System ASC Research Articles

Glutamine transport and human hepatocellular transformation.

Among other functions, the liver serves to regulate both glucose and nitrogen economy in the body, and in humans, the amino acid glutamine is a major gluconeogenic substrate and the primary extrahepatic ammonia shuttle. Accordingly, the liver acinus possesses a unique heterogeneous metabolic architecture suited to carry out these functions with glutamine-consuming urea cycle and gluconeogenic enzymes in the periportal hepatocytes and a high capacity for glutamine synthesis in the perivenous hepatocytes, resulting in net glutamine balance across the hepatic bed under most conditions. Cytoplasmic levels of glutamine are significantly governed by the activity of the System N transporter in the plasma membrane of parenchymal cells; in this capacity, this glutamine carrier has been shown to represent a rate-limiting step in metabolism via glutaminase. The unique properties of System N allow it to rapidly adapt in support of the dynamic demands of whole body ammonia and glucose homeostasis. In contrast to System N in normal hepatocytes, human hepatoma cells take up glutamine at rates several-fold faster through a broad-specificity higher affinity transporter with characteristics of System ASC or B0. It is currently hypothesized that the expression of this high activity carrier by hepatoma cells combined with accelerated metabolism and tumor-induced derangements in hepatocellular architecture result in net glutamine consumption, and may underlie the diminished plasma glutamine levels observed in patients with hepatocellular carcinoma (HCC). The transport of glutamine through System ASC has been shown to regulate growth in some human hepatoma cells, which suggests this transporter may warrant consideration as a therapeutic target for HCC.

Rat liver endothelial cell glutamine transporter and glutaminase expression contrast with parenchymal cells.

Despite the central role of the liver in glutamine homeostasis in health and disease, little is known about the mechanism by which this amino acid is transported into sinusoidal endothelial cells, the second most abundant hepatic cell type. To address this issue, the transport of L-glutamine was functionally characterized in hepatic endothelial cells isolated from male rats. On the basis of functional analyses, including kinetics, cation substitution, and amino acid inhibition, it was determined that a Na+-dependent carrier distinct from system N in parenchymal cells, with properties of system ASC or B0, mediated the majority of glutamine transport in hepatic endothelial cells. These results were supported by Northern blot analyses that showed expression of the ATB0 transporter gene in endothelial but not parenchymal cells. Concurrently, it was determined that, whereas both cell types express glutamine synthetase, hepatic endothelial cells express the kidney-type glutaminase isozyme in contrast to the liver-type isozyme in parenchymal cells. This represents the first report of ATB0 and kidney-type glutaminase isozyme expression in the liver, observations that have implications for roles of specific cell types in hepatic glutamine homeostasis in health and disease.

Neutral amino acid transport in bovine articular chondrocytes.

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.

A Na+-DEPENDENT SYSTEM A AND ASC-INDEPENDENT AMINO ACID TRANSPORT SYSTEM STIMULATED BY GLUCAGON IN RAT HEPATOCYTES

The effects of glucagon on amino acid transport in rat hepatocytes are not fully understood. We examined the effect of this hormone on alanine, serine and cysteine preferring system (system ASC)-mediated amino acid transport in rat hepatocyte monolayers using 2-aminoisobutyric acid (AIB) and l -cysteine. Glucagon induced a time and protein synthesis-dependent stimulation of Na+-dependent alanine preferring system (system A)-independent AIB transport. The glucagon-induced increase in transport activity was not modified by substrate starvation and not related to changes in the intracellular pool of amino acids. Glucagon did not modify system ASC activity measured by l -cysteine. Therefore the transport activity of AIB independent of system A stimulated by glucagon cannot be attributed to system ASC. This suggests a Na+-dependent transport system in rat hepatocytes not identified until now.

Effects of pH changes on systems ASC and B in rabbit ileum.

