Cystine transport activity of heterozygous rBAT mutants expressed in Xenopus oocytes

The rBAT gene encodes a transport protein for cystine and dibasic amino acids. It is a candidate gene for type I cystinuria, a genetic disorder inherited as an autosomal-recessive trait. Recently, several mutations in rBAT from Japanese patients with cystinuria have been reported from our laboratory. Some of these patients were heterozygous, which appears to be inconsistent with the previous concept that mutations in rBAT are recessive. To investigate the function of heterozygous mutants, we introduced these mutations into rBAT gene and analyzed the transport activity of cystine associated with the mutants in Xenopus oocytes. Co-injection of the mutant T1037C (L346P) and the polymorphism G1854A (M6181) into Xenopus oocytes produced a transport activity of 67.9% of the wild type. Oocytes co-injected with T2017C (C673R) and wild type had a transport activity of 70.3% of the wild type. These findings indicate that the heterozygous mutants show decreased transport activity compared to wild-type rBAT. Further, some mutants in rBAT may show decreased cystine transport activity even in heterozygous condition, which may contribute to stone-forming cystinuria.     

The molecular basis of cystinuria: an update

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal proximal tubule and small intestine. In 1992, a cDNA (rBAT) was isolated from kidney which induced high-affinity, sodium-independent uptake of cystine and dibasic amino acids when expressed in Xenopus oocytes. The rBAT gene was mapped to a region of chromosome 2p known to contain a cystinuria locus, and rBAT expression was demonstrated in the straight (S3) portion of renal proximal tubule and small intestine. Over 30 distinct rBAT mutations have been described in patients who inherit two fully recessive (type I) cystinuria genes. Recently, the second cystinuria gene (SLC7A9) on chromosome 19q was identified; SLC7A9 mutations were shown to cause the incompletely recessive form of cystinuria (types II and III). Patients who inherit two mutant SLC7A9 genes have recurrent nephrolithiasis comparable to those with two rBAT mutations. In some cystinuria families, patients inherit a fully recessive allele from one parent and an incompletely recessive allele from the other parent; patients with this 'mixed type' of cystinuria have somewhat milder disease. It is not yet clear whether this form of cystinuria involves rBAT as well as SLC7A9 mutations. Current evidence suggests that the transmembrane channel mediating uptake of cystine and dibasic amino acids at the luminal surface is encoded by SLC7A9; the smaller rBAT protein forms a heterodimeric complex with this channel and is critical for its targetting to the luminal membrane.     

Cationic amino acids involved in dicarboxylate binding of the flounder renal organic anion transporter

Three conserved cationic amino acids in predicted transmembrane domains 1, 8, and 11, respectively, of the flounder renal organic anion transporter, fROAT, were changed by site-directed mutagenesis and the resulting mutants functionally characterized in Xenopus laevis oocytes. Uptake of p-aminohippurate (PAH) in oocytes that expressed mutant H34I, K394A, or R478D was markedly reduced compared with oocytes that expressed wild-type fROAT, but was still several-fold higher than that in water-injected control oocytes. Immunocytochemically, no decrease in cell surface expression of the mutants could be detected. Only mutant R478D appeared to have a lower PAH affinity than the wild type. Similar to wild-type-dependent PAH transport, uptake induced by mutant H34I was sensitive to glutarate (GA) cis-inhibition. In contrast, mutants K394A and R478D could not be significantly affected by up to 10 mM GA, although the cRNA-dependent PAH uptake could still be almost completely suppressed by probenecid. Moreover, again in contrast to the wild type, neither PAH influx nor PAH efflux mediated by these two mutants could be trans-stimulated by GA, nor did they induce GA transport. These data suggest that amino acids K394 and R478 in fROAT are required for dicarboxylate binding and PAH/dicarboxylate exchange.     

Influence of rBAT-mediated amino acid transport on cytosolic pH

rBAT, together with its subunit b(0,+) AT mediates the hetero- and homoexchange of neutral and dibasic amino acids. Since the heteroexchange of dibasic amino acids against neutral amino acids is coupled to net transport of positive charge, this transport is electrogenic. Extracellular addition of histidine could create an inward or an outward current depending on extracellular pH (pH(e)) and cell membrane potential. It has been concluded that histidine may be transported in both its protonated and its neutral form. In this study measurements of cytosolic pH (pH(i)) were performed to test this hypothesis. As a result, addition of protonated histidine at acidic pH(e) to Xenopus oocytes expressing rBAT creates an inward current which is paralleled by cytosolic acidification. Both can be reduced by increase of pH(e). At alkaline pH(e) and simultaneous depolarization of the cell membrane the effect of histidine on pH(i) is virtually abolished. The neutral amino acid leucine does not alter cytosolic pH at neither pH 6.0 nor at pH 8.0. In conclusion, histidine can be transported in either its neutral or its protonated form. Transport of the protonated form is facilitated by extracellular acidification and hyperpolarization of the cell membrane.     

