Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.     

Role of proton dissociation in the transport of cystine and glutamate in human diploid fibroblasts in culture

The effect of extracellular pH on the transport interaction of cystine and glutamate in cultured human diploid cells was examined over the pH range of 5.8-8.0. The initial rates of uptake of cystine increased with an increase in pH and glutamate potently inhibited the cystine uptake independently of pH. The uptake of glutamate was almost invariable within the pH range, but it was inhibited by cystine in a pH-dependent manner; the inhibition increased with an increase in pH. Regardless of pH, the uptake of cystine and glutamate was strongly inhibited by alpha-aminoadipate, alpha-aminopimelate, and homocysteate. From the pK values of cystine and other amino acids, it is suggested that cystine is transported in the same ionic form as is glutamate.     

A novel function of glutamine in cell culture: utilization of glutamine for the uptake of cystine in human fibroblasts

Transport and metabolism of glutamine has been investigated in human diploid fibroblasts, IMR-90. Glutamine was taken up via System ASC (Na+-dependent amino acid transport system especially reactive with short or polar side chain amino acids). In the routine culture medium the cells contained a large quantity of glutamate; its major source was shown to be glutamine in the medium. Previously we described a transport system that mediates the entrance of cystine in exchange for the exit of glutamate (Bannai, 1986). Since the cystine taken up is reduced to cysteine and the cysteine readily exits to the medium where it is oxidized to cystine, a cystine-cysteine cycle across the plasma membrane has been postulated. When the cells were cultured in glutamate/glutamine-free medium, intracellular glutamate decreased, depending on the amount of cystine in the medium; in the absence of cystine, glutamate decreased very slowly. When the cells were cultured in ordinary medium, glutamine in the medium decreased, and glutamate in the medium increased. Both changes were well correlated with cystine concentration in the medium. These results are consistent with the view that the intracellular glutamate, of which the source is glutamine in the medium, is released from the cells into the medium in order to take up cystine and thereby to rotate the cystine-cysteine cycle. In the routine culture one-third to one-half of the total consumption of glutamine seems to be used for the uptake of cystine.     

Cystine and dibasic amino acid uptake by opossum kidney cells

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.     

Transport of cystine in isolated rat hepatocytes in primary culture

Uptake of cystine and factors affecting the transport were investigated in adult rat hepatocytes in primary monolayer culture. The cystine uptake was initially mediated by Na+-dependent route(s). However, the activity of Na+-dependent uptake decreased markedly during the culture, and Na+-independent uptake emerged with a lag period of 12 h in response to insulin and dexamethasone in the culture medium. After 48 h in culture, cystine was mainly transported into the cells through this Na+-independent route. The action of insulin and dexamethasone on the enhancement of the Na+-independent uptake was apparently additive, and the enhancement was completely blocked by cycloheximide or actinomycin D. Emergence of the Na+-independent uptake of cystine was also regulated by cell density; at lower density, the uptake tended to be elevated. The transport of cystine through the Na+-independent system was pH sensitive and was inhibited by some anionic amino acids, such as glutamate and homocysteate, but not by aspartate. These results suggest that the emerging system is similar to the ones reported in fibroblasts and in some hepatoma cell lines; the anionic form of cystine is transported through the system.     

Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine-free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na⁺-independent, glutamate-sensitive route previously found in various types of cells and designated as system x⁻_c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.     

A novel approach to enhancing cellular glutathione levels

GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of toxic stress. Of the three amino acids that comprise GSH, cysteine is limiting for GSH synthesis. As extracellularly cysteine is readily oxidized to form cystine, cystine transport mechanisms are essential to provide cells with cysteine. Cystine uptake is mediated by system x(c)(-), a Na(+)-independent cystine/glutamate antiporter. Inhibition of system x(c)(-) by millimolar concentrations of glutamate, a pathway termed oxidative glutamate toxicity, results in GSH depletion and nerve cell death. Recently, we described a series of compounds derived from the conjugation of epicatechin (EC) with cysteine and cysteine derivatives that protected nerve cells in culture from oxidative glutamate toxicity by maintaining GSH levels. In this study, we characterize an additional EC conjugate, cysteamine-EC, that is 5- to 10-fold more potent than the earlier conjugates. In addition, we show that these EC conjugates maintain GSH levels by enhancing the uptake of cystine into cells through induction of a disulfide exchange reaction, thereby uncoupling the uptake from system x(c)(-). Thus, these novel EC conjugates have the potential to enhance GSH synthesis under a wide variety of forms of toxic stress.     

