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The cystine content of cystinotic fibroblasts increases with the length of time in cultivation. Four of five cystinotic cell strains showed an increase in cystine content with increased passage number. Cystinotic fibroblasts did not increase in cell number after 3 days in dialyzed fetal bovine serum. However, their cystine content increased almost 2-fold on a per cell number basis. Cystinotic cells incubated in cystine-free medium lost approximately 50% of their free cystine over a period of 12 hr and subsequently remained at about this level for 12 additional hr. Treatment of cystinotic cells with 1.0 mM dithiothreitol in complete medium reduced their free cystine content to about 25% of the initial level in 5 hr. The free cystine remained at this level after incubation overnight incystine-free medium without dithiothreitol. When dithiothreitol is added in the presence of a cystine-free medium, even more free cystine is removed from cystinotic cells (90%), and the free cystine content remains at this low level for as long as 48 hr if the cells are kept in a cystine-free medium. Cystinotic cells depleted of more than 50% of their intracellular free cystine by incubation in cystine-free medium reaccumulate cystine rapidly after replacement of cystine-containing medium. When cystinotic cells were depleted of their free cystine by treatment with dithiothreitol in a cystine-free medium and were then replaced in complete medium the intracellular free cystine reaccumulated rapidly, regaining its initial value in about 4 hr. The intracellular free cystine content found after 10 hr in complete medium reflected the cystine content of the medium. Cells in medium containing 0.266 mM cystine reaccumulated almost 60% more cystine than cells in medium with 0.066 mM cystine. When cystinotic and normal control fibroblasts were incubated for 2, 4, and 24 hr in either [35S]methionine or [35S]cystathionine, no radioactivity was recovered as cystine, cysteine, or glutathionine.
Cystine and HOCl (a neutrophil-derived oxidant) react to form an intermediate that has a half-life of ca. 5 min at pH 7.5. The intermediate subsequently decomposes to eventually yield a mixture of cystine, higher oxides of Cys, and other uncharacterized species. Spectral titrations, transitory (1)H NMR and UV-vis spectra, and the reaction properties of the intermediate are consistent with a formulation of N,N'-dichlorocystine {NDC = [-SCH(2)CH(NHCl)(CO(2)H)](2)}. The reaction of equimolar amounts of HOCl with cystine at pH 11.3 does not yield N-chlorocystine [NCC = (-O2C)(H3N+)CHCH(2)SSCH(2)CH(NHCl)(CO(2)H)] but rather a 1:1 mixture of NDC and cystine. This result could be explained by two mechanisms: rapid disproportionation of NCC to produce NDC and cystine or a faster reaction of the second equivalent of HOCl with NCC than the first equivalent of HOCl reacts with cystine. The latter mechanism is favored because of our observation by NMR spectroscopy that NDC decomposes via a species that we have assigned as NCC. Thus, disproportionation of NCC is apparently a relatively slow process. The rates of reaction of cystine(0) = [-SCH(2)CH(NH(3)(+))(CO(2)(-))](2) degrees , cystine(1-) = [((-)O(2)C)(H(2)N)CHCH(2)SSCH(2)CH(NH(3)(+))(CO(2)(-))](-), and cystine(2-) = [-SCH(2)CH(NH2)(CO2)(-))]2(2-) have been investigated, and it is clear that cystine(0) is unreactive, whereas cystine(2-) is about four times more reactive than cystine(1-). Accordingly, the following mechanism is proposed (constants for 5 degrees C): HOCl = H+ + OCl-, pK1 = 7.47; cystine(0) = cystine(1-) + H+, pK2 = 8.15; cystine(1-) = cystine(2-) + H+, pK3 = 9.00; cystine(1-) + HOCl --> NCC(1-) + H2O, k4 = 4.3(2) x 10(6) M(-1) s(-1); cystine(2-) + HOCl --> NCC(2)(-) + H2O, k5 = 1.6(2) x 10(7) M(-1) s(-1); NCC(1-) --> NCC(2-) + H+, k6 = fast; NCC(2-) + HOCl --> NDC(2-) + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. The generation of long-lived chloramine derivatives of cystine may have physiological consequences, since such compounds are known to react with nucleophiles via mechanisms that are also characteristic of HOCl, electrophilic transfer C+.