Modulation of cellular proliferation alters glutamine transport and metabolism in human hepatoma cells.

OBJECTIVE: The authors determined the effects of growth inhibition on glutamine transport and metabolism in human hepatoma cells. SUMMARY BACKGROUND DATA: Hepatoma cells exhibit markedly higher (10- to 30-fold) glutamine uptake than normal human hepatocytes, via a disparate transporter protein with a higher affinity for glutamine. Currently, little is known about the effects of growth arrest on glutamine transport and metabolism in hepatoma cells. METHODS: The authors determined proliferation rates, glutamine transport, and glutaminase activities in the human hepatoma cell lines HepG2, Huh-7, and SK-Hep, both in the presence and absence of the chemotherapeutic agents novobiocin and sodium butyrate. The transport activities for alanine, arginine, and leucine also were determined in both treated and untreated cells. Glutaminase activity was determined in normal human liver tissue and compared with that present in hepatoma cells. RESULTS: Glutaminase activities were similar in all three cell lines studied, despite differences in proliferation rates, and were sixfold higher than the activity in normal human liver. In contrast to normal hepatocytes, which expressed the liver-specific glutaminase, hepatomas expressed the kidney-type isoform. Sodium butyrate (1 mmol/L) and novobiocin (0.1 mmol/L) inhibited cellular proliferation and reduced both glutamine transport and glutaminase activity by more than 50% after 48 hours in the faster-growing, less differentiated SK-Hep cells. In contrast, the agents required 72 hours to attenuate glutamine uptake by 30% and 50% in the slower-growing, more differentiated HepG2 and Huh-7 cell lines, respectively. Treatment of all three cell lines with novobiocin/butyrate also resulted in a 30% to 60% attenuation of the transport of alanine, arginine, and leucine, and glutamine, indicating that inhibition of cellular proliferation similarly affects disparate amino acid transporters. CONCLUSIONS: Hepatocellular transformation is characterized by a marked increase in glutamine transport and metabolism. Inhibition of cellular proliferation attenuates glutamine transport and metabolism, especially in fast-growing, relatively undifferentiated hepatoma cells. Because the uptake of other amino acids is similarly reduced under cytostatic conditions, plasma membrane amino acid transport activity in hepatoma cells is regulated by the proliferation state of the cells.     

Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b^0,+AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S³⁵) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P = 0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b^0,+ amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches. Gunnar Wendt-Nordahl, Sreedhar Sagi contributed equally to this work.     

Cystinuria phenotyping by oral lysine and arginine loading.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acids transport that results in urolithiasis because of poor cystine solubility. Three cystinuria phenotypes, differentiated according to urinary amino acid excretion in obligate heterozygotes, were regarded as allelic variants of a monogenic disease. Two mutated amino acid transporter genes, however, have been recently identified as responsible for cystinuria. Mutations in the SLC3A1 gene. encoding for the heavy subunit of the transporter protein rBAT, were associated with type I cystinuria, whereas type II and III cystinuria were associated with mutations in the SLC7A9 gene, encoding for a light subunit of rBAT. Lysine and arginine metabolism have, therefore, been evaluated in cystinuria homozygotes and heterozygotes to better define the cystinuria phenotypes and their correlations with these emerging genotypes. PATIENTS AND METHODS: Lysine and arginine intestinal absorption and renal excretion were assessed by oral loading and compared to normal controls. Seven cystinuria homozygotes and 7 obligate heterozygotes belonging to the different types received alternately an oral dose of 0.5 mmol/kg body weight lysine or arginine. Plasma concentrations of lysine, arginine, ornithine (derived from rapid arginine conversion) were measured 0, 1, 2, and 3 hours after loading. Their urinary concentrations were measured in morning urine and in urine collected 0-6 hours after loading. RESULTS: Gut lysine absorption was deficient in type II and III, and normal in type I cystinuria homozygotes. Impaired arginine intestinal absorption, as well as massive lysine, arginine, and ornithine hyperexcretion were shared by all homozygotes, irrespective of the type. All heterozygotes shared normal lysine absorption, whereas arginine absorption was slightly impaired in type II and III heterozygotes, which also displayed high lysine, arginine, and ornithine urinary excretion after loading. CONCLUSIONS: Two cystinuria phenotypes, type I and non-type I, can be identified in both homozygous and heterozygous cystinuric subjects by oral loading tests with lysine and arginine. In agreement with recent molecular findings, non-type I cystinuria comprises mentioned type II and type III, which constitute allelic variants of a cystine and dibasic amino acid transport disorder distinct from type I cystinuria.     

