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Role of glutathione in hepatic trans...

Lee, Thomas Keywon.

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Role of glutathione in hepatic transport of xenobiotics.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Role of glutathione in hepatic transport of xenobiotics./
Author:	Lee, Thomas Keywon.
Description:	144 p.
Notes:	Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4148.
Contained By:	Dissertation Abstracts International63-09B.
Subject:	Health Sciences, Toxicology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3064818
ISBN:	049383785X

Role of glutathione in hepatic transport of xenobiotics.
Lee, Thomas Keywon.

Role of glutathione in hepatic transport of xenobiotics. - 144 p.

Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4148.

Thesis (Ph.D.)--The University of Rochester, 2002.

The liver is the major site for the removal and detoxification of various compounds from the blood. Some of the transport proteins involved have recently been identified at the molecular level, but the functional mechanisms for many of these proteins are not well understood. Studies in this laboratory revealed that Oatp1 mediates uptake of organic anions in a GSH-dependent manner when expressed in Xenopus oocytes; however, the nature of this interaction remains unclear. The present study tested for the presence of a GSH-sensitive organic anion transport mechanism in HepG2 cells, a human hepatoma-derived cell line, and used the bile salt taurocholate as a prototypical substrate. Intracellular GSH depletion inhibited 3H-taurocholate uptake, and conversely, the release of GSH from HepG2 cells was stimulated in the presence of extracellular taurocholate and other bile acids, consistent with a role for intracellular GSH in stimulating organic anion uptake. Efflux of 3H-taurocholate from HepG2 cells was also sensitive to intracellular GSH concentration. The expression of several transport proteins was analyzed by RT-PCR. These results provide direct evidence that intracellular GSH levels modulate transport of taurocholate, and suggest that GSH plays a regulatory role in the hepatobiliary transport of xenobiotics. To further characterize the intracellular substrate site of Oatp1 and to circumvent some of the limitations of whole cell systems, a yeast model system was adopted. Although high level of Oatp1 expression was achieved in S. cerevisiae secretory vesicles, no transport activity was detected. It was hypothesized that the lack of glycosylation of Oatp1 in yeast may be a factor contributing to the lack of function. Therefore, Oatp1 was expressed in X. laevis oocytes and glycosylation was prevented by mutating the four potential glycosylation sites on Oatp1, and by treating oocytes with tunicamycin. In addition, site-directed mutagenesis was employed. The results indicate that underglycosylated Oatp1 expressed in oocytes has decreased transport activity, and that transport is abolished when all four N-glycosylation residues are removed. These results indicate that glycosylation of Oatp1 plays an important role in the functional expression of this transport protein.

ISBN: 049383785XSubjects--Topical Terms:

1017752
Health Sciences, Toxicology.

Role of glutathione in hepatic transport of xenobiotics.
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245 1 0 $a Role of glutathione in hepatic transport of xenobiotics.
300 $a 144 p.
500 $a Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4148.
500 $a Supervisor: Ned Ballatori.
502 $a Thesis (Ph.D.)--The University of Rochester, 2002.
520 $a The liver is the major site for the removal and detoxification of various compounds from the blood. Some of the transport proteins involved have recently been identified at the molecular level, but the functional mechanisms for many of these proteins are not well understood. Studies in this laboratory revealed that Oatp1 mediates uptake of organic anions in a GSH-dependent manner when expressed in Xenopus oocytes; however, the nature of this interaction remains unclear. The present study tested for the presence of a GSH-sensitive organic anion transport mechanism in HepG2 cells, a human hepatoma-derived cell line, and used the bile salt taurocholate as a prototypical substrate. Intracellular GSH depletion inhibited 3H-taurocholate uptake, and conversely, the release of GSH from HepG2 cells was stimulated in the presence of extracellular taurocholate and other bile acids, consistent with a role for intracellular GSH in stimulating organic anion uptake. Efflux of 3H-taurocholate from HepG2 cells was also sensitive to intracellular GSH concentration. The expression of several transport proteins was analyzed by RT-PCR. These results provide direct evidence that intracellular GSH levels modulate transport of taurocholate, and suggest that GSH plays a regulatory role in the hepatobiliary transport of xenobiotics. To further characterize the intracellular substrate site of Oatp1 and to circumvent some of the limitations of whole cell systems, a yeast model system was adopted. Although high level of Oatp1 expression was achieved in S. cerevisiae secretory vesicles, no transport activity was detected. It was hypothesized that the lack of glycosylation of Oatp1 in yeast may be a factor contributing to the lack of function. Therefore, Oatp1 was expressed in X. laevis oocytes and glycosylation was prevented by mutating the four potential glycosylation sites on Oatp1, and by treating oocytes with tunicamycin. In addition, site-directed mutagenesis was employed. The results indicate that underglycosylated Oatp1 expressed in oocytes has decreased transport activity, and that transport is abolished when all four N-glycosylation residues are removed. These results indicate that glycosylation of Oatp1 plays an important role in the functional expression of this transport protein.
590 $a School code: 0188.
650 4 $a Health Sciences, Toxicology. $3 1017752
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791 $a Ph.D.
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