Accumulation Of Cystine Research Articles

Cystine accumulation and loss in normal, heterozygous, and cystinotic fibroblasts.

Cystinotic fibroblasts contain approximately 100 times more cystine than do normal control fibroblasts. When cystinotic fibroblasts were placed in the presence of 30 mM cysteine-glutathione mixed disulfide (CSSG) for 24 hr, their cystine content increased about 3-fold. Similar treatment of normal fibroblasts and fibroblasts from patients heterozygous for cystinosis resulted in a 6- to 7-fold increase in cystine content. In all three cell types, the intracellular free cystine is located within lysosomes. When placed in cystine-free medium after 24 hr in CSSG-containing medium, the normal and heterozygous fibroblasts rapidly lost their lysosomal cystine (t 1/2 = 20 min), but the cystine content of the cystinotic cells remained stable for over 90 min. In contrast to the findings in intact fibroblasts, cystine loss could not be demonstrated from isolated, cystine-loaded lysosomes from any of the three cell types.

766 CYSTINOTIC FIBROBLASTS ACCUMULATE CYSTINE FROM CYSTINE BUT NOT CYSTEINE RESIDUES OF EXTRACELLULAR PROTEINS

Cystine depleted cystinotic fibroblasts reaccumulate cystine when incubated in cystine free medium supplemented with a variety of disulfide (cystine)-containing proteins. The cystine accumulation is linear with the concentration of added protein. Correlation of the amount of degradation of 125I labelled protein with cystine accumulation yields a significant correlation (r=0.97 for bovine serum albumin [BSA], and r=0.81 for bovine insulin). Similar results have been found for human and rat albumin, and bovine y globulin & thyroglobulin. This cystine accumulation is inhibited by inhibitors of proteolysis, and inhibitors of selective endocytosis. Reduced BSA (RBSA) produced by pre-treatment with mercaptoethanol in 6 M urea yields diminished cystine accumulation even though more of the reduced protein is degraded:0.18 nmol/106 cells cystine accumulated and 0.50 mg/106 cells RBSA degraded in 24 hrs; 0.70 nmol/106 cells cystine accumulated and 0.20 mg BSA degraded in 24 hrs. Ovalbumin (OVA), which contains 4 mol of cysteine/mol of protein and 1 mol cystine yields no cystine accumulation after 24 hrs incubation in which 0.86 mg/106 cells OVA was degraded. Addition of OVA or RBSA to non-depleted cystinotic fibroblasts produces cystine depletion (30% in 4 hrs, 55% in 24 hrs). We conclude that cystinotic cells accumulate cystine through the lysosomal retention of cystine, but not cysteine residues following their release from peptide chains during proteolysis. Supported by USPHS AM 25548.

The effect of chloroquine on the intralysosomal accumulation of cystine in cystinotic and normal fibroblasts.

The effect of chloroquine on the intralysosomal accumulation of cystine in cystinotic and normal fibroblasts.

Cystine depletion of cystinotic tissues by phosphocysteamine (WR638)

NEPHROPATHIC CYSTINOSIS is an inborn error of metabolism which is characterized by the intralysosomal accumulation of the disulfide amino acid cystine in most body tissues, which leads to the renal Fanconi syndrome and progressive renal failure; end-stage renal disease occurs bY the end of the first decade of life? The cause for the cystine accumulation and renal failure is not known; however, since sulfhydryl reactive agents and disulfides can induce the Fanconi syndrome in experimental animals,'-' reduction of the intracellular cystine content in these patients has been suggested as a potentially beneficial therapy. We 3 and others ~ a have suggested cysteamine (2-aminoethanthiol) as a possible therapeutic agentl following the demonstration that cysteamine produces rapid depletion of Cystine in cystinotic tissues both in vivo and in vitro. Since cysteamine is a free-thiol compound, it has a highly.disagreeable odor and taste which have precluded its use in some patients. We here report satisfactory cystine depletion of cystinotic tissues using the phosphorothioester of cysteamine, WR 638. This agent lacks the Objectionable odor of cysteamine and is less toxic; it appears to be as efficient in producing cystine depletion as the parent compound.

Treatment of cystinosis with cysteamine

C v s T I N 0 S I S is a recessively inherited metabolic disorder characterized by an accumulation of nonprotein cystine within the lysosomes. 1 ! n the juvenile nephropathic form, the disease presents with tubular renal dysfunction and progressive glomerular damage, leading to end-stage renal failure and eventual death. Although the primary defect is not known, attempts to reduce the content of !ntracellular cystine have been reported, using dithiothreitol in two patients, 2 and cysteamine in one? The present study was performed to evaluate the possible benefit of cysteamine in a patient with Fanconi syndrome and cystinosis.

Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticoids to the medium. The degree of stimulation was directly related to glucocorticosteroids potency. Sex steroids, certain peptide hormones and prostaglandins E 2 and F 2 α did not influence zinc accumulation. Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since 45Ca, 14C-labelled amino acids and [ 35S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatorgraphy confirmed that this additional cytosol zinc was bound to metallothionein. [ 35S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.

Depletion of cystine in cystinotic fibroblasts by drugs enclosed in liposomes.

Summary and degradable by lysosomal enzymes after their uptake by phagocytic cells. Lysosomally compartmentalized cystine can be removed from cystinotic cells by the use of reducing agents. In further investigation of means for converting cystine to a form or forms cystinosis is a recessive, autosomally inherited disease, the capable of penetrating the l~sosomal membrane, a number of fundamental cause of which remains unknown. There is an chemical agents either enclosed in phospholipid vesicles (li~o- accumulation of cystine in lysosomes, with particularly heavy wmes) or free in solution were presented to c~stinotic cells in concentrations in actively endocytic or phagocytic tissues such tissue culture. After a 2-hr incubation, cystine content of as kidney, liver, spleen, and white blood cells (25-27). These cultured cystinotic cells was generally reduced more effectively elevated cystine concentrations are apparently toxic to certain by such agents in liposomes than in the medium. The most cellular functions. During the first few years of life, the principal effective combination was cysteamine (MEA) in liposomes: a effects of abnormal cystine storage are growth retardation, 0-5 mM dose of MEA rduced the cystine content of cystinotic photophobia, hypothyroidism, and, most importantly, the renal cells 86% more when enclosed in liposomes than when dissolved Fanconi syndrome with subsequent renal failure (classic nephroat the equivalent dose in the medium. This observation could phathic cystinosis) (24). Adolescent onset and benign adult not be repeated when serum was omitted from the incubation cystinosis also shows intracellular accumulation of cystine but medium, indicating that serum binds or otherwise inactivates generally of lesser magnitude, with clinical symptoms correcystearnine and that the li~some-en~losed cystearnine is Pro- spondingly less severe or absent altogether. The existence of tected from this action. Other liposome-entra~~ed com~ounds these variant forms of cystinosis suggests that a partial reduction tested showed little if any depletion of intracellular cystine of intracellular cystine content in the nephropathic cystinotic beyond that caused by non-liposome-enciosed drug action. Some patient might therefore prove to be of clinical value. agents increased the intracellular cystine content. Others of low Cystine can be removed from cystinotic fibroblasts by agents molecular weight proved to be poorly retained by li~osomes, a such as MEA, cystamine, DTT, and vitamin C (4, 7, 11, 15, factor which may have been responsible for their relative 32). The first three compounds have known toxicity in vivo (13). ineffectiveness. Cysteamine, which is positively charged at neU- we have explored the action of these and other compounds on tral pH, was retained effectively when enclosed in negative cystinotic cells in tissue culture and the distribution of liposomes made by inclusion of phosphatidic acid in the lipid [35slcysteamine in normal mice, the free drug to its

Transformation of human cystinotic fibroblasts by SV40: Characteristics of transformed cells with limited and unlimited growth potential

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.

Pediatric kidney transplantation for cystinosis

In an experience of more than 700 kidney transplants, three recipients are known to have had congenital cystinosis. A cadaver kidney transplant in a 10 yr old child with nephropathic cystinosis functioned well for 7 1 2 yr before it was rejected and the graft was free of recurrent cystinosis after that interval. This prolongation of life was associated with marked accumulation of cystine in the patient's thyroid gland, presenting as a mass in the neck. If additional longevity is achieved in this patient, abnormal intracellular cystine accumulation may be expected in other host tissues. Retransplantation was carried out with the father as donor, accepting the possibility of increased cystine levels in the retransplant. This patient's age of 18 yr identifies him as one of the longest known survivors of infantile nephropathic cystinosis.

Cystinotic fibroblasts accumulate cystine from intracellular protein degradation.

Fibroblasts derived from patients with cystinosis, an autosomal recessive condition, accumulate the disulfide amino acid cystine within lysosomes. The metabolic defect leading to the cystine accumulation and the source from which the cystine is derived are unknown. In this report we present data showing that cystine in these cells accumulates from the degradation of endogenous protein. This conclusion is based upon: (i) no demonstrable synthesis of cystine from serine; (ii) no difference in cystine reaccumulation between glutathione-depleted and non-glutathione-depleted cystinotic cells; (iii) recovery of labeled cystine only when the protein pool is labeled; (iv) reversible inhibition of cystine reaccumulation by known inhibitors of lysosomal protein degradation (chloroquine and NH4Cl).

