A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis.

Lysosomes constitute only 4% of the intracellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 microM [35S]cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactivity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A lysosomal transport system, highly specific for cysteine, appears to facilitate this rapid lysosomal cysteine sequestration. Time courses of [35S]cysteine uptake into isolated, Percoll-purified fibroblast lysosomes at pH 7.0 and 37 degrees C are linear for the first 4-5 min and attain a steady state by 10 min. Lysosomal cysteine uptake displays a Km of 0.05 mM at pH 7.0 and an activation energy of 21 kcal/mol, corresponding to a Q10 of 3.2. The role of this transport system in delivering cysteine into lysosomes is supported by its pH curve showing a slow rate of cysteine transport at the acidic pHs between 5 and 6, but then increasing sevenfold between pH 6 and 7.5 to be maximally active near the cytosolic pH of 7. Carrier mediation by this lysosomal transport route demonstrates a high specificity for cysteine as indicated by the inability of the following amino acids to significantly inhibit at 5 mM the lysosomal uptake of 0.035 mM [35S]L-cysteine: ala, ser, pro, val, gly, homocysteine, D- or L-penicillamine, arg, asp, or leu. Similarly, D-cysteine and beta-mercaptopropionate were poor inhibitors, suggesting that both the L-isomer and alpha-amino group of cysteine appear to be required for recognition by the cysteine-specific transport system. In contrast, cysteamine, which lacks an alpha-carboxyl group, was able to strongly inhibit lysosomal cysteine uptake. The physiological importance of this cysteine-specific lysosomal transport system may be to aid lysosomal proteolysis by delivering cysteine into the lysosomal compartment to (a) maintain the catalytic activity of the thiol-dependent lysosomal enzymes and (b) break protein disulfide bridges at susceptible linkages, thereby allowing proteins to unfold, facilitating their degradation.

Impact of sex and glucose-lowering treatments on hypoglycaemic symptoms in people with type 2 diabetes and chronic kidney disease. The French Chronic Kidney Disease - Renal Epidemiology and Information Network (CKD-REIN) Study.

AIM: To describe current practices of glucose-lowering treatments in people with diabetes and chronic kidney disease (CKD), the associated glucose control and hypoglycaemic symptoms, with an emphasis on sex differences. METHODS: Among the 3033 patients with CKD stages 3-5 recruited into the French CKD-REIN study, 645 men and 288 women had type 2 diabetes and were treated by glucose-lowering drugs. RESULTS: Overall, 31% were treated only with insulin, 28% with combinations of insulin and another drug, 42% with non-insulin glucose-lowering drugs. In CKD stage 3, 40% of patients used metformin, 12% at stages 4&5, similar for men and women; in CKD stage 3, 53% used insulin, similar for men and women, but at stages 4&5, 59% of men and 77% of women used insulin. Patients were reasonably well controlled, with a median HbA1c of 7.1% (54mmol/mol) in men, 7.4% (57mmol/mol) in women (P=0.0003). Hypoglycaemic symptoms were reported by 40% of men and 59% of women; they were not associated with the estimated glomerular filtration rate, nor with albuminuria or with HbA1c in multivariable analyses, but they were more frequent in people treated with insulin, particularly with fast-acting and pre-mixed insulins. CONCLUSION: Glucose-lowering treatment, HbA1c and hypoglycaemic symptoms were sex dependent. Metformin use was similar in men and women, but unexpectedly low in CKD stage 3; its use could be encouraged rather than resorting to insulin. Hypoglycaemic symptoms were frequent and need to be more closely monitored, with appropriate patient-education, especially in women.

[DOPPS estimate of patient life years attributable to modifiable hemodialysis practices in Spain]. / Estimación, según el estudio DOPPS, de los años de vida de pacientes atribuibles a las prácticas de hemodiálisis modificables en España.

