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PubMed2004

L-cystine inhibits aspartate-beta-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by L- and D-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, beta-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK(a) of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [35S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [35S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)(2)TVSL, thus demonstrating that the essential 135Cys is the amino acid residue modified by the treatment with cystine.

Aspartate-Semialdehyde Dehydrogenase↗
PubMed2005

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex.

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.

Adenosine Diphosphate↗
PubMedDate not supplied

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.

Amino Acids↗
PubMed1982

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes. I. 2-Mercaptoethanol-induced stimulation of the uptake of cystine, an essential amino acid.

The mechanism of augmentation of the primary antibody response in vitro by 2-mercaptoethanol (2-ME) was investigated. By using cystine-free RPMI 1640 medium, it was demonstrated that cyst(e)ine was absolutely required for eliciting the following murine lymphocyte reactions: antibody response to sheep erythrocytes, proliferative response to concanavalin A or lipopolysaccharide (LPS), and polyclonal antibody response induced by LPS. The maximal antibody response was attained with 2.5-5 mM cysteine or half-cystine. The serial feeding of fresh cysteine markedly amplified its capacity to support antibody response particularly when cysteine concentration was suboptimal. Such an effect was not observed in the serial addition of cystine. On the other hand, the dose-response curve of cystine was dramatically shifted to lower concentrations by the addition of 2-ME (1 x 10(-5) M), which alone could not elicit the antibody response in the absence of cystine, nor could it augment furthermore the maximal response induced by 2.5 mM half-cystine. Commercially available RPMI 1640 medium contains 0.41 mM half-cystine, which proved to be a suboptimal concentration for eliciting the maximal response. 35S-cystine was incorporated into murine lymphocytes five to six times more slowly than 35S-cysteine. The rate of cystine uptake, however, was accelerated by 2.5-fold in the presence of 1 x 10(-5) M 2-ME. A close correlation was observed between dose-response profiles of 2-ME in augmenting the antibody response and the stimulation of cystine uptake. These results strongly suggest that one of the roles of 2-ME in augmenting the antibody response in vitro is to facilitate the use of cystine contained in RPMI 1640 medium only at a suboptimal concentration.

Amino Acids, Essential↗
PubMed1987

Induction of cystine transport activity in mouse peritoneal macrophages.

Uptake of cystine was investigated in mouse peritoneal macrophages. The rates of the uptake of cystine in resident macrophages or macrophages elicited by some irritants were very low, but a drastic increase was observed when the cells were cultured in vitro. This increase was time-dependent and required protein synthesis. In macrophages elicited by thioglycollate broth, the rate of the uptake of cystine increased by about 40-fold after 16 h in culture. Contrary to the uptake of cystine, the rates of uptake of some neutral amino acids did not change markedly during culture. We characterized the induced activity of the cystine uptake in macrophages elicited by thioglycollate broth. Cystine was taken up in an Na+-independent and pH-sensitive manner, and the uptake was potently inhibited by extracellular glutamate and the analogous anionic amino acids, but not by aspartate. The activity of the glutamate uptake was also induced during the culture in a way similar to that of cystine uptake, and the uptake of glutamate was potently inhibited by cystine. From these results we concluded that the uptake of cystine and glutamate in macrophages was mostly mediated by a single transport system similar to the ones previously reported in human fibroblasts and some other cells. As a consequence of the induction of the activity of the cystine uptake, glutathione levels in macrophages doubled during culture, and a thiol compound, presumably cysteine, was released into the culture medium and accumulated there. When the macrophages were cultured hypoxically, the induction of the cystine uptake activity was markedly depressed, suggesting an involvement of oxygen in the induction.

Amino Acids↗
PubMed2003

PKC isozyme S-cysteinylation by cystine stimulates the pro-apoptotic isozyme PKC delta and inactivates the oncogenic isozyme PKC epsilon.

