischemic Day 3. Factor VIII-positive regenerating capillaries were first observed on Day 3 without P-gp expression when the brain edema reached a maximum. P-gp positive endothelial cells began to reappear on Day 5, and were detected in all endothelial cells by Day 8. The time kinetics of P-gp expression in the endothelial cells showed a similar pattern as that of gamma-GTP, and its induction is associated with GFAP-positive reactive astrocytes. These results suggest that P-gp might play an important role in maintaining the BBB function in conjunction with glial cells.
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PMID:P-glycoprotein expression in brain capillary endothelial cells after focal ischemia in rat. 797 60

Small cell lung cancer (SCLC) is treated primarily with combination chemotherapy. Despite high initial response rates, most patients eventually die with drug resistant disease. In some tumours, resistance to multiple chemotherapeutic agents is attributed to overexpression of P-glycoprotein (P-gp). However, this does not appear to be a frequent occurrence in drug resistant SCLC. Increased levels of glutathione (GSH) and related enzymes may play a role in resistance to alkylating agents as well as natural product drugs. We measured levels of GSH, glutathione S-transferase (GST), glutathione reductase (GSH Red), glutathione peroxidase (GSH Px), and gamma-glutamyl transpeptidase (gamma-GT) in a panel of 20 SCLC cell lines. Most of these lines were established from patients treated at this centre. Each cell line had a characteristic and reproducible profile of GSH and related enzyme levels. Immunoblot analysis indicated that the predominant GST in the cell lines was the anionic pi isoenzyme. The relative sensitivity of each of these cell lines to 16 different chemotherapeutic agents was measured using a modified MTT assay. Spearman rank correlation analysis was used to determine the relationships between the relative chemosensitivity of these cell lines and the levels of GSH and related enzymes. The number of positive correlations was no greater than expected by chance alone. Furthermore, there was no correlation with the treatment history of the patients from whom the cell lines were derived. These data suggest that alterations in glutathione metabolism do not play a major role in resistance to chemotherapeutic agents in these human SCLC cell lines.
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PMID:Do glutathione and related enzymes play a role in drug resistance in small cell lung cancer cell lines? 810 44

The blood-brain barrier is formed by the cerebral capillary endothelial cells, joined together by tight junctions. These cells express the general endothelial cell markers as well as specific markers found on endothelial cells forming physiological barriers such as gamma-glutamyltranspeptidase, the glucose transporter Glut1 and the neutral amino-acid transporter. Using the monoclonal antibodies C219 and MRK16, we have revealed by Western blot and immuno-histochemistry the expression of the multidrug resistance P-glycoprotein on isolated rat cerebral cortex capillaries. On the other hand, P-glycoprotein was not detectable in brain cortex homogenates. P-glycoprotein thus appears to be a blood-brain barrier endothelium-specific marker which could regulate brain penetration of xenobiotics and thus participate in the neuroprotection of the brain.
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PMID:Detection of the multidrug resistance of P-glycoprotein in healthy tissues: the example of the blood-brain barrier. 873 93

A hydroquinone-resistant derivative of the M1 cell line, designated M1HQ, was generated and used to evaluate the biochemical mechanism responsible for resistance to oxidative stress-inducing agents. The hydroquinone concentrations that were cytotoxic to 50 and 90% of the parental M1 cell line in 48 hr were 25 and 90 microM, respectively, whereas exposure to 500 microM hydroquinone did not decrease M1HQ viability significantly. M1HQ cells grew slower than M1 cells and exhibited significantly higher resistance to colchicine, doxorubicin, hydrogen peroxide, 4-hydroperoxycyclophosphamide, and 1,3-bis (2-chloroethyl)-1-nitrosourea but not to benzoquinone, vinblastine, or gamma-radiation. M1HQ cells possessed significantly higher levels of total thiols, glutathione, glutathione peroxidase, glutathione reductase, quinone reductase, and gamma-glutamyl transpeptidase than the parental M1 cell line. Steady-state gamma-glutamylcysteine synthetase mRNA expression also was 1.6-fold higher in M1HQ cells. P-glycoprotein transcripts were detectable in both M1 and M1HQ cells, but were 2-fold higher in M1HQ. Multidrug resistance-associated protein transcripts were not detectable in either M1 or M1HQ. Hydroquinone resistance in M1HQ cells was partially reversible with a combination of inhibitors of quinone reductase, gamma-glutamylcysteine synthetase, glutathione peroxidase, and the multidrug resistance-associated protein, but not with inhibitors of P-glycoprotein, gamma-glutamyl transpeptidase, or glutathione-S-transferase. When treated with [14C]hydroquinone, M1HQ cells did not generate significant hydroquinone-protein adducts but did release an adduct similar to N-acetylcysteinyl-benzoquinone. In contrast, numerous [14C]hydroquinone-protein adducts were produced in M1 cells, while the N-acetylcysteinyl-benzoquinone-like molecule was undetectable. Thus, hydroquinone resistance in M1HQ cells appeared to result from a glutathione-dependent detoxification and export mechanism.
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PMID:Hydroquinone resistance in a murine myeloblastic leukemia cell line. Involvement of quinone reductase and glutathione-dependent detoxification in nonclassical multidrug resistance. 878 15

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 GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.
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PMID:GSH transport in immortalized mouse brain endothelial cells: evidence for apical localization of a sodium-dependent GSH transporter. 1038 92

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

In contrast to other vascular beds, the endothelial cells in brain capillaries, which constitute the blood-brain barrier, are sealed together by continuous tight junctions and have little transcellular vesicular transport. In addition to these morphological properties, the presence of specific enzymes and proteins highly restricts the passage of molecules from the blood to the brain. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and glial cells on the other. In these culture conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier, including gamma-glutamyl transpeptidase and P-glycoprotein activities. Moreover, the close correlation between the results obtained in vitro with our model and in vivo allows us to conclude that our in vitro blood-brain barrier model is a relevant model for the screening of new molecules to the brain.
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PMID:[Cerebral transfer and neuroprotection]. 1535 11

The availability of an in vitro blood-brain barrier model would represent a powerful alternative to experimental animals in pharmacological and toxicological research. This overview collects the various current approaches to build an in vitro model of the blood-brain barrier for these purposes. Purified bovine, porcine and human brain microcapillary endothelial cells as well as several immortalized cell lines have been used to model the blood-brain barrier in vitro, partly in co-culture with astrocytes of various species, or various cell lines such as C6 glioma or N2a neuroblastoma cells. The collected data indicate that functional parameters often can be induced by soluble and membrane-bound factors in such cell systems. Relevant barrier-specific parameters are reviewed: electrical resistance, and structure and function of the multidrug resistance P-glycoprotein and the y-glutamyl transpeptidase. Both P-glycoprotein and gamma-glutamyl transpeptidase have great influence on the pharmacodynamics, toxicology and metabolic capacity of the blood-brain barrier (drug efflux, oxidative damage, detoxification of endotoxins, etc.). Several available in vitro models appear to be suited for pharmacotoxicological screening, if the functional parameters gamma-glutamyl transpeptidase, P-glycoprotein as well as transendothelial resistance are monitored.
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PMID:Co-culture blood-brain barrier models and their use for pharmatoxicological screening. 2065 44


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