CYSTINOTIC FIBROBLASTS ACCUMULATE CYSTINE FROM INTRACELLULAR PROTEIN CATABOLISM

Nephropathic cystinosis is an autosomal recessive disease characterized clinically by renal tubular and glomerular dysfunction and biochemically by intralysosomal cystine accumulation. We have recently reported that aminothiol treatment of cultured fibroblasts from patients with cystinosis produces rapid and complete removal of the stored cystine (1). Subsequent studies (2) showed that such cystine-depleted cells can reaccumulate cystine during incubation in culture medium lacking cystine. Thus, cystine appears to accumulate in these cells from an intracellular source. Potential intracellular sources include (1) GSH, (2)protein, and (3) cystathionine. No activity of the cystathionase pathway has been found by previous investigations in these cells, and none was found under the conditions of cystine depletion in the current investigations. Cells depleted of >80% of their GSH content reaccumulate cystine to the same extent as non-GSH depleted controls. Cells selectively labelled with 35S-cystine in either the GSH + protein pools or GSH pool alone only reaccumulate labelled cystine when the protein pool is labelled. Known inhibitors of protein degredation (NH4Cl and chloroquin) reversibly inhibit cystine reaccumulation in cystine-depleted cells. We conclude that the intralysosomal cystine accumulation observed in cells cultured from patients with cystinosis is derived from protein catabolism. (1) Thoene, J.G. et al. J.Clin.Invest.58:180, 1976. (2) Oshima, R.G. et al. J.Biol.Chem. 251:4287, 1976.

Cystinosis and the Fanconi Syndrome

Cystinosis is a recessively inherited metabolic disorder characterized biochemically by a high intracellular content of free (nonprotein) cystine which appears to be compartmentalized within lysosomes. This results in crystal deposition in the cornea, conjunctiva, bone marrow, lymph nodes, leukocytes, and internal organs. The primary metabolic defect which leads to cystine accumulation is unknown.

4Recent progress in our knowledge of cystinosis

Summary Cystinosis is a recessively inherited metabolic disorder characterised biochemically by a high intracellular content of free (non-protein) cystine which results in crystal deposition in the cornea, conjunctiva, bone marrow, lymph nodes, leucocytes and other internal organs. The primary abnormal gene product which leads to the cystine accumulation is unknown. Clinical expression ranges widely from one family to another with three different forms of cystinosis having been clearly delineated. The most severe form, nephropathic cystinosis, is characterised by generalised renal tubular dysfunction (the Fanconi syndrome) and by progressive glomerular damage which leads to end-stage kidney disease within the first decade of life. In other families, cystinosis is diagnosed by an ophthalmologist who sees tinsel-like refractile opacities in the cornea and conjunctiva during a slit lamp examination. Although these patients generally have cystine crystals in both their conjunctiva and bone marrow, their renal function is completely normal. In a third type of cystinosis, there is both renal tubular and glomerular damage, but clinical symptoms are not noted until about five years of age. This has been called the intermediate or late-onset type of cystinosis. The intracellular content of free (non-protein) cystine is about 100 times normal in white blood cells and cultured skin fibroblasts from patients with nephropathic cystinosis and about 30 times normal in these tissues from patients with benign cystinosis. The values in patients with the late-onset type fall between these ranges. Heterozygotes for all three types show modest increases in cystine concentration. All three forms of cystinosis appear to be transmitted by an autosomal recessive pattern of inheritance. The fact that all three types of cystinosis run true in families demonstrates the genetic heterogeneity of this disorder. Experimental treatment with a diet restricted in cystine and methionineand by administration of d -penicillamine failed to influence the course of the disease. Dithiothreitol has been suggested as a useful form of therapy but it is still in an experimental stage. At present, renal homotransplantation in children with nephropathic and intermediate types of cystinosis appears to offer the best opportunity for extended survival and is the only effective treatment in terminal renal failure. Prenatal diagnosis of nephropathic cystinosis has been accomplished by measurement of the elevated cystine content in cultured amniotic fluid cells by a pulse-labelling technique, making preventive control of the disease possible. Cystinotic fibroblasts have been fused with non-cystinosic cells by the technique of somatic cell hybridisation. In every instance, the hybrid cells had a normal cystine content.

The proximal tubule and the podocyte in cystinosis.

The belief that the swan-neck lesion or the Fanconi syndrome in cystinosis are related to the accumulation of cystine in epithelium of the proximal tubule is questioned. The vulnerability of the visce

Cystine, cysteine and glutathione metabolism in normal and cystinotic fibroblasts in vitro and in cultured normal amniotic fluid cells

The intracellular concentrations of reduced cysteine and glutathione are normal in cystinotic fibroblasts, even though they contain a marked increase in cystine. Cystine accumulation is thus not attributable to an elevated concentration of cysteine with secondary cystine formation, nor can it result from a decreased concentration of reduced glutathione, which acts as a hydrogen donor in transhydrogenation reactions for maintaining intracellular cyst(e)ine in its reduced state. The rates of turnover of glutathione in cystinotic and normal fibroblasts are similar, with half-lives of about 6–8 h. The intracellular cystine pool in cystinotic cells has a half-life of approximately 6 h, indicating that while compartmentalized, it remains in a state of dynamic equilibrium with exogenous cystine. The intracellular cysteine and glutathione levels in normal amniotic fluid cells are similar to those in fibroblasts. This suggests that the metabolism of these substances is similar in both types of cells, and strengthens earlier suggestions that cultured amniotic fluid cells may be used for the prenatal diagnosis of cystinosis, as fibroblasts are now used for its diagnosis postnatally.