The possibility that the enzymatic reduction of cystine involves a multi-enzyme system led to re-evaluation of cystine reduction by fibroblasts from normal and cystinotic patients. Lineweaver-Burk plots of data with extracts of a normal cell line, representative of seven normal cell lines, under conditions of increasing cystine with variable levels of reduced glutathione (GSH) resulted in a two-limbed curve above 100 micronM cystine. Two cell lines from children with nephropathic cystinosis containing 1-6 micronmol half-cystine/g protein gave a family of curves similar to those of the normal. With six fibroblast lines containing more than 6 micronmol half-cystine/g protein, increasing cystine resulted in a family of lines without two-limbed curves. Plots of the data as activity against increasing cystine concentrations at ratios of cystine to GSH of 1:2 and 1:1 showed that two of the three lines from cystinotic subjects reduced cystine at a faster rate than the normal line. The third line from a cystinotic patient reduced cystine at a slightly slower rate when the substrate concentration in the assay was less than 80 micronM cystine. When the cystine to GSH ratio was maintained at 2:1, normal cells showed a linear increase in the rate of cystine reduction up to 100 micronM cystine, no increase in the rate between 100 micronM and 200 micronM cystine, and an increase again when the concentration of cystine was raised above 200 micronM. Such a stepwise phenomenon was absent with six cell lines containing more than 6 micronmol half-cystine/g cellular protein. A possible mechanism of control of cystine reduction is discussed.
Factors affecting cystine solubility were sought in an effort to derive a reliable method for estimating urinary saturation with respect to cystine. In synthetic solutions, cystine solubility was measured from the maximum ambient cystine concentration reached after incubation with an excess of solid cystine. It rose progressively as the ionic strength increased from addition of mineral electrolytes. The increment in cystine solubility between ionic strength of 0.005 and 0.3 ranged from 13 mg./1. for potassium chloride to 70 mg./1. for calcium chloride. Urinary macromolecules (less than 5,000 daltons, obtained by ultrafiltration), at concentrations normally expected to be found in urine, increased cystine solubility by as much as 45 mg./1. Because of these actions of electrolytes and macromolecules, cystine solubility in urine (from control subjects and patients with cystinuria) exceeded solubility in synthetic solution containing 5 mM sodium cacodylate by 68 to 89 mg./1. Thus, the saturation of cystine in individual urine samples cannot be estimated by comparing the original cystine concentration with a defined cystine solubility curve. It may be more accurately measured as the ratio of original cystine concentration and the directly determined cystine solubility in urine. The estimated assessment of urinary saturation with respect to cystine was shown to deviate from the empirically derived measure from -33 to +44 per cent. In conclusion, cystine is more soluble in urine because of the effects of electrolytes and macromolecules. Thus, urinary saturation of cystine cannot be estimated from solubility curves but must be empirically derived from the direct assessment of cystine solubility in individual urine samples.
The mechanism of uptake of cystine into human umbilical vein endothelial (HUVE) cells was further investigated. Previous experiments have suggested that the uptake of cystine into HUVE cells is mediated by the Xc- anionic amino acid transporter. However, in our experiments, the accumulation of cystine into confluent HUVE cells was not fully inhibited by anionic amino acids, such as glutamate, aminoadipate and homocysteate. On the other hand, inhibitors of gamma-glutamyl transpeptidase (gamma-GT), such as anthglutin and AT-125, were also effective inhibitors of cystine accumulation in these human cells. Indeed, incubation of HUVE cells with anthglutin effectively depleted cellular glutathione (GSH) in association with inhibition of cystine uptake. A dose-dependent inhibitory effect of anthglutin on the initial rate of accumulation of cystine into control HUVE cells was also confirmed, which was also dependent on the carrier concentration of the disulfide. Additionally, in an effort to relate the uptake of cystine and the levels of GSH in these human cells, the incubation of HUVE cells with diethylmaleate (DEM, 25 microM) was shown to stimulate the uptake of cystine by up to 200% over the first hour of incubation, irrespective of whether DEM was coincubated or preincubated with the cells for 60 min before the addition of cystine. Under these conditions the stimulation was not associated with extensive depletion of cellular GSH. Depletion of HUVE cell GSH by an 18 h incubation in M199 medium lacking sulfur amino acids (M199-medium) also stimulated the uptake of cystine by up to 300%, which was dependent on de novo protein biosynthesis. On the other hand, the depletion of HUVE cell GSH by an 18 h preincubation with buthionine sulfoximine did not stimulate cystine uptake above controls. Again, both the anionic amino acids and the inhibitors of gamma-GT were effective in inhibiting the accumulation of cystine into M199-medium-pretreated HUVE cells, in association with impaired resynthesis of GSH. Anthglutin also dose-dependently inhibited the initial cystine uptake rate into these M199-medium-pretreated cells by a proportionally similar extent to its effects in control cells. This also indicates that gamma-GT-dependent uptake of cystine accounts for a portion of the induced uptake of this disulfide into these M199-medium-pretreated cells. These results indicate that both the Xc- carrier and gamma-GT are involved in the uptake of cystine into these human endothelial cells, illustrating another important physiological role for gamma-GT in the uptake of cystine from the circulation into the human endothelium.(ABSTRACT TRUNCATED AT 400 WORDS)
