Proc. Natl. Acad. Scd. USA Vol. 75, No. 10, pp. 5048-5051, October 1978
Cell Biology
Sodium-dependent amino acid transport by cultured hamster cells: Membrane vesicles retain transport changes due to glucose starvation and cycloheximide (Nil cells/a-aminoisobutyrate/fructose feeding/regulation)
HOYOKU NISHINO*, C. WILLIAM CHRISTOPHERt, ROBERT M. SCHILLER*, MAUREEN T. GAMMON*, DONNA ULLREYt, AND KURT J. ISSELBACHER* * Gastrointestinal Unit and t The Huntington Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Contributed by Kurt J. Isselbacher, July 31, 1978
ABSTRACT Enhanced a-aminoisobutyric acid transport by hamster cells cultured in the absence of D-glucose has been demonstrated in isolated membrane vesicles. The observed enhancement was seen in the presence but not in the absence of Na+. Kinetic analysis of transport using both the intact cells and the membrane vesicles showed that the overall enhancement was associated with an increase in Vmax. Decreases in transport activity by intact cells resulting from extended exposure of the cells to inhibitors of protein synthesis, such as cycloheximide, were also evident in membrane vesicles. The use of metabolically inactive membrane vesicles demonstrated that amino acid uptake by intact cells is a transport property of the plasma membrane. In addition this study shows that membrane vesicle preparations can be expioited for the purpose of studying the regulation of amino acid transport. Taken together, the data suggest that carrier turnover is involved in the regulation of amino acid transport in animal cells. Uptake of nutrients by animal cells maintained in artificial media can be enhanced by a number of simple manipulations of culture conditions. For example, it has been reported that depriving cultured cells of glucose results in increases in hexose and amino acid uptake (1-5). When hamster fibroblasts (Nil cells) are maintained in media containing fructose instead of glucose, their hexose and amino acid uptake rates increase as if the cells were starved of glucose (6, 7).t In fact, the enhancements of uptake that develop with fructose feeding are often higher and more sustained than when the cells are totally deprived of hexoses (6). At least in Nil cells, the glucose starvation that results from extended fructose feeding is probably related to the inability of these cells to transport fructose (unpublished observation) and their failure to process this sugar through the glycolytic pathway (4). However, the extent of the involvement of glycolysis in the control of amino acid transport is not clear and this is due, in part, to a paucity of information about the characteristics of the transport system itself. Even when nonmetabolizable analogs such as a-aminoisobutyric acid (AIB) are used, transport by whole cells is often complicated by other metabolic considerations. However, the use of metabolically inactive plasma membrane vesicles that retain uptake activity has permitted a more detailed study of the possible mechanisms involved in nutrient transport. Thus, it seemed reasonable to use membrane vesicles to determine whether or not the increased uptake of amino acids that is associated with glucose starvation would be retained at the level of the cell membrane. Using AIB as a model amino acid substrate, we find that membrane vesicles prepared from glucose-starved (i.e., fructose-fed) Nil cells reflect the changes in amino acid uptake observed with intact cells.