Cystine and glutamate transport in renal epithelial cells transfected with human system x(-) (c)

BACKGROUND: System x(-) (c) is a heterodimeric transporter, comprised of a light chain, xCT, and heavy chain, 4F2hc, which mediates the sodium-independent exchange of cystine and glutamate at the plasma membrane. In the current study we tested the hypothesis that stable transfection of Madin-Darby canine kidney (MDCK) cells with human xCT and 4F2hc results in the expression of functional system x(-) (c). METHODS: MDCK cells were transfected stably with human clones for xCT and 4F2hc. Analyses of time- and temperature-dependence, saturation kinetics, and substrate specificity of l-cystine and l-glutamate transport were carried out in control and xCT-4F2hc-transfected MDCK cells. We also measured the uptake of l-cystine in Xenopus oocytes expressing human xCT and/or 4F2hc or xCT and/or rBAT (a heavy chain homologous to 4F2hc). RESULTS: All of the different sets of data revealed that transport of l-cystine and l-glutamate increased significantly (twofold to threefold) in the MDCK cells subsequent to transfection with xCT-4F2hc. Moreover, uptake of l-cystine also increased (about tenfold) in Xenopus oocytes expressing hxCT and h4F2hc. Biochemical analyses of l-cystine uptake in oocytes verified our findings in the transfected MDCK cells. Interestingly, in oocytes injected with rBAT with or without xCT, uptake of l-cystine was significantly greater than that in water-injected oocytes. CONCLUSION: Our findings indicate that stable transfection of MDCK cells with xCT and 4F2hc results in a cell-line expressing a functional system x(-) (c) transporter that can utilize l-cystine and l-glutamate as substrates. This study apparently represents the first stable transfection of a mammalian cell line with system x(-) (c).     

Tubular handling of amino acids after intravenous infusion of amino acids in healthy humans.

To examine the fractional excretion of amino acids after an increase in the filtered load, and to study a possible coupling between proximal tubular reabsorption of individual amino acids and sodium/water, eleven healthy subjects were examined before and during intravenous infusion of a mixture of essential and non-essential amino acids. Thirteen healthy subjects, who received isotonic glucose instead, participated in an identical time-control study. Glomerular filtration rate (GFR), renal plasma flow (RPF), and proximal and distal absolute and fractional tubular reabsorption of sodium and water (PARNa, PFRNa/water, DARNa, and DFRNa) evaluated by the lithium clearance method were determined during four clearance periods of 30 min each. After amino acid infusion, GFR and RPF increased, whereas filtration fraction (FF) was unchanged. PARNa was unchanged, but lithium clearance increased significantly (P less than 0.05) and PFRNa/water fell, indicating an increased delivery of sodium and water out of the proximal tubules. DARNa increased, but DFRNa was unchanged, thus no net increase was recorded in urinary sodium and water output. In the time-control study, no changes in kidney function were seen. Absolute excretion of amino acids increased for glutamic acid, serine, glutamine, glycine, threonine, histidine, alanine, arginine, tyrosine, valine, methionine, isoleucine, phenylalanine, leucine, and lysine (P less than 0.01), and fractional excretion increased for all but glutamic acid, tyrosine, arginine, isoleucine, and leucine. Reabsorption of amino acids was enhanced uniformly and almost paralleled the filtered load without any sign of saturation of the reabsorption mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human cystinuria-related transporter: localization and functional characterization

BACKGROUND: Cystinuria has been proposed to be an inherited defect of apical membrane transport systems for cystine and basic amino acids in renal proximal tubules. Although the mutations of the recently identified transporter BAT1/b(0,+)AT have been related to nontype I cystinuria, the function and localization of human BAT1 (hBAT1)/b(0,+)AT have not been well characterized. METHODS: The cDNA encoding hBAT1 was isolated from human kidney. Fluorescence in situ hybridization was performed to map the hBAT1 gene on human chromosomes. Tissue distribution and localization of expression were examined by Northern blot and immunohistochemical analyses. hBAT1 cDNA was transfected to COS-7 cells with rBAT cDNA, and the uptake and efflux of 14C-labeled amino acids were measured to determine the functional properties. The roles of protein kinase-dependent phosphorylation were investigated using inhibitors or activators of protein kinases. RESULTS: The hBAT1 gene was mapped to 19q12-13.1 on the human chromosome, which is the locus of nontype I cystinuria. hBAT1 message was expressed predominantly in kidney. hBAT1 protein was localized in the apical membrane of proximal tubules in human kidney. When expressed in COS-7 cells with a type II membrane glycoprotein rBAT (related to b(0,+)-amino acid transporter), hBAT1 exhibited the transport activity with the properties of amino acid transport system b(0,+), which transported cystine as well as basic and neutral amino acids presumably via a substrate exchange mechanism. BAT1-mediated transport was reduced by the protein kinase A activator and enhanced by the tyrosine kinase inhibitor. CONCLUSIONS: hBAT1 exhibited the properties expected for a transporter subserving the high-affinity cystine transport system in renal proximal tubules. The hBAT1 gene was mapped to the locus of nontype I cystinuria, confirming the involvement of hBAT1 in cystinuria.     