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.     

Exchange of cystine and glutamate across plasma membrane of human fibroblasts

It is found that both the inward and outward transport of cystine and glutamate through the plasma membrane of cultured human fibroblasts is mediated mostly by a single transport system. Cystine and glutamate at one side of the membrane stimulate the passage of these amino acids present at the other side of the membrane. When the concentration of intracellular glutamate is reduced to near zero, cystine hardly enters the cell, and likewise the release of glutamate from the cell ceases when cystine is absent in the medium. Homocysteate and alpha-aminoadipate share this transport system and, when added, similarly participate in the transport process. Since the intracellular pool of cystine is negligibly small whereas that of glutamate is very large, the physiologic flows via this system are the entry of cystine and the exodus of glutamate coupled together. Measurements of the rate of uptake of cystine into the cells and the rate of release of glutamate from the cells indicate that the entry of cystine and the exodus of glutamate occur at a ratio close to 1:1. Since cystine is known to behave as an anionic form in this transport, it is concluded that the transport system for cystine and glutamate in plasma membrane of human fibroblasts is a kind of an anion-exchanging agency.     

A cystine-cysteine shuttle mediated by xCT facilitates cellular responses to S-nitrosoalbumin

We have shown previously that extracellular cysteine is necessary for cellular responses to S-nitrosoalbumin. In this study we have investigated mechanisms involved in accumulation of extracellular cysteine outside vascular smooth muscle cells and characterized the role of cystine-cysteine release in transfer of nitric oxide (NO)-bioactivity. Incubation of cells with cystine led to cystine uptake, reduction, and cysteine release. The process was inhibitable by extracellular glutamate, suggesting a role for system x(c)(-) amino acid transporters. Smooth muscle cells express this transporter constitutively and induction of the light chain component (xCT) by either diethyl maleate or 3-morpholino-sydnonimine (SIN-1) led to glutamate-inhibitable cystine uptake and an increased rate of cysteine release from cells. Likewise, overexpression of xCT in smooth muscle cells or endothelial cells led to glutamate-inhibitable cysteine release. The resulting extracellular cysteine was found to be required for transfer of NO from extracellular S-nitrosothiols into cells via system L transporters leading to formation of cellular S-nitrosothiols. Cysteine release coupled to cystine uptake was also found to be required for cellular responses to S-nitrosoalbumin and facilitated S-nitrosoalbumin-mediated inhibition of epidermal growth factor signaling. These data show that xCT expression can constitute a cystine-cysteine shuttle whereby cystine uptake drives cysteine release. Furthermore, we show that extracellular cysteine provided by this shuttle mechanism is necessary for transfer of NO equivalents and cellular responses to S-nitrosoablumin.     

Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes

The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na(+)-dependent and a Na(+)-independent system. The Na(+)-dependent system was inhibited by aspartate and was consistent with the commonly occurring system designated system X-AG. The Na(+)-independent system was inhibited by cystine and was consistent with system x-c described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na(+)-independent glutamate uptake. In C-6 cells, the glutamate uptake via systems X-AG and x-c accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes, the glutamate uptake via system X-AG was very potent, whereas the uptake via system xc- was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system xc- was necessary for the uptake of cystine, another substrate of system xc-. Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration.     

