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PubMed1980

Methionine-cystine interrelationships in chicks and rats fed diets containing suboptimal levels of methionine.

Studies were continued with both chicks and rats on the previously reported (featherston and Rogler, 1978) growth depression observed when a crystalline amino acid diet adequate in all nonsulfur-containing amino acids and containing .2% DL-methionine and .2% L-cystine was supplemented with an additional .2% L-cystine. In the present investigation, an average growth depression of 37% was observed in chicks fed a diet containing .4% L-cystine and .2% DL-methionine as compared with chicks fed a diet containing .2% L-cystine and the same level of methionine. A comparable depression was noted when cystine was replaced by L-cysteine. In contrast, an additional .2% of another amino acid (L-tryptophan) did not depress growth. When the high and low cystine diets were meal-fed, results obtained were similar to those observed with ad libitum feeding, but the magnitude of the difference was reduced. Foot-pad lesions, similar to those described in turkey poults fed methionine-deficient diets, were observed in two experiments. Factorial analysis of plasma amino acids indicated that the total of all amino acids analyzed was significantly (P < .05) higher in chicks fed the lower cystine or cysteine diets. Plasma methionine was slightly lower (P < .10) but plasma cystine was significantly (P < .05) higher in chicks fed the cystine or cysteine supplemented diets. A study with .2 and .4% L-cystine and 200, 600, and 2000 mg/kg of choline (factorial design) demonstrated a significant (P < .05) depression in weight gain due to the added L-cystine, no significant (P < .05) differences due to choline, and the absence of a cystine-choline interaction. No detrimental effects were observed when weanling rats were fed a crystalline amino acid diet containing .1% DL-methionine and .2% L-cystine supplemented with additional L-cystine.

Animal Nutritional Physiological Phenomena↗
PubMed1976

Cystine metabolism in human fibroblasts. Comparison of normal, cystinotic, and gamma-glutamylcysteine synethetase-deficient cells.

Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of gamma-glutamylcysteine synthetase (gamma-glutamylcysteine synthetase-deficient) in culture medium containing L-[35S]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione, gamma-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [35S]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione. Cystinotic fibroblasts incorporated [35S]cystine into the large intracellular cystine pool not found in normal or gamma-glutamylcysteine synthetase-deficient cells and also appeared to synthesize glutathione more slowly than normal cells. However, the radioactivity recovered as cystine was reduced greatly and the rate of [35S]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [35S]cystine. This suggests that the apparent reduced rate of glutathione synthesis observed in untreated cystinotic cells was a secondary effect caused by dilution of the [35S]cystine by the large pool of nonradioactive cystine. Cystinotic cells depleted of cystine by treatment with mercaptoethylamine reaccumulate 30 to 50% of their initial cystine in 24 hours in the absence of extracellular cystine. Both normal and cystinotic cells lose more than 90% of their intracellular glutathione in 24 hours in cystine-free medium. Both cell types can reutilize cysteine from glutathione for protein synthesis.

Amino Acid Metabolism, Inborn Errors↗
PubMed1991

Direct analysis of the disulfide content of proteins: methods for monitoring the stability and refolding process of cystine-containing proteins.

So far, the cystine (disulfide) content of proteins has been routinely determined by indirect methods, i.e., cystines are first converted to more stable half-cystines prior to analysis. We present a study which demonstrates that the cystine content can be directly and accurately analyzed at low picomole to femtomole levels. The method involves (a) the employment of a vacuum during sample hydrolysis which permits quantitative recovery of cystine, and (b) the dabsyl chloride precolumn derivatization method which yields stable DABS-cystine that is subsequently analyzed by HPLC. Direct analysis of the cystine residue is important in numerous areas of protein research. It allows, by a single analysis, simultaneous determination of the sulfhydryl (cysteine, as S-carboxymethyl derivative) and disulfide (cystine) contents. The technique can be used to follow the refolding process of fully reduced, cystine-containing proteins. Direct analysis of the cystine content also serves to monitor the extent of inactivation of cystine-containing proteins caused by alkaline pH and heat treatments. In this mode of protein inactivation, cystines are selectively destroyed and converted to lanthionine and lysinoalanine. Both the decrease of cystine and the recoveries of lanthionine and lysinoalanine can be simultaneously evaluated by the proposed method. Examples of these applications are presented here.

Alanine↗
PubMed2003

Regulation of L-cystine transport and intracellular GSH level by a nitric oxide donor in primary cultured rabbit conjunctival epithelial cell layers.