The increased mortality risk in hemodialysis (HD) patients unable to meet six targets in different areas of HD practice has been reported previously. Using a prevalent cross-sectional sample of Spanish HD patients (n = 613) from the second stage of the Dialysis Outcomes and Practice Patterns Study to determine the percentage with low dialysis dose, hyperphosphatemia, hypercalcemia, hypoalbuminemia, anemia, and catheter use and based on the mortality hazard ratios and the total HD population in Spain, according to the Spanish Society of Nephrology Report, we estimated the number of patient life years that could potentially be gained in our country. These characteristics of HD practice were selected because each is modifiable through changes in practice, each is associated with mortality, and each has a large number of patients outside the target guidelines. The targets that define "within guidelines" are as follows: dialysis dose (single pool Kt/V >1.2), anemia (hemoglobin >110 g/L), albumin after standardization (>40 g/L), serum phosphorus (1.1-1.5 mmol/L), serum calcium (2.1-2.4 mmol/L), and facility catheter use (<10%). Cox proportional hazards regression models were used to calculate the relative risk of mortality for all patients outside each guideline. In all models, calcium values were adjusted for low serum albumin. A separate Cox survival model adjusted for all six HD practices simultaneously to account for correlation that may exist between some facility practices. All models were adjusted for age, sex, race, time on ESRD, and 14 summary comorbid conditions. Patient years attributable to each of the six practice patterns were estimated and are reported here as the potential patient years gained. Comparison of the estimates by individual guideline shows that, in Spain, increasing patient albumin above 40 g/L in all patients would lead to an estimated gain of 9,269 patient years (a 7.9% increase). Additionally, if all facilities could decrease catheter use to less than 10%, 2,842 patient years could be gained (a 2.4% increase). Though it may be an unrealistic goal, if all Spanish patients currently outside the guidelines achieved all six target levels, an estimated 17,300 life years could be gained over the next five years (a 15% increase). A more achievable goal of bringing 50% of patients who are currently outside targets within targets would result in 9,266 life years gained. In conclusion, this analysis suggests large opportunities to improve HD patient care in Spain.

Evidence for an essential histidine residue located in the binding site of the cysteine-specific lysosomal transport protein.

Previously, we observed that the activity of the cysteine-specific lysosomal transport system increases 7-10-fold between pH 6 and 7.3 to be maximally active in the neutral pH range. To understand what factors contribute to this pH dependence, different chemical modifying agents were used to probe the nature of amino acid residues residing in the transport protein binding site. Diethyl pyrocarbonate (1 mM) and N-ethylmaleimide (5 mM) each strongly inactivated lysosomal cysteine uptake > or = 88%, whereas dicyclohexyl-carbodiimide (2.5 mM), phenylisothiocyanate (2 mM), N-acetylimidazole (33 mM), and phenylglyoxal (2 mM) had a moderate to small effect. Maximal inactivation by DEPC occurs within 12-15 min upon exposure to DEPC concentrations > or = 1 mM. DEPC inactivation is consistent with modification of a histidine residue, displaying no inactivation at pH < 6, half-maximal inactivation at pH 6.6, and maximal inactivation at pH > or = 7.3. The close correspondence of DEPC inactivation to the pH activity curve of cysteine uptake suggests the large increase in lysosomal cysteine transport activity between pH 6 and 7.3 reflects deprotonation of an essential histidine residue. The substrate, L-cysteine (4 mM), fully protects the transport protein from DEPC inactivation suggesting that this histidine residue is located in the carrier's substrate binding site. Finally, part of the pH dependence of the lysosomal cysteine carrier appears to be due to responsiveness to the lysosomal transmembrane proton gradient as indicated by lysosomal membrane vesicles which display a 1.5-fold greater rate of cysteine uptake when pH 7.4out > pH 5.3in than when pH 7.4out = pH 7.4in.

Elevated temperature produces cystine depletion in cystinotic fibroblasts.

Increasing the incubation temperature of cystinotic fibroblasts to 40 or 43 degrees C produces a 70-80% decrease in lysosomal cystine content within 24-48 h. This effect is probably mediated by an altered substrate affinity for another lysosomal transport protein.

Characterization of a transport system for anionic amino acids in human fibroblast lysosomes.