PURPOSE: Many variables are known to be associated with the formation of calcium oxalate stones. We noted that on analysis a number of patients with calcium oxalate calculi also had cystine in the stones. Some but not all of these patients showed urinary cystine excretion slightly above the normal limits, resembling heterozygous carriers. This finding raised the question of whether some recurrent stone formers may be at risk for calcium oxalate calculi when they excrete cystine in above normal concentrations. MATERIALS AND METHODS: Pooled urine obtained from 3 pairs of age and sex matched controls was independently analyzed. Each urine sample was divided into spun and filtered, and ultrafiltered urine. A Multisizer II (Coulter Electronics Ltd, Beds, England) was used to measure particle number, diameter and volume. The metastable limit of each specimen was determined. Promotion activity was measured in spun and filtered, and ultrafiltered urine using 3 concentrations of cystine (80, 160 and 320 micromol./l.). Results were confirmed by measuring the incorporation of (14)C-oxalate into the crystals. Scanning electron microscopy was performed to study further the agglomerates as well as exclude cystine crystals. Each experiment was repeated 6 times. Crystalline material was collected for x-ray powder diffraction analysis. RESULTS: The urine metastable limit did not change with increasing cystine concentrations. Particle diameter increased significantly from 10.6 microm in ultrafiltered urine alone to 11.6 and 13.5 microm (p < 0.05) at 160 and 320 micromol/l. cystine, respectively. In addition, particle volume also increased proportionally in a dose response manner to cystine concentration. The dose of 320 micromol/l. cystine increased the crystal growth rate 52%. 14C-oxalate experiments confirmed these results. Scanning electron microscopy at 500x magnification revealed no cystine crystals in any experiments performed. Furthermore, x-ray powder diffraction analysis of samples revealed that experimentally determined parameters matched reference values for calcium oxalate trihydrate but not for cystine, again confirming absent cystine in the samples. CONCLUSIONS: Adding cystine to undiluted human urine resulted in the marked enhancement of calcium oxalate crystal precipitation. When considered with the finding of cystine in calcium oxalate stones in the noncystinuric population, this result implies that urinary cystine may be a risk factor for calcium oxalate calculi. Cystine was not observed in any calcium oxalate crystals, suggesting that the mechanism of crystal formation was a salting out effect.
Four stones each from 2 populations of cystine calculi, 1 with a rough external surface (cystine-R) and the other smooth (cystine-S), were studied for their crystal structure with stereoscopic and scanning electron microscopy. Two stones each of cystine-R and cystine-S, calcium oxalate monohydrate, calcium oxalate dihydrate, struvite plus apatite and brushite were fragmented with extracorporeal shock wave lithotripsy and the fragmentability was compared. Fragments resulting from cystine-R and cystine-S extracorporeal shock wave lithotripsy were examined under the stereoscope to assess the plane of cleavage or fracture. Results show that cystine-R stones are comprised of well formed blocks of hexagonal crystals, whereas cystine-S calculi have small, irregular and poorly formed interlacing crystals. The center of cystine-R stones was similar to that of the periphery but the center of cystine-S stones was formed of blocks of hexagons similar to but smaller than the cystine-R calculi. Fragmentation with extracorporeal shock wave lithotripsy revealed that cystine-S stones are the least fragile, calcium oxalate dihydrate and struvite plus apatite were the most fragile, and cystine-R, brushite and calcium oxalate monohydrate calculi were in the intermediate fragility range. The possibility of the patient having a cystine-R calculus should be considered during therapeutic procedures.
Two folded polypeptides, designated R1 and R2, respectively, combine in an as yet undefined stoichiometry to form ribonucleoside diphosphate reductase (ribonucleotide reductase) from Escherichia coli. Two pairs of cysteines in each R1 protomer have been implicated in the enzymatic mechanism. One pair, cysteines 225 and 462, is located in the active site of the enzyme and forms a cystine concomitant with the reduction of the ribonucleotide. The other pair, cysteines 754 and 759, is located near the carboxy terminus and is thought to reduce the cystine in the active site by disulfide interchange; either thioredoxin or glutaredoxin is then thought to reduce the cystine that results. Rapid quenching and site-directed immunochemistry have been used to follow the formation of the cystine in the active site and the peripheral cystine simultaneously during the pre-steady state. Prereduced R1 dimer of ribonucleoside diphosphate reductase, in the presence of ATP and CDP, was mixed with R2 dimer in an apparatus for quench flow. The reaction was quenched with a solution of acetic acid and N-ethylmaleimide, the protein was then precipitated with trichloroacetic acid, and the precipitate was separated into two portions. The percent of the cystine in the active site in one of the portions was determined as described previously [Erickson, H. K. (2000) Biochemistry 39, 9241-9250]. A similar method was employed to determine the percent of the peripheral cystine in the other portion of the precipitate. It was found that while the formation of both of these cystines was initiated by the addition of R2 dimer, presumably as products of the reduction of CDP, the peripheral cystine appeared to form more rapidly and in a higher yield than the cystine in the active site. These results demonstrate that the formation of the cystine between cysteines 754 and 759 of ribonucleotide reductase from E. coli is kinetically competent. A mechanism consistent with the prior formation of the cystine between cysteine 225 and cystene 462 as well as the kinetics for the formation of each cystine with time is presented. Because twice as much of the peripheral cystine than cystine in the active site had formed during the pre-steady state, it follows that the enzymatically competent complex between R1 dimers and R2 dimers cannot be symmetric.