PURPOSE: Metabolism and transport of cysteine are critical for maintenance of the intracellular glutathione (GSH) level. In this study, transport mechanisms of L-cystine and regulation of GSH biosynthesis in the absence or presence of NO-induced oxidant stress were investigated in primary cultured rabbit conjunctival epithelial cells (RCECs). METHODS: RCECs were grown in membrane filters to exhibit tight barrier properties. Uptake and transepithelial transport of L-cystine were determined in the presence or absence of extracellular Na(+). Uptake was determined at 10 minutes after (14)C-L-cystine instillation into apical (a) or basolateral (b) bathing fluid. The effect of nitric oxide (NO) on L-cystine uptake, cellular GSH level, and expression level of two subunits of the rate-limiting enzyme gamma-glutamylcysteine synthetase (GCS) was examined after a 24-hour incubation of primary cultured RCECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP; N-acetyl-3-(nitrosothio)-D-valine. RESULTS: Cellular uptake of L-cystine by RCECs occurred through both Na(+)-dependent and -independent mechanisms. Uptake from apical fluid was higher than that from basolateral fluid, except for the highest concentration of L-cystine tested (200 microM). Transepithelial permeability (P(app)) of L-cystine (at 2.5 microM) was three times higher in the a-to-b direction than in the b-to-a direction in the presence of Na(+), whereas the reverse was true in the absence of Na(+). Na(+)-dependent L-cystine uptake from apical fluid was significantly elevated in primary cultured RCECs treated for 24 hours with various concentrations (0.1-2.0 mM) of SNAP, with maximum uptake observed at 1 mM. A similar pattern of SNAP-induced increase of Na(+)-independent L-cystine uptake from apical fluid was observed, whereas no significant difference was observed for basolateral uptake. Concomitantly, a significant elevation of intracellular GSH (up to fivefold versus the control) was recorded, with the highest increase occurring at 0.1 to 0.25 mM SNAP. A parallel increase in the expression levels of both catalytic and regulatory subunits of GCS was observed by Western blot analysis of lysates from RCECs treated with 0.25 mM SNAP for 24 hours. CONCLUSIONS: L-Cystine is transported by both Na(+)-dependent and -independent amino acid transport systems in RCECs. At low substrate concentrations, L-cystine uptake was higher from apical than basolateral fluid. Permeability studies indicated net absorption of L-cystine across RCECs. SNAP caused significant increases in both L-cystine uptake and intracellular GSH level, which occurred concomitantly with elevation of both catalytic and regulatory subunits of GCS. Understanding sulfur amino acid precursor-dependent cellular mechanisms of GSH homeostasis would be of value in devising GSH-based treatment for conjunctival or other ocular disorders.

Animals↗
PubMed1992

A mechanism for glutamate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion.

We have demonstrated that addition of L-glutamate in millimolar amounts to a culture of C6 glioma cells induced cell death within 24 h. The glutamate-induced toxicity in the C6 glioma cells was completely suppressed by adding L-cystine (0.4-1.0 mM), while the C6 cells degenerated in L-cystine-deprived culture medium. Kinetic studies of [35S]cystine and [3H]glutamate uptake showed that cystine competitively inhibited glutamate uptake, and conversely glutamate inhibited cystine uptake competitively, suggesting that C6 cells have a cystine/glutamate antiporter (system CG or Xc) similar to that already described in the periphery. Exogenous cystine (1 mM) stimulated a release of endogenous glutamate from C6 cells in a Na(+)-independent Cl(-)-dependent fashion. Thus, the antiporter normally transports glutamate out of and cystine into the cells. With the glutamate analogues tested, there was a good correlation between cytotoxicity and inhibition of cystine uptake. The de novo synthesis of glutathione was largely dependent upon the uptake of extracellular cystine. Intracellular levels of glutathione were dramatically decreased within 8-10 h by culture in glutamate-added or cystine-free medium. Vitamin E (100 microM), an antioxidant, rescued the death of C6 cells induced by glutamate exposure or by culture in cystine-deprived medium, but did not restore the apparent decrease of intracellular glutathione. Taken together, the present data strongly indicate that glutamate-induced cell death is initially due to inhibition of cystine uptake through the antiporter Xc system; such inhibition leads to glutathione depletion exposing the cells to oxidative stress. Excess of extracellular glutamate introduced from endogenous or exogenous roots might disorder this mechanism, resulting in cell death.