Influx of D-aspartate (D-Asp), L-glutamate (L-Glu), and serine (Ser) across the brush-border membrane of the intact mucosa from rabbit ileum has been examined. L-Glu influx is chloride independent and completely sodium dependent. D-Asp and L-Glu share a transport system with a maximum transport rate of 1 micromol. cm-2. h-1 and an apparent affinity constant (K1/2) of approximately 0.3 mM. The function of this transport system is pH insensitive between pH 5.65 and 8.2, and bipolar amino acids do not affect the way in which the transport system handles D-Asp and L-Glu. The characteristics of this transport system match those of system X-AG. L-Glu and Ser share a transporter for which the inhibitor constant (Ki) of L-Glu against Ser decreases from 54 to 10 mM when pH is reduced from 7.2 to 5.65, while the maximum rate of transport remains unaffected at approximately 10 micromol. cm-2. h-1. The Ki values (5 mM) of Ser against L-Glu influx and the L-Glu-sensitive contribution to Ser influx (0.8 micromol. cm-2. h-1 at 1 mM Ser) are the same at both pH values. The L-Glu-sensitive transport of Ser together with the contribution of system bo,+ account for approximately 50% of Ser influx at pH 7.2. The remaining 50% can be ascribed to system B. Transport of Ser by system B is reduced by >95% at pH 5.65. At pH 7. 2 Ki of Ser against transport of leucine (Leu) by system B is 18 mM and Ki of Leu against transport of Ser is 1.7 mM. The low-affinity transport of L-Glu and the L-Glu-sensitive transport of Ser are performed by an equivalent of system ASC. Supplementary experiments using the jejunum confirm the validity of these results for a major portion of the rabbit small intestine.

Determinants of glutamine dependence and utilization by normal and tumor-derived breast cell lines.

A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth. GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media. However, the GLN requirements of tumor cell lines and the factors that determine a cell's need for GLN have not been comprehensively studied. Also, it remains unclear how various metabolic pathways contribute to GLN consumption. In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin. These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates. GLN uptake was facilitated by a single, common transporter functionally defined as System ASC. However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines. Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity. The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor-derived cells. The rates of CO2 production from GLN were similar for each cell lines. Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32-80% of media GLN converted to glutamate. Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system x(c)-. These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis.

Adaptive alterations in cellular metabolism with malignant transformation.

The authors studied the differences between glutamine and glucose utilization in normal fibroblasts and in fibrosarcoma cells to gain insights into the metabolic changes that may occur during malignant transformation. The process of malignant transformation requires that cells acquire and use nutrients efficiently for energy, protein synthesis, and cell division. The two major sources of energy for cancer cells are glucose and glutamine. Glutamine is also essential for protein and DNA biosynthesis. We studied glucose and glutamine metabolism in normal and malignant fibroblasts. Studies were done in normal rat kidney fibroblasts and in rat fibrosarcoma cells. We measured glutamine transport across the cell membrane, breakdown of glutamine by the enzyme glutaminase (the first step in oxidation), glutamine and glucose oxidation rates to CO2, rates of protein synthesis from glutamine, and glutamine-dependent growth rates. Glutamine transport rates were increased more than sixfold in fibrosarcomas compared to normal fibroblasts. In fibroblasts, glutamine transport was mediated by systems ASC and A. In malignant fibrosarcomas, only system ASC was identifiable, and its Vmax was 15 times higher than that observed in fibroblasts. Despite an increase in transport, glutaminase activity was diminished and glutamine oxidation to CO2 was reduced in fibrosarcomas versus normal fibroblasts. In fibroblasts, glutamine oxidation was 1.8 times higher than glucose oxidation. In contrast, glucose oxidation was 3.5 times greater than glutamine oxidation in fibrosarcomas. Protein synthesis from glutamine transported by fibrosarcomas was threefold greater than that observed in normal fibroblasts. Despite marked increases in glutamine utilization and glucose oxidation in fibrosarcoma cells, growth rates were higher in the normal fibroblasts. The process of malignant transformation is associated with a marked increase in cellular glutamine transport, which is mediated by a single high-affinity, high-capacity plasma membrane carrier protein. In normal fibroblasts, the transported glutamine is used primarily for energy production via oxidation of glutamine carbons to CO2. In fibrosarcomas, glutamine oxidation falls and glutamine is shunted into protein synthesis; simultaneously, the malignant cell switches to a glucose oxidizer. The increased glutamine transport and glucose oxidation in fibrosarcomas appears to be related to the malignant phenotype and not merely to an increase in cell growth rates.

Characterization of multiple cysteine and cystine transporters in rat alveolar type II cells.

Cysteine availability is rate limiting for the synthesis of glutathione, an important antioxidant in the lung. We used rat alveolar epithelial type II cells to study the mechanism of cysteine and cystine uptake. Consistent with carrier-mediated transport, each uptake process was saturable with Michaelis-Menten kinetics and was inhibited at 4 degrees C and by micromolar levels of amino acids or analogs known to be substrates for a specific transporter. A unique system XAG was found that transports cysteine and cystine (as well as glutamate and aspartate, the only substrates previously described for system XAG). We also identified a second Na(+)-dependent cysteine transporter system, system ASC, and two Na(+)-independent transporter systems, system xc for cystine and system L for cysteine. In the presence of glutathione at levels measured in rat plasma and alveolar lining fluid, cystine was reduced to cysteine and was transported on systems ASC and XAG, doubling the transport rate. Cysteinylglycine, released from glutathione at the cell surface by gamma-glutamyl transpeptidase, also stimulated uptake after reduction of cystine. These findings suggest that, under physiological conditions, cysteine and cystine transport is influenced by the extracellular redox state.