Protein kinase C (PKC) is a family of ten isozymes that play distinct and in some cases opposing roles in cell growth and survival. We recently reported that diamide, a diazene carbonyl derivative which oxidizes thiols to disulfides through addition/displacement reactions at the diazene bond, induces potent GSH-dependent inactivation of several PKC isozymes, including the oncogenic isozyme PKC epsilon, via S-glutathiolation. PKC delta, a pro-apoptotic isozyme, was distinguished by its resistance to inactivation. In this report, we show that PKC-regulatory S-thiolation modifications produced by physiological disulfides elicit opposing effects on PKC delta and PKC epsilon activity. We report that PKC delta is stimulated 2.0-2.5 fold by GSSG, (Cys-Gly)(2) and cystine, under conditions where PKC gamma and PKC epsilon are fully inactivated by cystine, and PKC alpha activity is affected marginally or not at all by the disulfides. Focusing on cystine, we show that DTT quenches cystine-induced PKC delta stimulation and PKC gamma and PKC epsilon inactivation, indicative of oxidative regulation. By analyzing DTT-reversible isozyme radiolabeling by [(35)S]cystine, we demonstrate that PKC gamma, PKC delta and PKC epsilon are each [(35)S] S-cysteinylated in association with the concentration-dependent regulation of isozyme activity by cystine. The restricted reactivity of cystine, together with the effects of DTT and thioredoxin on cystine-induced PKC isozyme regulation reported here, indicate that the cystine-induced PKC-regulatory effects entail isozyme S-cysteinylation. We recently hypothesized that antagonism of tumor promotion/progression by small cellular thiols may involve PKC regulation via oxidant-induced S-thiolation reactions with PKC isozymes. The findings of cystine-induced PKC isozyme regulation by S-cysteinylation reported here offer correlative support to the hypothetical model. Thus, PKC delta, a potent antagonist of DMBA-TPA-induced tumor promotion/progression in mouse skin, is stimulated by S-cysteinylation, PKC epsilon, an important mediator of the tumor promotion/progression response, is inactivated by S-cysteinylation, and PKC alpha, which is not influential in DMBA-TPA-induced tumor promotion/progression, is not regulated by cystine. Furthermore, PKC gamma has oncogenic activity, and S-cysteinylation inactivated PKC gamma and PKC epsilon similarly. These findings provide evidence that S-cysteinyl acceptor-sites in PKC isozymes may offer attractive targets for development of novel cancer preventive agents.

Apoptosis↗
PubMed1995

Urinary excretion of free cystine and the tiopronin-cysteine-mixed disulfide during long term tiopronin treatment of cystinuria.

We report the results of a biochemical evaluation of long-term treatment of cystinuria with the SH compound tiopronin (2-mercaptopropionylglycine). The effects of tiopronin were studied by monitoring the urinary excretion of free cysteine and the mixed disulfide between tiopronin and cysteine. Thirty-one patients with homozygous cystinuria were treated with tiopronin for 0.4-12 years (mean 7.8 years). The urinary concentration of free cysteine was used to adjust the tiopronin dose. In 28 of the 31 patients a mean urinary cystine concentration of less than 1,200 mumol/1(288 mg/l) was achieved with the final dose. The final daily doses of tiopronin ranged from 250 mg (1.5 mmol) to 3,000 mg (18.4 mmol; mean 1,540 mg; 9.4 mmol). In a majority of the patients the treatment reduced the 24-hour urinary free cystine excretion effectively, on average by 0.61 mumol (0.15 mg)/mg of tiopronin administered. No changes in the efficacy of tiopronin over time were observed, and the frequency of adverse effects was acceptable. To evaluate the effects of tiopronin on the metabolism of cystine we calculated the total urinary excretion of cystine as the sum of free cystine and the amount of cystine corresponding to the cysteine content of the tiopronin-cysteine disulfide. At low doses of tiopronin there was an increase in urinary excretion of the mixed disulfide as well as of total cystine. Monitoring urinary cystine concentration is necessary to achieve adequate individualized doses of tiopronin. Assessment of the mixed tiopronin-cysteine disulfide and the urinary excretion of total cystine shows that tiopronin may interfere with cystine metabolism in a more complex way than through a simple disulfide exchange reaction with urinary cystine.