Cystine, cysteine, and glutathione metabolism in normal and cystinotic fibroblasts in vitro, and in cultured normal amniotic fluid cells

The intracellular concentrations of reduced cysteine and glutathione are normal in cystinotic fibroblasts, even though they contain a marked increase in cystine. Cystine accumulation is thus not attributable to an elevated concentration of cysteine with secondary cystine formation, nor can it result from a decreased concentration of reduced glutathione, which acts as a hydrogen donor in transhydrogenation reactions for maintaining intracellular cyst(e)ine in its reduced state. The rates of turnover of glutathione in cystinotic and normal fibroblasts are similar, with half-lives of about 6–8 h. The intracellular cystine pool in cystinotic cells has a half-life of approximately 6 h, indicating that while compartmentalized, it remains in a state of dynamic equilibrium with exogenous cystine. The intracellular cysteine and glutathione levels in normal amniotic fluid cells are similar to those in fibroblasts. This suggests that the metabolism of these substances is similar in both types of cells, and strengthens earlier suggestions that cultured amniotic fluid cells may be used for the prenatal diagnosis of cystinosis, as fibroblasts are now used for its diagnosis postnatally.

Characteristics of cystine and lysine transport in renal and jejunal tissue from cystinuric dogs

Canine cystinuria differs from the human situation in two respects. Lysinuria was present in 10 of 14 cystinuric dogs, however the molar ratio of lysine excretion did not exceed that of cystine, although it commonly does in the human disease. Excessive excretion of other dibasic amino acids, ornithine and arginine, was not found in the dogs. Using conditions which demonstrate an in vitro transport defect in intestinal epithelial cell accumulation of cystine and lysine by human cystinuric jejunal mucosa, cystine and lysine accumulation by jejunal mucosa from cystinuric dogs was normal. No defect in cystine or lysine accumulation by renal cortical slices was demonstrated. It appears that isolated cystinuria occurs more frequently in the canine population than in humans, and epithelial cell transport defects of cystine and lysine cannot be demonstrated.

Benign cystinosis: The clinical, biochemical and morphologic findings in a family with two affected siblings

Benign cystinosis is an autosomal recessive disorder characterized by the presence of cystine crystals in the eye and the bone marrow. There are no associated renal or peripheral retinal abnormalities which are typical of nephropathic cystinosis. Patients affected with the benign disorder are asymptomatic and presumably have a normal life expectancy. The youngest known patients, a girl aged eleven and a boy aged sixteen, are described. Biochemical and morphologic evidence suggests that in both forms of cystinosis storage of cystine occurs within the lysosomes, primarily of actively endocytic, mesodermally-derived cells. Although the two forms of cystinosis show points of similarity in the distribution of cystine crystals, they differ quantitatively in the amount of cystine accumulated, substantially less intracellular cystine being present in the benign than in the nephropathic form of the disorder. The abnormal gene products responsible for the accumulation of cystine in the two forms of the disease remain to be demonstrated.

Amino Acid Uptake by Kidney and Jejunal Tissue from Dogs with Cystine Stones

Cystine and lysine accumulation in vitro in intestinal and renal tissue was studied in eight dogs that form cystine stones. Under conditions which demonstrate in vitro defects in tissue obtained from humans with cystinuria, normal amino acid accumulation occurred in six dogs with the canine disorder. Normal amino acid uptake in tissue and the demonstration of normal to minimum increases in excretion of lysine suggest that the canine disorder is not similar to the human syndrome.

Increased free-cystine content of fibroblasts cultured from patients with cystinosis

The presence of a significantly increased content of free-cystine in skin fibroblasts from both homozygotes and heterozygotes for cystinosis emphasizes the central role of cystine in this disease, even though the primary defect responsible for cystine accumulation is yet to be determined. The studies described in this communication provide evidence that cystine is compartmentalized in a subcellular location in cystinotic cells. In fact, the very growth of cystinotic fibroblasts in the presence of more than 100 times the usual content of free-cystine is evidence that the accumulated cystine is not freely dispersed throughout the cell, since it would otherwise inhibit many enzymes requiring free sulfhydryl groups for activity ( Patrick, 1965). We have no evidence as to whether the cystine is located in a known subcellular organelle or in a previously unrecognized location. Skin fibroblasts may provide a convenient tool to pursue these questions.