L-Aspartate and L-glutamate are transported into human fibroblast lysosomes by a single, low Km, Na(+)-independent transport system, which has been provisionally named lysosomal system d. This system resembles the Na(+)-dependent plasma membrane system chi-AG, although these differences have been observed: (1) lysosomal system d recognizes the D- as well as the L-isomers of both aspartate and glutamate, whereas only for aspartate is the D-isomer recognized by system chi-AG; (2) the anion L-homocysteate is not accepted by system chi-AG, but is an effective inhibitor of lysosomal system d; (3) N-methyl, alpha-methyl, and omega-hydroxamate derivatives of both aspartate and glutamate inhibit lysosomal system d, but only the aspartate derivatives are accepted by system chi-AG; (4) lysosomal system d shows a preference for the substrate amino group in the alpha-position, a preference not seen for system chi-AG. These points imply differences at the two recognition sites with respect to substrate length, size, and rotation, with the lysosomal site generally being the less restrictive.

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids.

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.

Biosynthesis of proteochondroitin sulfate: substrate requirements for formation of two independent species.

Formation of two species of [14C]proteochondroitin sulfate has previously been demonstrated with UDP-D-[14C]glucuronic acid and UDP-N-acetylgalactosamine as substrates with a microsomal preparation from chick-embryo epiphyseal cartilage. A large species of [14C]proteoglycan that appeared at an earlier stage of synthesis was excluded on Sepharose CL-2B, indicating that it was larger than proteoglycans found in cartilage matrix. Another newly synthesized, smaller [14C]proteoglycan species also formed was retarded on Sepharose CL-2B, and appeared to be at a later stage of synthesis. A 6-h pulse-chase experiment using UDP-[14C]GlcA and UDP-GalNAc followed by cold UDP-GlcA demonstrates that there was no conversion of the large [14C]proteoglycan to the small [14C]proteoglycan. Sulfation of the newly formed large and small [14C]proteoglycans with adenylyl sulfate 3'-phosphate also did not alter their chromatographic patterns, indicating that sulfation did not trigger any post-synthetic size modification. Synthesis with lower concentrations of the sugar nucleotides resulted in a disproportionate diminution in formation of the large proteoglycan. The apparent Km values for UDP-GlcA for the formation of large and small proteoglycans were 0.055 and 0.015 mM, respectively. Concentration requirements for UDP-GalNAc also showed a similar 4-fold difference. These results indicate that, even though the large proteoglycan was at an earlier stage of synthesis, it was not a precursor to the small proteoglycan, and that these proteoglycans represent two separately synthesized species.

Dopps estimate of patient life years attributable to modifiable haemodialysis practices in Belgium.

BACKGROUND: Various organizations have published clinical practice guidelines for the care of haemodialysis patients. However, it is unknown to what extent improving or even reaching perfect compliance with guidelines would improve the survival of HD patients in Belgium. METHODS: Using data from the second phase of the Dialysis Outcomes and Practice Patterns Study (DOPPS), the proportion of haemodialysis patients failing to meet six key practice targets (Kt/V > or = 1.2, haemoglobin > or =11 g/dl, phosphate 1.1-1.5 mmol/l, calcium 2.1-2, 4 mmol/l, albumin > or =40 g/l, and facility catheter use < or =10%) was calculated along with the relative risk of mortality associated with being outside these targets. The life years potentially gained from adherence to the six targets, both separately and all six together were then estimated. RESULTS: The percentage of patients outside the targets were as follows: 30.3%, Kt/V; 33.6%, haemoglobin; 56.2%, phosphate; 58.2%, calcium; 67.1%, albumin; and 91.1%, catheter. Estimated patient life years gained with improved compliance with guidelines was highest for albumin (3.670) and catheter use (2.331) but still substantial for the other four targets (ranging from 551 to 1.258). The total of patient years gained if 100% of patients have all six practices brought within target reaches 7.516 years. A conservative estimate of 50% of patients within all targets still yields an improvement of survival of 3.958 patient years. CONCLUSION: This analysis suggests large opportunities to improve HD patient care in Belgium. The avoidance of HD catheters, with the use of AV fistulas whenever possible, should be given a high priority. Admittedly, these calculations assume causality or partial causality that has not been definitively proven. Still, if causality is only partial, the results emphasize that the improvement of patient care through adherence to targets of clinical guidelines might be substantial and all Belgian nephrologists and staff members of dialysis units should carefully pursue every potential effort.

