High-affinity glutamate transport activity is induced by stress in NBL-1 cells. Exposure of cells to hyperosmotic medium led to an induction of the EAAC1 glutamate transporter, preceded by a large increase in EAAC1 mRNA levels. Culture of cells in amino acid-free medium also caused a protein synthesis-dependent increase in glutamate transport activity, but this was not accompanied by an increase of either EAAC1 mRNA or protein. Indirect evidence suggests that the increase in EAAC1 activity in the latter case may be due to the synthesis of an activator protein in response to decreased intracellular glutamate concentrations.
- cellular stress
- amino acid deprivation
the bovine renal epithelial cell line NBL-1 has been used extensively in our laboratory for the study of mechanisms involved in the regulation of amino acid transport (1, 2, 6, 8, 10). The cells form a polarized confluent monolayer. They express high activities of a Na+-dependent broad specificity amino acid transport system (system Bo) and also of high-affinity glutamate transport. The cells do not express Na+-dependent glucose or nucleoside transport, nor the proximal tubule-specific enzymes aminopeptidase or alkaline phosphatase. Their general properties resemble those of MDCK cells, and they may provide a good model for regulatory responses of cells in the kidney medulla. Na+-dependentl-glutamate transport in NBL-1 cells has a K m of 5–10 μM, is inhibited by bothd- andl-aspartate but notd-glutamate, and is catalyzed by the high-affinity transporter EAAC1. This article describes some of the characteristics of the regulation of glutamate transport in NBL-1 cells.
Induction of Glutamate transport by Hyperosmotic Stress and by Exposure of Cells to Tunicamycin
NBL-1 cells were cultured in Ham’s F-12 medium. EAAC1 activity was measured as uptake ofl-aspartate in the presence of aminooxyacetate to prevent metabolism. Addition of 200 mM sucrose resulted in an increase in Na+-dependent aspartate transport activity, which was observable after 6 h and reached a maximum after 18 h. After 18 h, theV max increased threefold, but theK m for aspartate was unchanged at 5–10 μM. A half-maximal effect was observed at 100 mM sucrose, and a similar effect was observed with mannitol. The effect of hyperosmotic medium was abolished by the protein synthesis inhibitor cycloheximide and by the glycosylation inhibitor tunicamycin, indicating that a protein glycosylation event was involved in the induction of glutamate transport (2).
The open reading frame of EAAC1 cDNA in NBL-1 cells was cloned by PCR (B. Nicholson, unpublished work) and found to have a sequence 89% identical to that of rabbit intestine EAAC1 (3). cDNA probes and a COOH-terminal polyclonal antibody were prepared and used to quantify the level of EAAC1 mRNA and protein, respectively, in NBL-1 cells. Prolonged exposure to 200 mM sucrose led to a large increase in EAAC1 mRNA after 9 h and a threefold increase in immunoreactive EAAC1 protein (2, 6).
Tunicamycin itself caused a lesser but still significant induction of glutamate transport activity (50% increase after 16 h), preceded by an increase in EAAC1 mRNA and a fourfold increase in immunoreactive EAAC1 protein. Tunicamycin inhibition of protein glycosylation is known to cause stress responses in cells, possibly mediated by accumulation of incomplete or unfolded proteins. This form of stress apparently stimulates transcription of the EAAC1 gene, but the increase in the level of mRNA and protein is not reflected in increased transport activity presumably because in the presence of tunicamycin the EAAC1 protein is not properly glycosylated and inserted into the membrane.