Animals↗
PubMed1976

Effect of selenium compounds on selenium content, growth and 35S-cystine metabolism of skin fibroblasts from normal and cystinotic individuals.

Kidney samples from children with the inborn metabolic disease cystinosis contain 4 times more selenium (Se) than do kidney samples from normal individuals (p = 0.1). However, when cultured skin fibroblasts from cystinotic patients and normal control individuals are incubated in Se-D,L-methionine, Se-D,L-cystine, Se-cystamine X HCl, Se-urea, selenite or in medium without added selenium, only the cystinotic fibroblasts grown in Se-urea or selenite (SeO3=) contain more selenium than do the corresponding normal cells (p less than 0.05). In both types of cultured fibroblasts, the order of descending toxicity per ppm selenium is: Se-urea greater than Se-cystamine greater than Se-cystine greater than or equal to SeO3= much greater than Se-methionine. High (apparently toxic) concentrations of Se-urea and Se-cystamine lower the elevated intracellular free (nonprotein) cystine content of cystinotic fibroblasts to less than 60% of control values; at lower concentrations, these compounds raise the cystine content of these cells to over 140% of control values. Appropriate concentrations of SeO3=, Se-cystine and Se-methionine also elevate the free cystine content of the cystinotic cells. During a 75 minute incubation in 35S-cystine, the incorporation of 35S into the acid precipitable (protein) fraction of both cell types is significantly inhibited by Se-cystamine (approximately 55% control; p less than 0.05). The incorporation of 35S-cystine into glutathione is inhibited by Se-cystine (approximately 40% control) in both fibroblast types (p less than 0.05). In cystinotic cells, Se-cystamine significantly reduces incorporation of 35S-cystine into the cystine pool (40% control) as does SeO3= (67% control; p less than 0.05). Protein and glutathione synthesis in cystinotic fibroblasts are more strongly inhibited by Se-cystine and SeO3=, respectively, than in normal fibroblasts (p less than 0.05). These studies demonstrate that selenium compounds exhibit a different sequence of toxicity in fibroblasts than in the intact animal and that some previously unreported metabolic effects (i.e. inhibition of glutathione synthesis) may contribute to their toxicity.

Amino Acids↗
PubMed1993

The effect of condensed tannins in Lotus pedunculatus on the digestion and metabolism of methionine, cystine and inorganic sulphur in sheep.

Two experiments were conducted with sheep fed on fresh Lotus pedunculatus containing 50-55 g condensed tannin (CT)/kg dry matter. Effects of CT were assessed by comparing control sheep (CT operating) with sheep receiving a continuous intraruminal infusion of polyethylene glycol (PEG) to bind and inactivate CT. Digestion of methionine and cystine was determined using a continuous intraruminal infusion of indigestible markers, whilst plasma irreversible loss (IRL) of methionine, cystine and inorganic sulphate was determined using 35S labelling. The proportion of microbial non-NH3-N (NAN) in whole rumen digesta NAN and the IRL of reducible S from the rumen were determined using a continuous intraruminal infusion of (NH4)2(35)SO4. The proportion of microbial NAN in whole rumen digesta NAN (0.44 v. 0.71) and the IRL of reducible S from the rumen (0.84 v. 2.49 g S/d) were lower in control than PEG sheep. PEG sheep lost 30% of ingested methionine and cystine across the rumen, whereas the control sheep lost no methionine and cystine across the rumen. Apparent absorption of methionine from the small intestine was 27% higher in control than PEG sheep, but both groups had a similar apparent absorption of cystine. The apparent digestibility of cystine in the small intestine was lower in control (0.42) than PEG (0.53) sheep, whereas the apparent digestibility of methionine was similar (0.78) for both groups. CT had no effect on plasma methionine IRL, but markedly increased the IRL of cystine (39.8 v. 22.4 mumol/min) and reduced the IRL of plasma inorganic sulphate (35.9 v. 50.2 mumol/min). A three-pool model comparing interconversions between the three plasma metabolites showed that CT increased the flow of cystine to body synthetic reactions (36.5 v. 17.3 mumol/min). This was due to trans-sulphuration of methionine to cystine being greater in control than in PEG sheep, whilst the oxidation of both methionine and cystine were reduced in control sheep. It was concluded that CT reduced the proteolysis of forage protein and the degradation of S amino acids to inorganic sulphide in the rumen, resulting in increased net absorption of methionine and increased utilization of cystine for body synthetic reactions in sheep with a high capacity for wool growth (and, hence, high cystine requirement).