Adolescent↗
PubMed1987

Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential.

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.

Adenosine Triphosphatases↗
PubMed1981

The role of glutathione turnover in the apparent renal secretion of cystine.

Previous studies with cystinuric dogs and humans have demonstrated that the amount of cystine excreted in the urine is, in some cases, larger than the amount of cystine removed from the plasma by glomerular filtration. It was concluded that the kidney must secrete cystine into the renal tubule. The present studies indicate that renal glutathione turnover constitutes a mechanism of cystine secretion which may account for a large fraction of the cystine burden in the mouse renal tubule. Mice administered L-arginine or L-lysine, inhibitors of cystine transport, excrete large amounts of cystine in their urine (approximately 15 mumol of cystine/mg of creatine). If the mice are pretreated with buthionine sulfoximine, an inhibitor of glutathione biosynthesis, glutathione turnover is substantially decreased, and the arginine- or lysine-induced cystinuria is reduced by 43 to 55%. The plasma cystine concentration following arginine or lysine administration is reduced less than 15% by buthionine sulfoximine. These findings suggest the in vivo operation of a cycle in which glutathione, synthesized from cysteine intracellularly, is transported into the tubule and oxidized to glutathione disulfide. Subsequent breakdown of glutathione disulfide by gamma-glutamyl transpeptidase and dipeptidase releases cystine within the tubule. In the absence of cystine transport defects or inhibitors, cystine is reabsorbed and reduced intracellularly to cysteine.

Amino Acids↗
PubMed1988

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.

Biological Transport, Active↗
PubMed2003

Cystine crystal volume determination: a useful tool in the management of cystinuric patients.

We prospectively determined cystine crystal volume (Vcys) in urine specimens from all consecutive patients with cystine urolithiasis followed at our institution over the past decade, in order to assess its predictive value as to the risk of recurrent cystine stone formation. A total of 57 patients (29 males, 28 females) with homozygous cystinuria entered in the study between January 1990 and December 2000, including 15 children aged less than 15 years and 42 patients aged 15 years or more. The clinical and radiological course was followed until December 2001, for a total of 243 patient-years of follow-up. From study entry until the end of follow-up, we serially examined first voided morning urine specimens in all patients, with determination of the number of cystine crystals per mm3, and the average size of crystals, thus allowing us to calculate Vcys using a simple formula based on crystal geometry. Recurrence was diagnosed on the basis of serial radiographic examinations using X-rays and echography. Overall, cystine crystals were present in 179 (39%) of the 460 examined urine specimens. Cystine crystalluria was significantly more frequent among the 27 patients who developed new cystine stones (SF) than in the other 30 who remained stone-free (63.3 vs 25.5% of samples, P<0.001). The presence of crystals in > or =50% of serially examined urine samples was more frequently found in patients with recurrent stone formation than in non-recurrent patients (24/27 vs 2/30, P<0.001). The average Vcys value was significantly higher in recurrent SF than in stone-free patients (8,173 +/- 1,544 vs 233 +/- 150 micro3/mm3, P<0.001) and there was no overlap in the individual values of recurrent vs stone-free patients. A Vcys value > or =3,000 micro3/mm3 was observed at least once prior to each of the 63 stone recurrences observed in 27 patients (2.3 per patient on the average). In addition, Vcys reflected the efficacy of treatment, with Vcys mean values of 12,097 +/- 3,214 micro3/mm3 at baseline, falling to 2,648 +/- 658 micro3/mm3 on basic therapy (hyperdiuresis plus alkalinization) alone, 1,141 +/- 522 micro3/mm3 on tiopronin therapy (median dose 1,000 mg/day) and 791 +/- 390 micro3/mm3 on D-penicillamine therapy (median dose 900 mg/day) whereas captopril had no effect (5,114 +/- 2,128 micro3/mm3). Based on the results of the present study, cystine crystalluria appears to accurately reflect active stone formation in cystinuric patients. Determination of total Vcys provides a simple, cheap and accurate means of predicting the risk of cystine stone recurrence with a Vcys value > or =3000 micro3/mm3 as the threshold risk value. We propose that serial Vcys determination be performed simultaneously with the measurement of urine pH and specific gravity to optimally monitor the medical treatment of cystine patients.