[DOPPS estimate of guideline impact on survival in hemodialysis in Italy]. / Stima DOPPS dell'impatto delle linee guida sulla sopravvivenza in emodialisi in Italia.

Knowing the relative risk (RR) of mortality associated with being outside the guideline targets and the percentage of patients in this situation, it is possible to estimate the number of patient life years that could be gained from adhering to guideline recommendations. We used a prevalent cross-sectional sample of 576 Italian patients from the Dialysis Outcomes and Practices Patterns Study (DOPPS) phase II (2002-2004) to determine the percentage of patients who failed to meet the Italian Society of Nephrology's targets for dialysis dose (spKt/V ≥ 1.3), anemia management (hemoglobin ≥ 11 g/dL), and mineral metabolism (serum calcium and phosphorus: ≤ 2.6 and ≤ 1.8 mmol/L, respectively), and the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative (K/DOQI) targets for nutritional status (serum albumin ≥ 4 g/dL) and vascular access (facility catheter use ≤ 10%). We used a larger random sample of DOPPS patients to establish the adjusted RRs of mortality associated with the 6 examined targets. The percentage of patients outside the targets and the adjusted RRs were 34% and 1.12 for dialysis dose, 37.7% and 1.20 for anemia management, 40.8% and 1.14 for phosphorus, 14.4% and 1.22 for calcium, 62.5% and 1.46 for albumin, and 40.1% and 1.20 for facility catheter use. The adjusted sum of life years potentially gained by complete adherence to all 6 guidelines was 25,156 over a period of 5 years (2006-2010); a more conservative estimate, modeling life years potentially gained by bringing half of all patients outside targets within them, was 13,382. In conclusion, this analysis suggests opportunities to improve hemodialysis patient care in Italy. The magnitude of potential savings in life years should encourage greater adherence to guidelines and practices that are significantly associated with better survival.

Incidence and risk factors for hip or other bone fractures among hemodialysis patients in the Dialysis Outcomes and Practice Patterns Study.

The available data on bone fractures in hemodialysis (HD) patients are limited to results of a few studies of subgroups of patients in the United States. This study describes the prevalence of hip fractures and the incidence and risk factors associated with hip and other fractures in representative groups of HD facilities (n=320) and patients (n=12 782) from the 12 countries in the second phase of the Dialysis Outcomes and Practice Patterns Study (2002-2004). Among prevalent patients, 2.6% had a prior hip fracture. The incidence of fractures was 8.9 per 1000 patient years for new hip fractures and 25.6 per 1000 for any new fracture. Older age (relative risk (RR)(HIP)=1.91, RR(ANY)=1.33, P<0.0001), female sex (RR(HIP)=1.41, P=0.02; RR(ANY)=1.59, P<0.0001), prior kidney transplant (RR(HIP)=2.35, P=0.04; RR(ANY)=1.76, P=0.007), and low serum albumin (RR(HIP)=1.85, RR(ANY)=1.45, per 1 g/dl lower, P<0.0001) were predictive of new fractures. Elevated risk of new hip fracture was observed for selective serotonin reuptake inhibitors and combination narcotic medications (RR=1.63, RR=1.74, respectively, P<0.05). Several medications were associated with risk of any new fracture: narcotic pain medications (RR=1.67, P=0.02), benzodiazepines (RR=1.31, P=0.03), adrenal cortical steroids (RR=1.40, P<0.05), and combination narcotic medications (RR=1.72, P=0.001). Parathyroid hormone (PTH) levels >900 pg/ml were associated with an elevated risk of any new fracture (RR=1.72, P<0.05) versus PTH 150-300. The results suggest that greater selectivity in prescribing several classes of psychoactive drugs and more efficient treatment of secondary hyperparathyroidism may help reduce the burden of fractures in HD patients.