Animal Feed↗
PubMed1975

Role of coprophagy in masking dietary deficiencies of cystine in the rat.

When a diet containing raw soybean was fed to rats, there was an increase in the synthesis of pancreatic protein, presumably exocrine protein, as evidenced by an increased uptake of [35S]cystine. There was also an increased transsulfuration of methionine sulfur as indicated by labeled sulfur transformation from methionine to cystine. This same pattern of events was produced in rats receiving a casein-containing diet when 50 mg of crystalline trypsin inhibitor was administered by gavage. However, if coprophagy was prevented, the increased uptake of [35S]cystine and [35S]methionine transsulfuration under both dietary conditions was blocked. It was found that prevention of coprophagy was without effect upon these two processes if supplementary dietary cystine was provided or if a dietary protein source with adequate cystine, i.e., heat-treated soybean, was provided. It was concluded that by practicing coprophagy, sufficient fecal cystine was being returned to the upper intestinal tract to permit some synthesis of pancreatic exocrine protein and with this stimulation of synthesis, transulfuration could proceed. This assumes that the biosynthesis of cystine is dependent upon the availability of sufficient cystine to permit active protein synthesis. In another situation where cystine requirement is high, namely, the rapidly growing rat, a limited amount of cystine was fed by providing a 12% casein diet. Either supplementary cystine or methionine was provided and it was found that both amino acids gave optimal growth in conventional rats, but when coprophagy was prevented, optimal growth was achieved only with the cystine-supplemented diet.

Animals↗
PubMed1999

Modulation of extracellular glutamate concentration in rat brain slices by cystine-glutamate exchange.

1. The role of cystine-glutamate exchange in controlling the extracellular glutamate concentration in the central nervous system was examined by whole-cell clamping neurons in rat brain slices, and using their glutamate receptors as sensors of extracellular glutamate concentration. 2. Applying cystine to cerebellar slices generated a membrane current in Purkinje cells which was abolished by glutamate receptor blockers. Similar cystine-evoked currents were seen in pyramidal cells of frontal cortex slices. 3. Control experiments on non-N-methyl-D-aspartate (non-NMDA) receptors in enzymatically isolated Purkinje cells showed that cystine did not produce a current in slice Purkinje cells by directly activating glutamate receptors, nor by potentiating the action of background levels of glutamate on receptors. Experiments on isolated salamander Muller cells showed that cystine did not block Na+-dependent GLAST glutamate transporters (homologous to the transporters in the Bergmann glia ensheathing the Purkinje cells), nor did it block the current produced by EAAT4 and EAAC1 glutamate transporters in Purkinje cells. Thus the cystine-evoked current in Purkinje cells is not due to a rise in extracellular glutamate concentration caused by block of Na+-dependent uptake. 4. The dependence of cystine-evoked current on cystine concentration in slice Purkinje cells could be fitted by a Michaelis-Menten relation with a Km of 250 microM. The Km predicted from this for cystine activating glutamate efflux is less than 140 microM, because of the non-linear dependence on glutamate concentration of the Purkinje cell current. The current evoked by 1 mM cystine was little affected by removal of extracellular chloride or addition of 1 mM furosemide (frusemide), but was potentiated by 1 mM 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS). 5. These data suggest that external cystine generates a current in slice Purkinje cells by activating cystine-glutamate exchange in cells of the slice, releasing glutamate which activates non-NMDA receptors in the Purkinje cell membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

A role for gamma-glutamyl transpeptidase and the amino acid transport system xc- in cystine transport by a human pancreatic duct cell line.

1. The roles of the gamma-glutamyl cycle and the anionic amino acid transport system xc- in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis-Menten constant (Km), 86 +/- 25 microM; maximal velocity (Vmax), 109 +/- 33 nmol (mg protein)-1 h-1) and reduced by 80-90% by a 50-fold excess concentration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system xc-, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma-GT, decreased gamma-GT activity from 2.58 +/- 0.96 to 0.97 +/- 0.11 mU (mg protein)-1 and decreased the initial rates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin (1-gamma-L-glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L-glutamate, a poor substrate for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system xc-, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma-GT. 6. Cystine transport was also 60-80% inhibited by a series of gamma-glutamyl amino acids (5 mM) including gamma-glutamyl-glutamate, gamma-glutamyl-glutamine and gamma-glutamyl-glycine. alpha-Dipeptides inhibited cystine transport by only 6-22%. 7. These findings demonstrate that in human pancreatic duct PaTu 8902 cells, cystine uptake is mediated by system xc- (50-60%) and the gamma-glutamyl cycle. Our results provide the first evidence linking gamma-GT with cystine transport in human epithelial cells and are of relevance in view of the importance of cystine as a sulphur amino acid source for GSH synthesis in cells exposed to oxidative stress.