Adolescent↗
PubMed1988

Promotion of cystine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetylcysteine.

Chinese hamster ovary (CHO) cells obtain a high capacity to utilize cystine from the growth medium by exposure to cysteamine (2-mercaptoethylamine, MEA) or N-acetylcysteine (NAC). For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM [35S]cystine was used. The uptake of cystine was dependent on the time of exposure (0-60 min) and the concentrations of MEA or NAC (0-8 mM). At high concentrations of MEA or NAC, the uptake of cystine became saturated. Half-maximal uptake of cysteine was observed at concentrations of 0.12 mM MEA and 0.66 mM NAC, respectively. Increase in temperature (37-44 degrees) or pH (6.0-8.0) during MEA or NAC exposure further increased the cystine uptake. The increased uptake of cystine was not affected in the presence of glutamate or homocysteate which both inhibited the cystine uptake of control cells. Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione showed a twofold increase in MEA- or NAC-treated CHO cells. DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis completely blocked the promotion of cystine uptake by MEA and NAC. By further analysis using reversed-phase HPLC of cell extracts, more than 90% of the [35S] radioactive cystine taken up by the cells could be recovered within the pool of GSH. The results demonstrate that exposure of CHO cells with MEA and NAC leads to a promoted uptake of cystine from the culture medium and its rapid utilization for cellular GSH biosynthesis.

Acetylcysteine↗
PubMed1987

Effect of cystine dimethylester on renal solute handling and isolated renal tubule transport in the rat: a new model of the Fanconi syndrome.

The effect of cystine dimethylester on the renal handling of phosphate, glucose, alpha-amino nitrogen, amino acids, and protein in vivo and on the uptake of lysine, glycine, taurine, and alpha-methyl glucoside by isolated renal tubules in vitro was studied in adult male rats. Parenteral administration of 400 mumol twice a day for four days of cystine dimethylester led to an increased urine volume, and excretion of phosphate, glucose, alpha-amino nitrogen, and the amino acids glutamine, proline, alanine, 1/2 cystine, ornithine, lysine, histidine, and glycine. Cystine dimethylester treatment did not affect the creatine clearance nor were any renal anatomic abnormalities noted. Intracellular cysteine, but not cystine, was increased in the kidney after the four days of treatment. Pre-incubation of isolated renal tubules with 2 mmol/L cystine dimethylester for ten minutes markedly inhibited the uptake of 0.025 mmol/L lysine, 0.1 mmol/L glycine, 0.01 mmol/L taurine, and 2 mmol/L alpha-methyl glucoside. Incubation with 2 mmol/L cystine dimethylester for ten minutes did not affect the ability of the renal tubule to exclude trypan blue dye, although longer incubation times did lead to significant staining. The intracellular cystine concentration of the renal tubule did rise significantly after incubation with cystine dimethylester, a biochemical correlate of the human disease cystinosis. These studies indicate that cystine dimethylester can induce an experimental form of the Fanconi syndrome both in vivo and in vitro and offers a new model for investigating the mechanisms underlying this enigmatic disorder.

Amino Acids↗
PubMed1988

Characteristics of cystine uptake by cultured LLC-PK1 cells.