MATERIALS AND METHODS Reagents and Buffers. a-Amino[14C]isobutyric acid, a[methyl-3H]aminoisobutyric acid, and L-[3H]glucose were purchased from New England Nuclear Corp. Culture media and fetal calf sera were obtained from Grand Island Biological Co. and Microbiological Associates. Corning roller flasks (no. 25130, 490 cm2 surface area) were obtained from A. H. Thomas Co. Nitrocellulose filters (type HA, 0.45 ,gm) were purchased from Millipore Filter Corp. Cycloheximide and puromycin-HCl were purchased from Sigma Chemical Co. Buffer S was 1 mM Tris-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (pH 7.5) containing 100 mM sorbitol and was used in the preparation of membrane vesicles and membrane vesicle assays. For whole cell assays, Dulbecco's phosphatebuffered saline, pH 7.2, was used (for sodium-free assays, the Na2HPO4 and NaCl of the phosphate-buffered saline were replaced with K2HPO4 and choline chloride). Cell Cultures and Membrane Vesicle Preparation. Conditions for the growth and maintenance of hamster fibroblasts (Nil strain) in Dulbecco's modified Eagle's minimal essential medium in plastic culture dishes have been described (2, 6). Cells used for membrane vesicle preparations were grown in the modified Eagle's medium containing 10% fetal calf serum in tightly sealed roller bottles under an atmosphere of approximately 10% C02/90% air. Cultures were allowed to grow to confluence before the medium was changed. The cells were then washed with sterile phosphate-buffered saline and refed with glucose-free Eagle's medium containing 10% dialyzed fetal calf serum and either 22 mM D-glucose (glucose-fed) or 22 mM D-fructose (glucose-starved) for 18-24 hr. Cells were harvested from the roller bottles by scraping, and membrane vesicles were prepared according to the method described by Nishino et al. (8). Compared with the cell homogenate, the membrane preparations were approximately 3-fold enriched for 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity. The vesicles could be stored at 0-40 for at least 4 days without loss of uptake activity. Uptake by these vesicles was sensitive to changes in osmotic pressure induced by changes in external sorbitol concentrations, and amino acid uptake was temperature dependent. Uptake Assays. Monolayers. Cultures, maintained as monolayers on dishes, were washed twice with 2 ml of sodium free Abbreviations: AIB, a-aminoisobutyric acid; buffer S, 1 mM Tris-4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes)/100 mM sorbitol. t Although Nil cells and 3T3 cells increase the uptake of amino acids in response to fructose feeding, we have found that chick embryo fibroblasts do not increase amino acid uptake when either totally starved of hexose or fructose-fed (unpublished).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
5048
Cell Biology: Nishino el al.
Proc. Natl. Acad. Sct. USA 75 (1978)
Table 1. Enhanced AIB uptake by glucose-starved cells
Culture conditions
Mean AIB uptake,* pmol/mg protein per min
Enhancement factor, -fold (1.00) 2.74 ± 1.21
Glucose-fed 286 + 188 Glucose-starved 637 ± 273 Confluent Nil hamster cells were maintained for 18-24 hr in 35-mm plastic culture dishes in Dulbecco's modified Eagle's medium containing 4 mg of D-glucose per ml (glucose-fed) or 4 mg of D-fructose per ml (glucose-starved). Twenty-eight duplicate cultures were assayed for AIB uptake by the monolayer assay. * Absolute AIB uptake values and enhancement factors varied from experiment to experiment. In this table, the mean i SD AIB uptake and the mean ± SD enhancement factors were calculated separately. Uptake data were grouped separately from enhancement data and the mean + SD was obtained for each parameter with a Texas Instruments SR-51A calculator.
