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Title: Substrate regulation of ascorbate transport activity in astrocytes

Abstract

Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

Authors:
; ; ;  [1]
  1. (Univ. of Western Ontario, London (Canada))
Publication Date:
OSTI Identifier:
5962553
Resource Type:
Journal Article
Resource Relation:
Journal Name: Neurochemical Research; (USA); Journal Volume: 15:10
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ASCORBIC ACID; MEMBRANE TRANSPORT; GLUCOSE; SUBSTRATES; BIOLOGICAL AVAILABILITY; CARBON 14 COMPOUNDS; CELL MEMBRANES; CEREBRAL CORTEX; HOMEOSTASIS; NERVE CELLS; RATS; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ALDEHYDES; ANIMAL CELLS; ANIMALS; BODY; BRAIN; CARBOHYDRATES; CELL CONSTITUENTS; CENTRAL NERVOUS SYSTEM; CEREBRUM; HEXOSES; HYDROGEN COMPOUNDS; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MAMMALS; MEMBRANES; MONOSACCHARIDES; NERVOUS SYSTEM; ORGANIC COMPOUNDS; ORGANS; RODENTS; SACCHARIDES; SOMATIC CELLS; VERTEBRATES; VITAMINS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Wilson, J.X., Jaworski, E.M., Kulaga, A., and Dixon, S.J.. Substrate regulation of ascorbate transport activity in astrocytes. United States: N. p., 1990. Web. doi:10.1007/BF00965751.
Wilson, J.X., Jaworski, E.M., Kulaga, A., & Dixon, S.J.. Substrate regulation of ascorbate transport activity in astrocytes. United States. doi:10.1007/BF00965751.
Wilson, J.X., Jaworski, E.M., Kulaga, A., and Dixon, S.J.. Mon . "Substrate regulation of ascorbate transport activity in astrocytes". United States. doi:10.1007/BF00965751.
@article{osti_5962553,
title = {Substrate regulation of ascorbate transport activity in astrocytes},
author = {Wilson, J.X. and Jaworski, E.M. and Kulaga, A. and Dixon, S.J.},
abstractNote = {Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.},
doi = {10.1007/BF00965751},
journal = {Neurochemical Research; (USA)},
number = ,
volume = 15:10,
place = {United States},
year = {Mon Oct 01 00:00:00 EDT 1990},
month = {Mon Oct 01 00:00:00 EDT 1990}
}
  • Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na{sup +}-dependent L-ascorbate transporter in the plasma membrane. The present study examined the effects of ascorbate deprivation and supplementation on the activity of the transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-({sup 14}C)ascorbate for 1 minute at 37C. They observed that the maximal uptake rate, V{sub max}, rapidly (<3 hours) increased when cultured cells were deprived of L-ascorbate. There was no change in the apparent affinity (K{sub m}) of the transport system for ascorbate. V{sub max} returned to normalmore » following addition of L-ascorbate, but not D-isoascorbate, to the medium. The authors conclude that astrocytes adapt ascorbate transport rates to changes in substrate availability. Furthermore, the data suggest that the transport system located in the astroglial plasma membrane regulates intracellular ascorbate concentration, because changes in transport rate may compensate for regional differences and temporal fluctuations in extracellular ascorbate levels.« less
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  • Syncytin-1 has a physiological role during early pregnancy, as mediator of trophoblast fusion into the syncytiotrophoblast layer, hence allowing embryo implantation. In addition, its expression in nerve tissue has been proposed to contribute to the pathogenesis of multiple sclerosis (MS). Syncytin-1 is the env glycoprotein of the ERVWE1 component of the W family of human endogenous retroviruses (HERV), located on chromosome 7q21-22, in a candidate region for genetic susceptibility to MS. The mechanisms of ERVWE1 regulation in nerve tissue remain to be identified. Since there are correlations between some cytokines and MS outcome, we examined the regulation of the syncytin-1more » promoter by MS-related cytokines in human U-87MG astrocytic cells. Using transient transfection assays, we observed that the MS-detrimental cytokines TNF{alpha}, interferon-{gamma}, interleukin-6, and interleukin-1 activate the ERVWE1 promoter, while the MS-protective interferon-{beta} is inhibitory. The effects of cytokines are reduced by the deletion of the cellular enhancer domain of the promoter that contains binding sites for several transcription factors. In particular, we found that TNF{alpha} had the ability to activate the ERVWE1 promoter through an NF-{kappa}B-responsive element located within the enhancer domain of the promoter. Electrophoretic mobility shift and ChIP assays showed that TNF{alpha} enhances the binding of the p65 subunit of NF-{kappa}B, to its cognate site within the promoter. The effect of TNF{alpha} is abolished by siRNA directed against p65. Taken together, these results illustrate a role for p65 in regulating the ERVWE1 promoter and in TNF{alpha}-mediated induction of syncytin-1 in multiple sclerosis.« less
  • Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigatedmore » the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.« less