The characteristics of the uptake of L-cystine by LLC-PK1 cells were examined. The uptake diminished with time in culture after passage of cells while the uptake of sugar increased. In 48-h-cultured cells at a range of cystine concentrations including physiological levels uptake occurred via a saturable process which was independent of medium sodium concentration and pH. No inhibition of cystine uptake occurred in the presence of lysine which is known to share the cystine transport system in uncultured renal proximal tubule cells and brush-border membrane vesicles. Glutamate was a potent inhibitor of cystine uptake and participated in heteroexchange diffusion with cystine. The cystine-glutamate transport process resembles that of cultured human fibroblasts. The inability of these cells to reflect the genetically determined cystine-lysine system which is altered in the kidney in human cystinuria makes them an inappropriate model of the renal tubule cell cystine transport system. On the other hand, they may provide a model system for examining the factors which determine the presence of the various cystine transport process.

Amino Acids↗
PubMed1982

A mechanism of the augmentation of antibody response in vitro by 2-mercaptoethanol: facilitation of cystine uptake in murine lymphocytes.

The mechanism of augmentation of antibody response in vitro by 2-mercaptoethanol (2-ME) was investigated. By employing cystine-free RPMI-1640 medium, it was demonstrated that cystine was absolutely required for eliciting the in vitro antibody response in murine lymphocytes. The augmentation by 2-ME was completely dependent on the presence of cystine. Maximal response was reached when cysteine or cystine (as half cystine) was added at 2.5 mM where 2-ME did not further enhanced the response. The serial feeding of cysteine, but not of cystine, amplified its potentiating activity at lower concentrations (less than or equal to 1 mM). The addition of 10(-5) M 2-ME to cystine shifted the dose-response curve to lower concentrations by about one order of magnitude. It was found that 35S-cystine was incorporated into lymphocytes one fifth more slowly than 35S-cysteine. Cystine uptake was accelerated by about 2.5-fold in the presence of 10(-5) M 2-ME. These data suggest that one of the roles of 2-ME in augmenting antibody response in vitro is to facilitate the uptake of cystine which is contained in RPMI-1640 medium and which is utilized less efficiently than cysteine.

Animals↗
PubMed2006

Cystine alters the renal and hepatic disposition of inorganic mercury and plasma thiol status.

In the present study, we determined whether cystine can inhibit, under certain conditions, the renal tubular uptake of inorganic mercury in vivo. We co-injected (i.v.) cystine with a non-toxic dose of mercuric chloride to rats and then studied the disposition of inorganic mercury during the next 24 h. We also determined if pretreatment with cystine influences the disposition of administered inorganic mercury. Moreover, plasma thiol status was examined after the intravenous administration of cystine with or without mercuric chloride. During the initial hour after co-injection, the renal tubular uptake of mercuric ions was diminished significantly relative to that in control rats. The inhibitory effects of cystine were evident in both the renal cortex and outer stripe of the outer medulla. In contrast, the renal accumulation of mercury increased significantly between the 1st and 12th hour after co-treatment. Urinary excretion and fecal excretion of mercury were greatly elevated in the rats co-treated with cystine and mercuric chloride. Thus, when cystine and mercury are administered simultaneously, cystine can serve as an inhibitor of the renal tubular uptake of mercury during the initial hour after co-treatment. In rats pretreated with cystine, the renal uptake of inorganic mercury was enhanced significantly relative to that in rats not pretreated with cystine. This enhanced accumulation of inorganic mercury correlated with the increased circulating concentrations of the reduced cysteine and glutathione. Additionally, the present findings indicate that thiol status is an important determinant of renal and hepatic disposition, and urinary and fecal excretion, of inorganic mercury.

Animals↗
PubMed2005

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development.

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Animals↗
PubMed2000

Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes.

The synaptosomal transport of L-[35S]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (Km 463 +/- 86 microM, Vmax 185 +/- 20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (Km 6.90 +/- 2.1 microM, Vmax 0.485 +/- 0.060 nmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (Km 327 +/- 29 microM, Vmax 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X(AG)- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[35S]cystine accumulated into synaptosomes. L-glutamate (Ki 11.2 +/- 1.3 microM) was a non-competitive inhibitor of this transporter, and at 100 microM L-glutamate, the Vmax for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed.

ATP-Binding Cassette Transporters↗