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.

Detection and characterization of a nucleoside transport system in human fibroblast lysosomes.

Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.

Demonstration of adenosine deaminase activity in human fibroblast lysosomes.

Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2'-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2'-3'-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase, which could assist in maintaining cellular energy requirements under conditions of nutritional deprivation.

Incorporation of [32P]orthophosphate into long chains of inorganic polyphosphate within lysosomes of human fibroblasts.

When isolated human fibroblast lysosomes are incubated with 4 microM [32P]phosphate at pH 7.0, orthophosphate is transported into lysosomes and is rapidly incorporated into low and high molecular weight products. We have characterized the high molecular weight (HMW) lysosomal material into which [32P]phosphate is incorporated and have found it to consist of long chains of inorganic polyphosphate based on the following observations. 1) greater than 97% of HMW 32P-lysosomal material is converted to [32P]orthophosphate when incubated with 1 N HCl for 20 min at 100 degrees C. 2) Incubation of HMW 32P-lysosomal material at pH 7.0 and 65 degrees C for 96 h results in the formation of [32P]trimetaphosphate, which is known to be produced only from linear chains of polyphosphate under these conditions. 3) HMW 32P-lysosomal material is resistant to degradation by proteinase K, ribonuclease, and deoxyribonuclease and extracts into the aqueous phase during phenol/chloroform extractions. 4) HMW 32P-lysosomal material displays heterogeneous mobility on polyacrylamide gels with most chains ranging in length from 100 to at least 600 phosphate residues. 5) HMW 32P-lysosomal material is partially hydrolyzed under alkaline conditions to yield a continuous ladder of polyphosphate species differing by one or several residues in length on polyacrylamide gels.

Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal?

The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. trans-Stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. In addition, we find cystine exodus from the lysosomal fraction of cystinotic human fibroblasts to be greatly retarded as compared to that of normal human fibroblasts with half-times of exodus similar to those reported for the lysosomes of cystinotic and normal human leukocytes (Gahl, W. A., Tietze, F., Bashan, N., Steinherz, R., and Schulman, J. D. (1982) J. Biol. Chem. 257, 9570-9575). In contrast, normal and cystinotic human fibroblasts did not show any differences with regard to lysine efflux or its trans-stimulation by cationic amino acids. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.

Cimetidine transport in brush-border membrane vesicles from rat small intestine.

In previous studies, sulfoxide metabolite was observed in animal and human intestinal perfusions of cimetidine and other H2-antagonists. A sequence of follow-up studies is ongoing to assess the intestinal contributions of drug metabolism and drug and metabolite transport to variable drug absorption. An evaluation of these contributions to absorption variability is carried out in isolated fractions of the absorptive cells to uncouple the processes involved. In this report, data is presented on the drug entry step from a study on [3H]cimetidine uptake into isolated brush-border membrane vesicles from rat small intestine. A saturable component for cimetidine uptake was characterized with a Vmax and Km (mean +/- S.E.M.) of 6.1 +/- 1.5 nmol/30s/mg protein and 8.4 +/- 2.0 mM, respectively. Initial binding, and possibly intravesicular uptake, was inhibited by other cationic compounds including ranitidine, procainamide, imipramine, erythromycin, and cysteamine but not by TEA or by the organic anion, probenecid. Initial uptake was not inhibited by amino acids methionine, cysteine, or histidine, by the metabolite cimetidine sulfoxide, or by inhibitors of cimetidine sulfoxidation, methimazole, and diisothiocyanostilbene-2,2'-disulfonic acid. Equilibrium uptake was inhibited by ranitidine, procainamide, and cysteamine but not by erythromycin or imipramine. Initial cimetidine uptake was stimulated by an outwardly directed H+ gradient, and efflux was enhanced by an inwardly directed H+ gradient. Collapse of the H+ gradient as well as voltage-clamping potential difference to zero significantly reduced initial cimetidine uptake. The data is supportive of both a cimetidine/H+ exchange mechanism and a driving-force contribution from an inside negative proton or cation diffusion potential.