The treatment of cystinuria is hampered by methods used to measure urinary lithogenicity. Most cystine assays cannot reliably distinguish cystine from soluble thiol drug-cysteine complexes. We used a solid-phase assay of urinary cystine capacity in a large sample of patients with cystinuria. A known amount of solid-phase cystine is added to urine. In supersaturated urine, cystine precipitates onto added crystals, so the solid phase recovered after incubation will be greater than that added. We studied the effect of cystine-binding thiol drugs (CBTD) to solubilize cystine and determined correlates of cystine capacity in patients who were and were not taking CBTD. Increasing concentrations of D-penicillamine, tiopronin and captopril dissolved cystine in urine with similar efficacy. A general linear model in which 24 h cystine excretion was the dependent variable showed that creatinine, urea nitrogen, and sodium excretions were associated with cystine excretion (P<0.02, all three). Urine volume, pH, and cystine excretion strongly correlated with cystine capacity (P<0.001). Tiopronin had no effect on supersaturation in a cross-sectional analysis. A subset of supersaturated samples, with negative cystine capacity, occurred mainly among women not taking CBTD. For this subset, capacity differed significantly between CBTD users and non-users; use of CBTD avoided extremes of supersaturation. Female enrichment in the supersaturated group was accounted for in part by underprescription of CBTD to women. This assay of cystine capacity was reliable in the presence of CBTD. It should be useful in monitoring patients' response to dietary interventions and administration of fluid, citrate, and CBTD.
The uptake of l-[(35)S]cystine was studied in six cystinotic and six normal fibroblast lines grown for five days either on cover slips or in 32-oz plastic flasks. Cystinotics showed greater uptake than normals. The apparent K(t) for cystine entry in both types of cells was 0.043 mM but cystinotic cells showed a higher maximum velocity of entry. A comparison of the fate of l-[(35)S]cystine incubated for 20 min with monolayers of cells showed 30% and 15% of the intracellular (35)S to be l-cystine in cystinotic and normal cells, respectively. The (35)S effluxed more slowly from cystinotic than from normal cells after a 20-min preloading with l-[(35)S]cystine. Identification of (35)S compounds in efflux media after 3 min showed 75% of the total (35)S was l-cystine with the remainder in cysteine and acidic sulfur metabolites of cystine with no essential difference between cystinotics and normals. In paired experiments, the specific activity of the effluxed l-[(35)S]cystine after both efflux periods was the same as that entering the cell, thus indicating that the free l-[(35)S]cystine had not exchanged with the pre-existing pool in the cystinotic cells. During 3 min efflux, the l-cystine pool in normal cells was depleted mainly by loss of free cystine. In cystinotic cells, a new steady state was attained after 21 min of efflux and the intracellular l-[(35)S]cystine had the same percentage of total radioactivity seen after the initial 20-min uptake. After the rapid efflux of l-[(35)S]cystine from normals, [(35)S]cysteine and other labeled cystine metabolites appeared in the efflux media. By the end of a 3-min efflux, cystinotic cells had incorporated more label into reduced glutathione than had normal cells. However, when the new steady state was attained in cystinotics, the amounts of (35)S in glutathione were not markedly different in the two types of cells. Approximately 95% of the total label could be accounted for in free sulfur compounds. The data show an increased uptake and decreased efflux of cystine from cystinotic cells. However, it is not possible to conclude if these differences are due to primary changes in membrane function or to the reflection of metabolic defects without further investigation.
It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.
Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.
In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC50 values for cystine for a 24-h exposure ranged from 2 to 65 microM. After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 +/- 13% of the control. Free radical scavengers (alpha-tocopherol, ascorbate, idebenone, and N-tert-butyl-alpha-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.