Induction of Glutamate Transport by Amino Acid Deprivation
Culture of NBL-1 cells in amino acid-free medium led to a protein synthesis dependent increase in the initial rate of glutamate transport (8). The V maxincreased up to threefold in 15 h with no change inK m. This induction was inhibited by cycloheximide and by tunicamycin. To gain more information about the mechanisms involved, cells were cultured in amino acid-free medium to which a single amino acid had been added. The presence of substrates of the glutamate transport system prevented induction. Induction was also prevented by alanine, glutamine, or asparagine and also byd-glutamate but not by methylaminoisobutyrate, leucine, or cysteine. Because alanine and glutamine and asparagine can be rapidly metabolized to glutamate or aspartate while in these cells cysteine and leucine cannot be rapidly metabolized, these results suggested that intracellular glutamate and/or aspartate levels may be important in regulating the induction of glutamate transport activity. In accordance with this, specific depletion of glutamate levels from 30 to 10 nmol/mg by incubation of cells in glutamine-free medium in the presence of 0.5 mM aminooxyacetate to prevent transamination also caused protein synthesis-dependent induction of glutamate transport activity (6).
The increase in the rate of glutamate transport as a result of prolonged incubation in amino acid-free medium was not accompanied by a significant increase in EAAC1 mRNA levels (8) or by an increase in the amount of EAAC1 protein in cell membrane extracts (6). The expression of various members of the highly-affinity glutamate transport family was assessed by using specific oligonucleotide primers for each of these in PCR reactions with NBL-1 cell cDNA as template. In NBL-1 cells cultured either in normal medium, in hyperosmotic medium or in amino acid-free medium, only EAAC1 (EAAT3) mRNA was expressed. mRNA for EAAT1, EAAT2, and EAAT4 was absent. Therefore, the induction of transport by glutamate deprivation must be due either to induction of a hypothetical EAAC1-activating glycoprotein or to a novel glutamate transporter that has not yet been cloned. Although no amino acid transporter activator protein has yet been identified, there is circumstantial evidence for the involvement of such proteins in the stimulation of system A activity by amino acid deprivation and by hyperosmotic stress (see Ref. 5 for a review).
To identify glycoproteins that might be involved in the regulation of EAAC1 activity, cells were incubated with [35S]methionine in normal and amino acid-free medium, and the glycoprotein fraction was purified using concanavalin A, separated by SDS-PAGE, and autoradiographed. Monoclonal antibodies were also prepared to glycoprotein fractions from normal and amino acid-starved cells. Three glycoproteins that are induced on amino acid starvation in NBL-1 cells were identified by these approaches. These were the stress protein grp78 (7), the endoplasmic Ca2+-binding molecular chaperone calreticulin (7), and the endoplasmic reticulum-associated enzyme glucosidase II (4). Both calreticulin and glucosidase II are involved in the glycoprotein maturation pathway, but so far the identity of the novel glycoprotein that is responsible for activating glutamate transport activity in NBL-1 cells remains elusive.
We have shown that expression of glutamate transport activity in NBL-1 cells is induced by various forms of cell stress and have partially characterized the mechanisms responsible (Fig.1). Induction of EAAC1 by hyperosmotic stress may require mechanisms that are specific to renal cells, since this phenomenon is not observed in several other cell types that express the EAAC1 transporter, including human fibroblasts, Chinese hamster ovary CHO cells, and the rat hepatoma cell line H-4-II-E. Interestingly, in NBL-1 cells the induction of EAAC1 is also specific in that the activities of the broad-specificity amino acid transporter and the phosphate transporter are not induced by hyperosmotic stress. Elucidation of these mechanism will require sequencing and analysis of the EAAC1 promoter. EAAC1 has been shown to be expressed in the medulla region of rat kidney (9), and it is possible that enhanced glutamate transport contributes to cell volume regulation in the renal medulla. Conversely, induction of glutamate transport activity by amino acid deprivation is observed in a number of different cell types that express EAAC1 and may be a more general phenomenon involved in maintaining intracellular amino acid concentrations when the external concentration is reduced.
We thank Prof. M. S. Kilberg for making available the primers for EAAT1, EAAT2, and EAAT4.
Address for reprint requests and other correspondence: J. D. McGivan, Dept. of Biochemistry, School of Medical Sciences, Univ. Walk, Bristol BS8 1TD, United Kingdom (E-mail:).
This article is the fourth of five in this forum, which is based on a series of reports on glutamate transport and glutamate metabolism that was first presented at Experimental Biology ’98 in San Francisco, CA.
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