The inhibitory effect of glutamate on the growth of a murine hybridoma is caused by competitive inhibition of the x- C transport system required for cystine utilization

Glutamic acid was found to be growth inhibitory to a murinelymphocyte hybridoma in a concentration-dependent manner from 3to 12 mM glutamate. At 12 mM glutamate there was a 70% decreasein the specific growth rate of the cells. Attempts to alleviateinhibition or adapt cells to growth in glutamate-based mediawere unsuccessful. It is proposed that elevated glutamate levelsimpair adequate uptake of cystine, a critical amino acid for thesynthesis of glutathione. Glutathione is required by cells toprevent intracellular oxidative stress. The measured rate ofuptake of U-¹⁴C L-cystine into the cells was found to havethe following parameters: K_m = 0.87 mM, V_max = 0.9nmole/mg cell protein per min. The uptake was sodiumindependent and resembled the previously described x^- _ctransport system, with elevated glutamate levels causingextensive inhibition. Glutamate at a concentration of 1.4 mMcaused a 50% decrease in cystine uptake from the serum-freegrowth medium. Glutamate was taken up from the external medium(K_m = 20 mM and V_max = 12.5 nmole/mg cell protein permin) by the same transport system in a stereo specific, sodiumindependent manner. Of the amino acids examined, it was foundthat cystine and homocysteic acid were the most extensiveinhibitors of glutamate uptake and that inhibition was competitive. Metabolic profiles of the cells grown in culturescontaining enhanced glutamate levels revealed an overallincrease in net production of alanine, serine, asparagine andaspartate. A substantially increased specific consumption ofglutamate was accompanied by a decreased consumption of cystine,valine and phenylalanine.The combined kinetic and metabolic results indicate thatglutamate and cystine are taken up by the anionic transportsystem x^- _c. The increasing levels of glutamate in themedium result in a decreased transport of cystine by this systemdue to competitive inhibition by glutamate.     

Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress

Glutamate binds to both excitatory neurotransmitter binding sites and a Cl(-)-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (N18-RE-105) are susceptible to glutamate-induced cytotoxicity. The Cl(-)-dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Lowering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and Ca2+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like N18-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.     

Redox imbalance in cystine/glutamate transporter-deficient mice

Cystine/glutamate transporter, designated as system x(-)(c), mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT(-/-) mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT(-/-) mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT(-/-) mice was lower than that in the xCT(-/-) mice. The embryonic fibroblasts derived from xCT(-/-) mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT(-/-)-derived cells and permitted growth. These results demonstrate that system x(-)(c) contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.     

Low membrane transport activity for cystine in resting and mitogenically stimulated human lymphocyte preparations and human T cell clones.

In order to determine whether the cysteine requirement of human T lineage cells is met primarily by extracellular cysteine or by cystine, amino-acid-transport activities were measured in resting and mitogenically stimulated human peripheral blood lymphocytes (PBL) and several human T cell clones and T cell tumors. The transport activity of the small neutral amino acids cysteine and alanine (ASC system) and the transport of the cationic amino acid arginine (y+ system) were found to be markedly increased after stimulation of PBL by the T cell mitogen phytohemagglutinin from Phaseolus vulgaris. The anionic transport activity for cystine and glutamate (Xc- system), in contrast, was extremely weak in both resting and activated human PBL and also in all human T cell lines under test. The weak system Xc- activity of human T lineage cells was further confirmed by an independent line of experiments showing that an increase of the extracellular concentration of glutamate, i.e. a competitive inhibitor of cystine transport, causes a decrease in the intracellular cystine levels in cells of the promonocytic line U937, but not in T lineage cells (Molt-4). A third set of experiments showed that the rate of DNA synthesis in mitogenically stimulated human PBL is strongly influenced by variations of the extracellular cysteine level, even in cultures with relatively high and approximately physiological concentrations of cystine. Cysteine cannot be replaced in this case by the addition of corresponding amounts of cystine or methionine. This demonstrates an important functional consequence of the weak cystine transport activity of human lymphocytes. The results may be relevant for the pathogenetic mechanism of the acquired immunodeficiency syndrome, since the mean plasma cysteine concentration of human-immunodeficiency-virus-1-seropositive persons was found to be strongly decreased in comparison with that of healthy blood donors, and since the cysteine level even of healthy persons is extremely low in comparison with all other protein-forming amino acids.     

Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

The Uptake and Metabolism of Cysteine by Giardia lamblia Trophozoites

ABSTRACT. The cysteine, cystine, methionine and sulfate uptake and cysteine metabolism of Giardia lamblia was studied. Initial experiments indicated that bathocuproine sulphonate (20 μM) added to Keister's modified TYI-S-33 medium supported the growth of G. lamblia at low L-cysteine concentration. This allowed the use of high specific activity radiolabeled L-cysteine for further studies. The analyses of L-cysteine uptake by G. lamblia indicate the presence of at least two different transport systems. The total cysteine uptake was non saturable, with a capacity of 3.7 pmoles per 10⁶ cells per min per μM of cysteine, and probably represent passive diffusion. However, cysteine transport was partially inhibited by L-methionine, D-cysteine and DL-homocysteine. indicating that another system specific for SH-containing amino acids is also present. Cysteine uptake was markedly decreased in medium without serum. In contrast to cysteine, the uptake of L-methionine and sulfate were carried out by saiurable systems with apparent K_m, of 71 and 72 μM, respectively, but the Vmax of the uptake of sulfate was six orders of magnitude lower than the Vmax of methionine uptake. Cystine was not incorporated into trophozoites. [³⁵S]-labeled L-cysteine and L-methionine, but not [³⁵S]sulfate, were incorporated into Giardia proteins, indicating that the parasite lacks the capacity to synthesize cysteine or methionine from sulfate. Neither cystathionine γ lyase nor crystathionine γ synthase activities was detected in homogenates of Giardia lamblia , suggesting that the transsulfuration pathway is not active and there is no conversion of methionine to cysteine. Our data indicate that cysteine is essential for Giardia because the parasite: a) cannot take up cystine, and b) cannot synthesize cysteine de novo.     

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.     

The reduction of cystine during its transport into rat jejunal everted segments and sacs

1. Everted segments and sacs of rat jejunum were incubated in buffer containing [(35)S]cystine. 2. Concentration gradients were achieved by both segments and sacs, and the effects of duration of incubation and of cystine concentration on the isotope distribution ratios were determined. 3. Kinetic constants were determined for the uptake of cystine by both segments and sacs, and the differences between the two systems are discussed. 4. Reduction to cysteine was virtually complete intracellularly and in the sac lumen. Extensive reduction in the medium occurred only when segments were incubated. 5. Anaerobiosis prevented a concentration gradient being obtained between the medium and the tissue, but had little effect on the extent of reduction to cysteine in the tissue and sac lumen. 6. It is concluded that cystine is transported by an active process into rat jejunum, where it is present almost entirely in the reduced form, and that efflux of cysteine occurs through the serosal surface.     

The x(c)- cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases

The x(c) (-) cystine/glutamate antiporter is a major plasma membrane transporter for the cellular uptake of cystine in exchange for intracellular glutamate. Its main functions in the body are mediation of cellular cystine uptake for synthesis of glutathione essential for cellular protection from oxidative stress and maintenance of a cystine:cysteine redox balance in the extracellular compartment. In the past decade it has become evident that the x(c) (-) transporter plays an important role in various aspects of cancer, including: (i) growth and progression of cancers that have a critical growth requirement for extracellular cystine/cysteine, (ii) glutathione-based drug resistance, (iii) excitotoxicity due to excessive release of glutamate, and (iv) uptake of herpesvirus 8, a causative agent of Kaposi's sarcoma. The x(c) (-) transporter also plays a role in certain CNS and eye diseases. This review focuses on the expression and function of the x(c) (-) transporter in cells and tissues with particular emphasis on its role in disease pathogenesis. The potential use of x(c) (-) inhibitors (e.g., sulfasalazine) for arresting tumor growth and/or sensitizing cancers is discussed.