An Expression System for Mammalian Amino Acid Transporters Using a Stably Maintained Episomal Vector

Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and XAG−, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein–Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4×) and EAAC1 (39×) transport activity than does the vaccinia/HeLa expression system (2.6× and 22×, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X−AG, or A; and (iii) the endogenous transport velocities of Systems ASC and XAG−in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.

L-Alanine uptake by frog ( Rana esculenta) red blood cells

l-Alanine uptake has been studied in frog red blood cells. The present study shows the presence of different carriers for this amino acid in these cells. In the physiological concentration range, most l-alanine is taken up through the Na +-dependent system ASC, although the sodium-independent systems asc and L are also active. The competitive inhibition data obtained makes difficult to differentiate the two Na +-independent activities in a clear contrast with data from fish or mammalian erythrocytes, indicating that despite its widespread occurrence in vertebrates, these carriers show characteristics that are species specific.

Seasonal variation in uptake of short-chain neutral amino acid by red blood cells and hepatocytes in trout (Salmo trutta).

The present study shows that the capacity of trout (Salmo trutta) red blood cells (RBCs) and freshly isolated hepatocytes to take up short-chain neutral amino acids changes according to a seasonal pattern. Maximal amino acid uptake rates in RBCs were obtained in winter and spring, while minima were seen in summer and autumn. In contrast, the maximal rates for the freshly isolated hepatocytes were obtained in autumn and winter, and the minima were seen in spring and summer. In addition, by studying the uptake of glycine, evidence was found that the activities of the amino acids carriers ASC, asc and Gly in RBCs varied according to a seasonal rhythm. The activity of the ASC and asc systems changed in parallel with the global uptake of amino acids. Moreover, the RBC:plasma concentration ratio for certain substrates of these carriers (alanine, serine and glycine) varied accordingly. In contrast, the activity of the Gly system was modified inversely with respect to the overall amino acid uptake. The activity of the ASC system in freshly isolated hepatocytes was also seasonally modified, reaching a maximum in autumn, shortly before the reproductive period.

Characterization of Glutamine and Glutamate Transport in Rat Lung Plasma Membrane Vesicles

Insufficient glutamine for the lungs during sepsis may contribute to an impairment in lung function. Lung glutamine metabolism is supported by both blood glutamine uptake andde novobiosynthesis using circulating glutamate as a precursor. Information regarding the specific plasma membrane carriers involved in this uptake is lacking. Furthermore, the effect of sepsis on amino acid transport in whole lung has not been studied. We isolated lung plasma membrane vesicles (LPMVs) from control and LPS-treated rats and assayed glutamine and glutamate transport activity in LPMVs. Vesicle purity and functionality were confirmed by time-dependent concentrative amino acid uptake in the presence of Na+, impoverishment of microsomal enzymes, and a 25-fold enrichment in the plasma membrane marker 5′-nucleotidase. Eighty percent of glutamine uptake in lung vesicles was mediated via the high affinity Na+-dependent carrier System ASC (Vmax= 80 ± 10 pmole/mg protein/15 sec;Km= 224 ± 30 μM) while 19% occurred via the Na+-independent System ASC (Vmax= 11 ± 2 pmole/mg/15 sec;Km= 141 ± 23 μM). Ninety percent of glutamate transport was mediated by the Na+-independent System xAG−. Treatment of rats with LPS resulted in a decrease in both glutamine and glutamate transport in LPMVs. LPMVs offer a novel method for characterizing lung amino acid transport and studying the effects of catabolic states on this activity. The effects of endotoxin on System ASC and xAG−activity may contribute to reduced lung glutamine availability during septic states which may impair cellular metabolism and function.

Interleukin-1β and interleukin-6 stimulate 2-methylaminoisobutyric acid uptake in HepG2 cells

The metabolic response to inflammation involves an increased uptake of amino acids in the liver. It has been suggested that cytokines, such as interleukin-1β and interleukin-6, could be involved in this increased amino acid uptake. We investigated the role of these two inflammatory cytokines in regulating hepatic amino acid transport systems in the human hepatoma cell line, HepG2. Uptake of methylaminoisobutyric acid, the most specific known substrate of system A, and of glutamine, both transported by other sodium-dependent transport systems ASC and N, was assayed after incubation of the cells for various times with cytokines, using the cluster-tray method. Interleukin-1β and interleukin-6 (1000 U/ml) stimulated methylaminoisobutyric acid uptake by 36 ± 6 and 41 ± 4%, respectively (per cent ± SD, n ≥ 6). Under our experimental conditions, these cytokines had no effect on glutamine uptake. The stimulatory effect on methylaminoisobutyric acid uptake was not increased by combining the cytokines or by the presence of dexamethasone. The cytokine effect was abolished by cycloheximide, suggesting the involvement of de novo protein synthesis in this activation of transport system A. These data demonstrate that, in our culture conditions, interleukin-1β and interleukin-6 indirectly exert a stimulatory effect on methylaminoisobutyric acid transport in HepG2 cells.