phosphate-buffered saline and then 0.5 ml of phosphate-buffered saline (or sodium-free phosphate-buffered saline) containing 0.1 mM [14C]AIB (0.5 MCi/ml), and 0.1 mM L-[3H]glucose (2 MCi/ml) was added to the culture dish. Cells were allowed to take up the AIB for various times at 22° and then were washed five times with 2 ml of ice-cold phosphate-buffered saline. The radioactivity of ethanol extracts was measured by liquid scintillation and normalized for the protein content of each dish as described (2, 6). Corrections for simple diffusion and interstitial (i.e., nonspecific) trapping were made by subtracting the amount of L-glucose associated with each sample. Suspended cells. Aliquots of cells suspended by scraping from dishes and roller bottles were twice washed with sodium-free phosphate-buffered saline and collected by centrifugation (900 X g, 10 min). The final cell suspension was divided into two equal volumes and collected by centrifugation, and the clear supernatant was removed by aspiration. The assay was initiated by addition of the radioactive solutions (in phosphate-buffered saline or sodium-free phosphate-buffered saline, as described above) at 22°. By a modification of the technique. described by 3 C
0
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Cell Biology
Sodium-dependent amino acid transport by cultured hamster cells: Membrane vesicles retain transport changes due to glucose starvation and cycloheximide (Nil cells/a-aminoisobutyrate/fructose feeding/regulation)
HOYOKU NISHINO*, C. WILLIAM CHRISTOPHERt, ROBERT M. SCHILLER*, MAUREEN T. GAMMON*, DONNA ULLREYt, AND KURT J. ISSELBACHER* * Gastrointestinal Unit and t The Huntington Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Contributed by Kurt J. Isselbacher, July 31, 1978
ABSTRACT Enhanced a-aminoisobutyric acid transport by hamster cells cultured in the absence of D-glucose has been demonstrated in isolated membrane vesicles. The observed enhancement was seen in the presence but not in the absence of Na+. Kinetic analysis of transport using both the intact cells and the membrane vesicles showed that the overall enhancement was associated with an increase in Vmax. Decreases in transport activity by intact cells resulting from extended exposure of the cells to inhibitors of protein synthesis, such as cycloheximide, were also evident in membrane vesicles. The use of metabolically inactive membrane vesicles demonstrated that amino acid uptake by intact cells is a transport property of the plasma membrane. In addition this study shows that membrane vesicle preparations can be expioited for the purpose of studying the regulation of amino acid transport. Taken together, the data suggest that carrier turnover is involved in the regulation of amino acid transport in animal cells. Uptake of nutrients by animal cells maintained in artificial media can be enhanced by a number of simple manipulations of culture conditions. For example, it has been reported that depriving cultured cells of glucose results in increases in hexose and amino acid uptake (1-5). When hamster fibroblasts (Nil cells) are maintained in media containing fructose instead of glucose, their hexose and amino acid uptake rates increase as if the cells were starved of glucose (6, 7).t In fact, the enhancements of uptake that develop with fructose feeding are often higher and more sustained than when the cells are totally deprived of hexoses (6). At least in Nil cells, the glucose starvation that results from extended fructose feeding is probably related to the inability of these cells to transport fructose (unpublished observation) and their failure to process this sugar through the glycolytic pathway (4). However, the extent of the involvement of glycolysis in the control of amino acid transport is not clear and this is due, in part, to a paucity of information about the characteristics of the transport system itself. Even when nonmetabolizable analogs such as a-aminoisobutyric acid (AIB) are used, transport by whole cells is often complicated by other metabolic considerations. However, the use of metabolically inactive plasma membrane vesicles that retain uptake activity has permitted a more detailed study of the possible mechanisms involved in nutrient transport. Thus, it seemed reasonable to use membrane vesicles to determine whether or not the increased uptake of amino acids that is associated with glucose starvation would be retained at the level of the cell membrane. Using AIB as a model amino acid substrate, we find that membrane vesicles prepared from glucose-starved (i.e., fructose-fed) Nil cells reflect the changes in amino acid uptake observed with intact cells.