Adenosine Triphosphate↗
PubMed1973

Cellular accumulation of L-cystine in rat kidney cortex in vivo.

Cellular accumulation of L-cystine in rat kidney cortex in vivo has been studied using L-[(35)S]cystine. The L-[(35)S]cystine radioactivity in plasma decreases to less than 10% of the initially calculated value by 15 min. Four (35)S-containing intracellular products of L-cystine metabolism were identified including cystine, cysteine, reduced glutathione, and an as yet unidentified compound. The latter is probably taurine, cysteinesulphinate, or cysteic acid. Cellular accumulation of these products was found to be more rapid in vivo than in vitro. Cellular accumulation of the products of L-cystine metabolism was found to be essentially unchanged in the presence of ureter ligation. Unlabeled L-lysine administered simultaneously with L-[(35)S]cystine, in both the presence and absence or ureter ligation, enhanced the cellular accumulation of intracellular metabolic products of L-[(35)S]cystine. Simultaneous (35)S cellular accumulation and L-cystine clearance studies were performed both in the presence and absence of L-lysine. L-Lysine enhanced cellular accumulation of (35)S-products despite an accompanying increase in L-cystine clearance. The results are interpreted as evidence for a dissociation between cellular accumulation and transepithelial transport. This evidence for independent luminal transport and peritubular cellular accumulation could explain the apparent paradox in the disease cystinuria where there appears to be a luminal transport defect for L-cystine, but no defect for cellular accumulation of L-cystine metabolic products in vitro.

Animals↗
PubMed1999

New method for determining cystine in leukocytes and fibroblasts.

BACKGROUND: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. METHODS: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. RESULTS: The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.

Adult↗
PubMed2000

Clinical use of cystine supersaturation measurements.

PURPOSE: We measured the concentration and solubility of cystine in urine from patients with cystinuria or calcium stones and from normal subjects to determine whether urine cystine supersaturation can be calculated from a standard nomogram of solubility versus pH or needs to be measured directly. We also evaluated whether increasing pH of the 24-hour collection recovered enough crystallized cystine to increase cystine supersaturation. MATERIALS AND METHODS: Cystine concentration, pH and usual stone risk factors were measured on 50 ml. aliquots of 24-hour collections from 24 patients with cystinuria, 22 calcium stone formers and 15 normal subjects. After 48 hours of incubation with sodium bicarbonate, a second aliquot was taken from the 24-hour collection for cystine concentration. The original urine at its ambient pH was incubated with an excess of cystine crystals for 24, 48, 72 or 96 hours at 37C to determine solubility and kinetics of equilibration. RESULTS: Cystine solubility varied so widely at any pH range that no predictive nomogram could be relied on for calculating supersaturation. Addition of sodium bicarbonate to the 24-hour urine significantly increased cystine concentration. Urine from stone formers had higher cystine solubility than urine from normal subjects. CONCLUSIONS: Clinical management of cystinuria can be improved by direct measurement of cystine solubility because it varies widely at any given pH. Increasing 24-hour collection pH with sodium bicarbonate additionally improves accuracy of supersaturation measurement by recovering crystallized cystine.

Calcium↗
PubMed1986

L-cystine transport by papain-treated rat renal brush-border membrane vesicles.

In papain-treated rat renal brush-border membrane vesicles, cystine uptake was enhanced under sodium gradient conditions. This effect was not observed when sodium was equilibrated across the vesicle membrane or when sodium was completely absent from the incubation medium. The increased rate of cystine uptake occurred within the first two minutes of incubation and coincided with the period of increased flux of sodium known to occur after papain treatment. Under sodium gradient conditions, the Vmax of cystine uptake by treated vesicles was 65% greater while the Km was 25% lower than the value observed in untreated membranes. The increased cystine uptake after papain treatment occurred when medium cystine was in the electroneutral form. In the absence of a sodium gradient, cystine uptake by control membranes was insensitive to changes in membrane potential and this was unaltered after papain treatment. Exposure of the membranes to papain also resulted in a profound decrease in cystine binding which occurs in native membranes incubated with cystine. The fact that cystine uptake is unchanged under sodium equilibration and even enhanced under sodium gradient conditions suggests that the component of cystine binding is not essential for cystine transport and may represent non-specific binding to membrane proteins.