Sodium-Dependent and -Independent Transport of L-Glutamate in the Rat Intestinal Crypt-like Cell Line IEC-17

Mechanisms of L-glutamate transport in intestinal crypts were investigated using the rat intestinal crypt-like cell line IEC-17. Kinetic analysis and competition experiments run in the presence or in the absence of extracellular sodium indicate that L-glutamate uptake occurs through three different transport components: (1) a high affinity Na+-independent component also carrying cystine, similar to system x−C; (2) a high affinity Na+-dependent component inhibited by D- and L-aspartate corresponding to the ubiquitous system X−A,G; and (3) a low affinity Na+-dependent system resembling the neutral amino acid transport system ASC. The simultaneous presence of these three components suggest that crypt cells are ready to face potential high variations of L-glutamate concentration in the intestinal villus environment.

Identification of a system N-like Na(+)-dependent glutamine transport activity in rat brain neurons.

Glutamine is a primary precursor for the biosynthesis of the neurotransmitters glutamate and gamma-aminobutyric acid. It is proposed that glutamine, synthesized and released by astrocytes, is transported into the neuron for subsequent conversion to neurotransmitters. To provide a more complete characterization of this process, we have delineated the transport systems for glutamine uptake in primary cultures of brain neuronal cells from 1-day-old rats. The Na(+)-dependent glutamine entry is mediated by system A, system ASC, and a third, previously unidentified, activity that has been tentatively designated as system Nb. System Nb activity can be monitored by assaying Na(+)-dependent [3H]glutamine uptake in the presence of 2 mM concentrations of both 2-(methylamino) isobutyric acid and threonine to block uptake by systems A and ASC, respectively. The newly identified transport activity exhibits an apparent substrate specificity that is unique compared with the hepatic system N, because it is inhibited by glutamine and asparagine, but not by histidine. Also, the affinity of system Nb for glutamine, as estimated from K(m) values, is significantly greater than that observed for the hepatic and muscle Na(+)-dependent glutamine transporters, systems N and Nm. In sharp contrast to the hepatic system N transporter, system Nb exhibits a relative insensitivity to pH and does not permit Li+ substitution for Na+ as the cosubstrate. The substrate specificity, kinetic analysis, pH sensitivity, and cation dependence of this transport activity indicate that it represents a glutamine transport system not previously identified.

Influence of hypo-osmolality on the activity of short-chain neutral amino acid carriers in trout (Salmo trutta) red blood cells.

The present study shows that in trout red blood cells the activity of some amino acid carriers, not directly involved in cell volume regulation, is affected by external osmolality. Glycine uptake has been used as the experimental approach because it was shown previously that it is effected by different carriers, namely the Na+-dependent ASC and Gly systems, as well as the Na+-independent asc and L systems. An increase in the uptake through the Gly system and the two Na+-independent carriers was found, while the ASC system appeared to be downregulated. Those systems whose activities were increased by hypo-osmolality did not share the mechanism by which this increase was obtained. Thus, the Gly system was sensitive to intracellular ionic changes, while the Na+- independent systems were mechanically stimulated, as assessed by the iso-osmotic swelling caused by ammonium chloride. On the other hand, a volume-sensitive transporter may be present in trout red blood cells, which is involved in the swelling-induced glycine movement, as can be deduced from the effect of some inhibitors such as pyridoxal phosphate, DIDS (4,4'-diisothiocyanate-stilbene-2,2'-disulfonic acid) and quinine.

Correlation between changes in cell ascorbate and growth of Lupinus albus seedlings

Summary The growth of Lupinus albus seedlings is stimulated when ascorbate (Asc) content is experimentally raised; on the contrary, when Asc content is lowered by lycorine — an alkaloid inhibiting Asc biosynthesis — the growth is inhibited. The stimulation of growth by Asc is due to the promotion of cell division and cell expansion. Redox enzymes of the Asc system respond in different manners to the Asc changes. Asc peroxidase is the enzyme that is most affected by Asc changes in the cell: its activity increases or decreases in parallel with the Asc content. Dehydroascorbic acid (DHA) reduction capability is affected in the opposite way: when Asc content rises, the DHA reduction ability of the cells drops, while it rises when Asc decreases. Ascorbate free radical (AFR) reductase activity does not vary with changing Asc content. A possible regulatory function of Asc redox enzymes is discussed.