MATERIALS AND METHODS Reagents and Buffers. a-Amino[14C]isobutyric acid, a[methyl-3H]aminoisobutyric acid, and L-[3H]glucose were purchased from New England Nuclear Corp. Culture media and fetal calf sera were obtained from Grand Island Biological Co. and Microbiological Associates. Corning roller flasks (no. 25130, 490 cm2 surface area) were obtained from A. H. Thomas Co. Nitrocellulose filters (type HA, 0.45 ,gm) were purchased from Millipore Filter Corp. Cycloheximide and puromycin-HCl were purchased from Sigma Chemical Co. Buffer S was 1 mM Tris-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (pH 7.5) containing 100 mM sorbitol and was used in the preparation of membrane vesicles and membrane vesicle assays. For whole cell assays, Dulbecco's phosphatebuffered saline, pH 7.2, was used (for sodium-free assays, the Na2HPO4 and NaCl of the phosphate-buffered saline were replaced with K2HPO4 and choline chloride). Cell Cultures and Membrane Vesicle Preparation. Conditions for the growth and maintenance of hamster fibroblasts (Nil strain) in Dulbecco's modified Eagle's minimal essential medium in plastic culture dishes have been described (2, 6). Cells used for membrane vesicle preparations were grown in the modified Eagle's medium containing 10% fetal calf serum in tightly sealed roller bottles under an atmosphere of approximately 10% C02/90% air. Cultures were allowed to grow to confluence before the medium was changed. The cells were then washed with sterile phosphate-buffered saline and refed with glucose-free Eagle's medium containing 10% dialyzed fetal calf serum and either 22 mM D-glucose (glucose-fed) or 22 mM D-fructose (glucose-starved) for 18-24 hr. Cells were harvested from the roller bottles by scraping, and membrane vesicles were prepared according to the method described by Nishino et al. (8). Compared with the cell homogenate, the membrane preparations were approximately 3-fold enriched for 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity. The vesicles could be stored at 0-40 for at least 4 days without loss of uptake activity. Uptake by these vesicles was sensitive to changes in osmotic pressure induced by changes in external sorbitol concentrations, and amino acid uptake was temperature dependent. Uptake Assays. Monolayers. Cultures, maintained as monolayers on dishes, were washed twice with 2 ml of sodium free Abbreviations: AIB, a-aminoisobutyric acid; buffer S, 1 mM Tris-4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes)/100 mM sorbitol. t Although Nil cells and 3T3 cells increase the uptake of amino acids in response to fructose feeding, we have found that chick embryo fibroblasts do not increase amino acid uptake when either totally starved of hexose or fructose-fed (unpublished).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
5048
Cell Biology: Nishino el al.
Proc. Natl. Acad. Sct. USA 75 (1978)
Table 1. Enhanced AIB uptake by glucose-starved cells
Culture conditions
Mean AIB uptake,* pmol/mg protein per min
Enhancement factor, -fold (1.00) 2.74 ± 1.21
Glucose-fed 286 + 188 Glucose-starved 637 ± 273 Confluent Nil hamster cells were maintained for 18-24 hr in 35-mm plastic culture dishes in Dulbecco's modified Eagle's medium containing 4 mg of D-glucose per ml (glucose-fed) or 4 mg of D-fructose per ml (glucose-starved). Twenty-eight duplicate cultures were assayed for AIB uptake by the monolayer assay. * Absolute AIB uptake values and enhancement factors varied from experiment to experiment. In this table, the mean i SD AIB uptake and the mean ± SD enhancement factors were calculated separately. Uptake data were grouped separately from enhancement data and the mean + SD was obtained for each parameter with a Texas Instruments SR-51A calculator.
phosphate-buffered saline and then 0.5 ml of phosphate-buffered saline (or sodium-free phosphate-buffered saline) containing 0.1 mM [14C]AIB (0.5 MCi/ml), and 0.1 mM L-[3H]glucose (2 MCi/ml) was added to the culture dish. Cells were allowed to take up the AIB for various times at 22° and then were washed five times with 2 ml of ice-cold phosphate-buffered saline. The radioactivity of ethanol extracts was measured by liquid scintillation and normalized for the protein content of each dish as described (2, 6). Corrections for simple diffusion and interstitial (i.e., nonspecific) trapping were made by subtracting the amount of L-glucose associated with each sample. Suspended cells. Aliquots of cells suspended by scraping from dishes and roller bottles were twice washed with sodium-free phosphate-buffered saline and collected by centrifugation (900 X g, 10 min). The final cell suspension was divided into two equal volumes and collected by centrifugation, and the clear supernatant was removed by aspiration. The assay was initiated by addition of the radioactive solutions (in phosphate-buffered saline or sodium-free phosphate-buffered saline, as described above) at 22°. By a modification of the technique. described by 3 C
0
0_.U 0,
U