Animals↗
PubMed1980

Transport interactions of cystine and dibasic amino acids in isolated rat renal tubules.

Isolated renal cortical tubules prepared from adult male Sprague-Dawley rats were used to study the nature of cystine entry into tubule cells and its transport interactions with dibastic amino acids. The uptake of cystine over time was progressive, reaching a steady-state after 60 min of incubation. Analysis of the intracellular pool after incubation revealed that a significant fraction of the transported cystine was reduced to cysteine. A kinetic analysis of uptake demonstrated that two systems for cellular entry of cystine existed with a Km1 of 0.012 mM and Km2 of 0.55 mM. Cystine uptake was sodium dependent with an apparent Km for sodium of 36 mEq/liter. Lysine inhibited cystine uptake via the low Km system, but appeared not to inhibit cystine uptake via the high Km system. Ornithine, leucine, and isoleucine each inhibited cystine uptake via the low Km system. Arginine appeared to affect both systems for cystine uptake. Cystine inhibited the uptake of lysine by isolated renal tubules. The data suggest that cystine at physiologic concentrations is transported into renal tubule cells nearly equally by two systems, of which, the low Km system is shared with the dibasic amino acids. A defective low Km system could in part explain human cystinuria.

Amino Acids, Diamino↗
PubMed1992

Parenchymal organ cystine depletion with long-term cysteamine therapy.

Nephropathic cystinosis is a lysosomal storage disorder characterized by renal failure, multisystem organ damage, and poor growth. Oral cysteamine therapy retards renal deterioration and enhances growth, but parenchymal organ cystine depletion has never been documented. We measured skeletal muscle cystine in 11 cystinosis patients not treated with cysteamine; analysis of their values plus 11 published values showed that muscle cystine increases linearly with age in cystinosis patients (slope, 0.074 nmol half-cystine/mg wet wt/year). In contrast, 15 patients treated for 4 to 11 years with oral cysteamine had a relatively constant muscle cystine content (slope, 0.004 nmol half-cystine/mg wet wt/year). The treated patients' mean muscle cystine, 0.091 +/- 0.064 (SD) nmol half-cystine/mg wet wt, was significantly less (P < 0.001) than that for the 11 youngest untreated patients, 0.754 +/- 0.534 nmol half-cystine/mg wet wt. On postmortem examination, a 9-year-old cystinosis patient treated for 8 years with oral cysteamine had liver, kidney, pancreas, lung, and spleen cystine values 5 to 90 times lower than those of an untreated age-matched control. We conclude that long-term oral cysteamine therapy routinely depletes cystinotic skeletal muscle of cystine; cysteamine is the treatment of choice for the prevention of both renal and nonrenal complications of cystinosis.

Adolescent↗
PubMed2006

Cystine inhibits creatine kinase activity in pig retina.

BACKGROUND: Cystinosis is an autosomal recessive disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Visual deficit is a possible consequence of cystine accumulation in cornea and retina. Fibroblasts from cystinotic patients present ATP deficit with intact mitochondrial energy-generating capacity by an unknown mechanism. Considering that creatine kinase is a thiol enzyme crucial for energy homeostasis in retina, and disulfides like cystine may alter thiol enzymes, the main objective of the present study was to investigate the effect of cystine and cysteamine, the drug used for treatment of cystinotic patients, on creatine kinase activity in cytosolic and mitochondrial fractions of the retina from adult pigs. METHODS: Retina was isolated from 6-month-old Landrace pigs, homogenized and mitochondrial and cytosolic fractions separated by centrifugation. Cytosolic and mitochondrial creatine kinase activities were determined in the presence of different concentrations of cystine and/or cysteamine. RESULTS: Cystine inhibited the enzyme activity in a dose- and time-dependent manner and cysteamine prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits creatine kinase activity by oxidation of the sulfhydryl groups of the enzyme. CONCLUSIONS: Considering that creatine kinase is a crucial enzyme for retina energy homeostasis, in case cystine leaves lysosome these results provide a possible mechanism for cystine toxicity and also another beneficial effect for the use of cysteamine in patients with cystinosis.

Animals↗
PubMed2004

Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart.

Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug.

Animals↗