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

Jonas, Adam J.; Greene, Alice A.; Smith, Micah J.; Schneider, Jerry A.

doi:10.1073/pnas.79.14.4442

Cited by 57 publications

(40 citation statements)

References 17 publications

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“…Cystinotic fibroblasts appear metabolically normal with the exception of the inability to transport cystine from lysosomes, which results from a defective lysosomal transport system for cystine (13,14). This defect is specific for cystine; no other aminoacid, including Fig.…”

Section: Resultsmentioning

confidence: 99%

Inhibitors of protein synthesis also inhibit lysosomal proteolysis. Studies using cystinotic fibroblasts.

Thoene¹,

Lemons²,

Boskovich³

et al. 1985

J. Clin. Invest.

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Cystine depleted cystinotic fibroblasts incubated in cystine-free medium accumulate lysosomal-free cystine from the degradation of cystine-containing intracellular and extracellular proteins. In this report we have used this characteristic of these cells to study lysosomal proteolysis. We find that inhibitors of protein synthesis (cycloheximide, emetine, and puromycin) inhibit cystine accumulation from endogenous proteins and therefore act to inhibit lysosomal proteolysis of these proteins. However, cycloheximide does not inhibit cystine accumulation derived from the degradation of the extracellular disulfide-rich proteins, albumin and RNase, but lysosomal cystine accumulation derived from insulin is inhibited by cycloheximide. We conclude that a rapidly turning over protein may be required for the lysosomal degradation of intracellular and some extracellular proteins.

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Section: Resultsmentioning

confidence: 99%

Inhibitors of protein synthesis also inhibit lysosomal proteolysis. Studies using cystinotic fibroblasts.

Thoene¹,

Lemons²,

Boskovich³

et al. 1985

J. Clin. Invest.

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“…Cystinosis is an autosomal recessive dis ease characterized by the excessive accumu lation of cystine in the lysosomes as a result of a defective cystine efflux [1][2][3][4][5]. Accumu lation of cystine occurs in almost all cells of the body but the kidney is the most vulnera ble organ [6].…”

Section: Introductionmentioning

confidence: 99%

“…concentrations of the relatively im permeable amino acid cystine into noncystinotic cells has limited initial investigations into the pathogenesis of the disease. This drawback was overcome in recent years when it was demonstrated that incubation of cells with the permeable methyl ester deriva tives of different amino acids leads to accu mulation of the respective amino acid in the lysosome [7], The lysosomal loading is due to the activity of intralysosomal esterases which cleave the methyl ester leaving high concentration of the less permeable free amino acid in the lysosomes [1][2][3][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Cystine Accumulation Impairs Glucose Transport in LLC-PK<sub>1</sub> Cells

Bennun¹,

Bashan²,

Potashnik³

et al. 1991

Cell Physiol Biochem

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We studied the effect of lysosomal loading with cystine (by incubation with cystine dimethyl ester, CDME) on the functions of renal epithelial cells in culture (LLC-PK₁). The concentrating capacity (the ratio of the intra- and extracellular concentrations) of the nonmetabolized glucose analog, α-methylglucoside (AMG), was reduced after incubation with CDME in a dose- and time-dependent manner. Consistent with these findings was a 30% reduction in the number of the sodium-coupled D-glucose transporters assessed by phlorizin binding. Since the impairment in glucose transport was less effective with other amino acid methyl ester analogues, LLC-PK₁ cells treated with CDME can be used to study the cellular mechanisms responsible for the impairment of kidney function in cystine accumulation diseases, e.g., cystinosis.

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“…Cystine was measured by the competitive binding assay (20). * Sample was obtained 5 hr after a dose of oral cysteamine.…”

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confidence: 99%

Cystine accumulation and clearance by normal and cystinotic leukocytes exposed to cystine dimethyl ester.

Steinherz¹,

Tietze²,

Gahl³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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Upon exposure to 0.25 mM cystine dimethyl ester, normal and cystinotic leukocytes accumulate substantially more intracellular cystine than is present endogenously in cystinotic cells. Leukocytes loaded by exposure to cystine dimethyl ester may have abnormally lucent and distended lysosomes, and the cystine is compartmentalized within the granular fraction of the cells. After the cells are exposed to cystine dimethyl ester, cystine clearance from normal leukocytes is much faster than from cystinotic cells. The ratios of labeled cysteine-N-ethylmaleimide to cystine are also greater in normals than in cystinotics 60 min after termination of loading. No overlap in ranges of cystine clearance half-times or cysteine-N-ethylmaleimide to cystine ratios was observed in normal compared to cystinotic leukocytes. Limited experiments with fibroblasts exposed to cystine dimethyl ester suggest a correspondingly prolonged cystine clearance for cystinotic cells. These experiments provide evidence for defective clearance of cystine from cystinotic lysosomes in situ.In classical cystinosis, an autosomal recessive disorder (1, 2), progressive kidney disease usually leads to death before age 10 unless renal transplantation is performed. Many cystinotic tissues, including leukocytes and cultured fibroblasts, have greatly increased concentrations offree cystine (1-4), with cystine compartmentalization in lysosomes (1, 2, 5-7).Although the fundamental metabolic defect remains unknown, one possibility is that cystinotic cells lack a mechanism for disposal ofcystine from their lysosomes. However, there is no direct evidence for defective efflux ofcystine from cystinotic lysosomes, despite attempts to assess transcellular uptake or efflux in whole cystinotic cells in comparison with normal cells (8,9). Examinations of subcellular transport have been limited by the inability to load normal cells or lysosomes to cystine concentrations approximating the cystinotic.Goldman and Kaplan (10) and Reeves (11) have demonstrated that certain amino acid methyl esters can be used to load isolated rat liver lysosomes with high concentrations of the corresponding amino acid. Apparently, the methyl esters penetrate lysosomal membranes and are hydrolyzed by esterases to free amino acids, which accumulate because of slow efflux from the lysosomes. Using radioactive amino acid methyl esters, Reeves characterized the efflux of certain amino acids from rat liver lysosomes (11). We have used related techniques to study amino acid efflux from isolated normal and cystinotic human leukocyte lysosomes (12)(13)(14).In this report, we demonstrate that intact normal and cystinotic human leukocytes incubated with cystine dimethyl ester accumulate cystine, primarily within lysosomes, in concentrations far exceeding those present endogenously in cystinotic cells. Loaded cystinotic leukocytes exhibited retarded cystine clearance when compared to normal cells. These observations strongly suggest that cystinotic lysosomes in situ are defective in their capacity t...

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

Abstract: Medical SciencesCystine accumulation and loss in normal, heterozygous, and cystinotic fibroblasts (nephropathic cystinosis/amino acid storage/lysosomes)

Cited by 57 publications

References 17 publications

Inhibitors of protein synthesis also inhibit lysosomal proteolysis. Studies using cystinotic fibroblasts.

Inhibitors of protein synthesis also inhibit lysosomal proteolysis. Studies using cystinotic fibroblasts.

Cystine Accumulation Impairs Glucose Transport in LLC-PK<sub>1</sub> Cells

Cystine accumulation and clearance by normal and cystinotic leukocytes exposed to cystine dimethyl ester.

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