Regulation of the ASC system and Na+/K + pump activities in brown trout (Salmo trutta) hepatocytes

The present study investigates the regulation of Na+/K+ pump activity and alanine uptake in trout hepatocytes. Pump activity increased when cells were incubated in an amino-acid-free medium, while it was reduced in cells from fasted animals. Short-term exposure (3 h) to glucagon modified the activity of the pump in a complex seasonally dependent pattern: in experiments carried out in autumn and winter there was some inhibition, while in spring the pump was activated by this hormone. Pharmacological modification of levels of two intracellular signal transducers, namely cyclic AMP and Ca2+, always led to a reduction in pump activity. These experiments were conducted in May, when activation of the pump by glucagon exposure occurred. There is no apparent explanation for the mechanism by which this hormone modifies the activity of the pump. Glucagon also regulates the activity of system ASC (a Na+-dependent amino acid carrier with short-chain neutral amino acids as preferred substrates). This regulation also showed a seasonally dependent pattern, although the pattern was opposite to that found for the regulation of Na+/K+ pump activity.

Modulation of transport systems for neutral and anionic amino acids in mesenchymal cells.

Introduction The concerted operation of several amino acidtransport systems ensures the optimal composition of the intracellular compartment as well as inter-organ amino acid flow [ l ] . The operation of these transport systems is linked through substrates accumulated by active transport systems (such as Systems A and X,;;) and then exchanged with other amino acids through exchange pathways (Systems L, ASC and &-) [2-41. Glutamine is an ideal substrate for the co-ordinated operation of transport systems for neutral amino acids. Indeed, this amino acid is a good substrate for System A but can also interact with Systems ASC and L [5,6] as well as with specific N-type transport systems, originally described in the liver [7] but also characterized in some mesenchymal models [8]. In the intracellular compartment, 1,-glutamine can be easily converted into 1.-glutamate. However, the intracellular pool of 1.-glutamate can also be fuelled through specific transport routes, such as System X,;; [9,10], an active transport mechanism which has the same operational features of the Na+,K+dependent glutamate carriers, cloned from nervous system and absorptive epithelia [ 1 1 131. Thus the high intracellular concentrations of glutamine and glutamate found in mesenchymal cells [ 141 are the result of the operation of both System A (through glutamine uptake) and System X,; (through glutamate uptake). In mesenchymal cells the kinetic features of these two mechanisms are very different; the V,,, of System A is much higher than that of System X,;; [2]; conversely, the affinity of System A for its substrates is much lower than that of System X,;;. Mammalian cells are usually cultured in media that contain high concentrations of glutamine and little glutamate, a situation resembling that observed in extracellular fluids in viva [ 15,161. Under these conditions, mesenchymal cells accumulate glutamine through System A

Glutamine as a regulator of DNA and protein biosynthesis in human solid tumor cell lines.

The transport of glutamine by six different human solid tumor-derived cell lines (e.g., breast, colon, liver) was characterized and the impact of glutamine deprivation on rates of tumor cell proliferation and DNA and protein synthesis was assayed. Glutamine is added routinely to cell culture media and its importance for cellular growth has been established. However, carrier-mediated glutamine transport by solid tumors has not been studied extensively, and the mechanisms by which glutamine contributes to cell growth regulation require further investigation. In a panel of different human solid tumor-derived cells, sodium-dependent glutamine transport was characterized in vitro and rates of cell proliferation, protein and DNA synthesis, as well as thymidine transport, were correlated with glutamine concentrations in the culture media. In all cells, regardless of tissue origin, sodium-dependent glutamine transport was mediated almost exclusively by a single carrier. There was a range of Michaelis constants (Km) and maximal transport velocities (Vmax) for the glutamine transporter in each cell type, but the amino acid inhibition profiles were nearly identical, consistent with uptake by the System ASC family of transporters. Rates of cell growth, DNA and protein synthesis, and thymidine transport correlated with the glutamine concentration in the culture media, indicating the central role of this amino acid in regulating cellular proliferation. These data indicate that glutamine transport by all solid tumors is mediated by the System ASC family of transporters. The variation in Km values suggests that some cancers may be better suited to survive in a low glutamine environment than others. The mechanism by which glutamine supports cell proliferation and regulates cell cycle kinetics involves its modulation of DNA and protein biosynthetic rates.