Recent advances in the treatment of cystinosis.

Cysteamine bitartrate capsules (Cystagon) have been approved by the US Food and Drug Administration for use in patients with nephropathic cystinosis. Plasma cysteamine concentrations were virtually identical at various times following ingestion of either cysteamine hydrochloride or Cystagon capsules in 24 normal control subjects. A transfer study was done with eight cystinosis patients who had been receiving either cysteamine hydrochloride or phosphocysteamine for many years. The plasma cysteamine concentration was significantly higher 2h after Cystagon and the leukocyte cystine content was significantly lower at all times after Cystagon compared to older forms of the drug. These differences are probably the result of greater patient compliance in taking the capsules compared to the older, liquid forms of the drug. A new method for following the course of renal glomerular deterioration in diseases such as cystinosis has been published recently. This method was used to re-analyse data on the efficacy of cysteamine treatment and to re-analyse new data on treating cystinosis patients with either of two doses of cysteamine (1.30 g/m2 per day and 1.95 g/m2 per day). This new method agrees well with other methods and shows that both doses of drug are equally effective in maintaining glomerular function.

Special transport and neurological significance of two amino acids in a configuration conventionally designated as D.

We point out an ability of certain amino acids to be recognized at a biological receptor site as though their amino group bore, instead of an alpha relationship to a carboxylate group, a beta, gamma or delta relationship to the same or a second carboxylate group. For aspartate, the unbalanced position of its amino group between a pair of carboxylates allows its occasional biorecognition as a beta-rather than as an alpha-amino acid, whereas for proline and its homologs, their cyclic arrangement may allow the imino group, without its being replicated, to be sensed analogously as falling at either of two distances from the single carboxylate group. The greater separation might allow proline to be seen as biologically analogous to gamma-aminobutyric acid. This more remote positioning of the imino group would allow the D-form of both amino acids to present its amino group in the orientation characteristic of the natural L-form. The dual modes of recognition should accordingly be signalled by what appears to be low stereospecificity, actually due to a distinction in the enantiorecognition of the two isomers. Competing recognition for transport between their respective D- and L-forms, although it does not prove that phenomenon, has been shown for proline and, significantly, even more strongly for its lower homolog, 2-azetidine carboxylate. Such indications have so far revealed themselves rather inconspicuously for the central nervous system binding of proline, reviewed here as a possible feature of a role suspected for proline in neurotransmission.

Biochemical and genetic analysis of a child with cystic fibrosis and cystinosis.

We have studied a child with cystic fibrosis (CF), nephropathic cystinosis, and manifestations of Bartter syndrome, a finding reported previously in both of these diseases (CF and cystinosis). The chance of an individual inheriting a mutant allele for both CF and cystinosis from each of his parents by independent segregation is very small. Therefore, other mechanisms of inheritance were investigated, including whether his diseases were caused by a chromosome deletion or rearrangement that caused defects in both genes, whether his phenotype was caused by a new mutation or variant of either disease, or whether both diseases were inherited together due to inheritance of 2 copies of the same chromosome from one of the parents (uniparental disomy). An investigation was made of whether having mutations for both CF and cystinosis resulted in a different phenotype for either disease and whether the child was a heterozygote rather than a homozygote for one of the mutations. The results suggest that neither disease influenced the expression of the defect in the other and that this child inherited a mutant allele for both diseases independently from each parent.

Characterization of the lysosomal cystine transport system in mouse L-929 fibroblasts.

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.

Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis.

Cystinosis is an autosomal recessive disease in which three clinical forms are recognized: infantile nephropathic, with renal tubular damage by 1 year of age and progressive glomerular insufficiency; intermediate, with tubular and glomerular insufficiency beginning at a later age; benign, with no kidney damage. Skin fibroblasts cultured from patients with all types of cystinosis show increased intralysosomal free (nonprotein) cystine; however, fibroblasts from heterozygotes have normal free-cystine values. To determine whether genetic complementation occurs between the different forms, somatic cell hybrids were constructed between cells from a patient with infantile nephropathic cystinosis and cells from patients with other types of cystinosis. If complementation occurred, the hybrids would be expected to have normal cystine levels. To construct hybrid cells, a "universal parent" cell type (TG1-neo), which was hypoxanthine/aminopterin/thymidine (HAT) sensitive and G418 resistant was constructed from an infantile nephropathic cystinosis fibroblast strain. Polyethylene glycol fusion of TG1-neo with other cells that are not HAT sensitive or G418 resistant allowed for selection of hybrid cells in a medium containing HAT and the aminoglycoside G418. As indicated by elevated cystine levels, complementation did not occur between TG1-neo and two different benign cystinosis strains, an intermediate cystinosis strain, or another nephropathic cystinosis cell strain. When a normal fibroblast strain was fused with TG1-neo, all 15 hybrid clones studied contained normal amounts of intracellular free cystine.

Cystine exodus from normal leucocytes is stimulated by MgATP.

Cystine exodus from partially purified granular fractions of normal leucocytes is stimulated by MgATP. N-Ethylmaleimide, an inhibitor of the lysosomal H+-translocating ATPase, eliminated the stimulated exodus, but had no effect on basal exodus. As the initial content of cystine was increased, the initial velocity of both the basal and ATP-stimulated egress increased. However, as saturation with substrate was approached, the ATP stimulation disappeared leaving only the N-ethylmaleimide-insensitive basal exodus. The increased initial velocity in the presence of ATP may represent improved binding of cystine to the partially saturated inner transporter as a result of conformational or charge optimization brought about by the action of the H+-translocating ATPase.

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.

Lysosomal cystine storage in cystinosis and mucolipidosis type II.

Cultured fibroblasts from mucolipidosis II (ML-II) patients demonstrated an elevated cystine content which increased with time in culture compared to fibroblasts from cystinotic patients or normal controls under the same conditions. In both cystinotic and ML-II cells the increased levels of cystine could be derived either from endogenous proteolysis or from in vitro supplementation of the cultured cells with cysteine-glutathione mixed disulfide. Cystine was depleted from both cell types by cysteamine. When cysteamine was replaced with complete medium, the cystine reaccumulated in both cystinotic and ML-II cells within 24 h, although a lag of 4 h was seen with ML-II cells. The intracellular location of the increased cystine in cultured fibroblasts was examined utilizing free-flow electrophoresis and found to be in the purified population of secondary lysosomes of both cystinotic and ML-II cells. White blood cell and hepatic cystine, which was greatly increased in cystinotic patients, was not elevated in ML-II patients. Compared to normal control fibroblasts the efflux of cystine from isolated granular fractions was virtually absent in cystinotic fibroblasts and considerably reduced in ML-II fibroblasts. The examination of such similarities and differences in cystine accumulation and transport in tissues from cystinotic and ML-II patients has provided some insight into the defects in these diseases.

Proton-translocating ATPase and lysosomal cystine transport.

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.

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.