Growth hormone enhances amino acid uptake by the human small intestine.

OBJECTIVE: The effects of growth hormone (GH) on the luminal transport of amino acids and glucose by the human small intestine were investigated. SUMMARY BACKGROUND DATA: The anabolic effect of growth hormone administration is associated with nitrogen retention and an increase muscle strength, but the impact of growth hormone on nutrient uptake from the gut lumen has not been examined. METHODS: Twelve healthy patients received a daily subcutaneous dose of low-dose GH (0.1 mg/kg), high-dose GH (0.2 mg/kg), or no treatment (controls) for 3 days before surgery. At operation, ileum (8 patients) or jejunum (4 patients) was resected, and brush border membrane vesicles (BBMVs) were prepared by differential centrifugation. Vesicle purity was confirmed by a 16-fold enrichment of marker enzymes. The carrier-mediated transport of glutamine (System B), leucine (System L), alanine (System B), arginine (System y+), MeAIB (methyl alpha-aminoisobutyric acid [System A]), and glucose (Na(+)-dependent glucose transporter) by BBMVs was measured by a rapid mixing/filtration technique. RESULTS: Treatment with low-dose GH resulted in a statistically insignificant increase in amino acid transport rates in jejunal and ileal BBMVs. High-dose GH resulted in a generalized 20%-to 70%-stimulation of amino acid transport, whereas glucose transport was not affected. The effects of GH were similar in ileum and jejunum. Kinetic analysis of the transport of glutamine (the most abundant amino acid in the body and the principal gut fuel) and the essential amino acid leucine revealed that the increase in transport was caused by a 50% increase in carrier Vmax, consistent with an increase in the number of functional carriers in the brush border membrane. Pooled analysis of transport velocities demonstrated that total rates of amino acid uptake from the gut lumen were increased significantly by 35% in GH-treated patients. CONCLUSIONS: The ability of GH to enhance amino acid uptake from the gut lumen provides energy and precursors for protein synthesis in the gut mucosa, as well as additional substrate for anabolism in other organs.     

Alterations in the activity of placental amino acid transporters in pregnancies complicated by diabetes

Alterations in placental transport may contribute to accelerated fetal growth in pregnancies complicated by diabetes. We studied the activity of the syncytiotrophoblast amino acid transporter system A and the transport of the essential amino acids leucine, lysine, and taurine. Syncytiotrophoblast microvillous plasma membranes (MVMs) and basal plasma membranes (BMs) were isolated from placentas obtained from normal pregnancies and pregnancies complicated by gestational diabetes mellitus (GDM) and type 1 diabetes, with and without large-for-gestational-age (LGA) fetuses. Amino acid transport was assessed using radio-labeled substrates and rapid filtration techniques. System A activity in MVM was increased (65-80%, P < 0.05) in all groups with diabetes independent of fetal overgrowth. However, MVM system A activity was unaffected in placentas of normal pregnancies with LGA fetuses. MVM leucine transport was increased in the GDM/LGA group. In BMs, amino acid transport was unaffected by diabetes. In conclusion, diabetes in pregnancy is associated with an increased system A activity in MVM, and MVM leucine transport is increased in the GDM/LGA group. We suggest that these changes result in an increased uptake of neutral amino acids across MVM, which may be used in placental metabolism or be delivered to the fetus. The increased MVM leucine uptake in the GDM/LGA group may contribute to accelerated fetal growth in these patients.     

Blood-brain barrier derangement in sepsis: Cause of septic encephalopathy?

Patients with sepsis often manifest disorientation, somnolence, asterixis and coma, symptoms also seen in portasystemic encephalopathy. Altered plasma concentrations of the neutral amino acids and increased blood-brain transport of these acids may play a role in portasystemic encephalopathy. Plasma amino acids and blood-brain barrier transport of neutral amino acids were investigated in a rat model of abdominal sepsis, cecal ligation and puncture. The blood-brain transport was studied by the technique of Oldendorf with carbon-14-amino acids 12 and 24 hours after the induction of sepsis. In similar groups of animals, isolation of brain capillaries was carried out by the technique of Hjelle and the capillaries were incubated with carbon-14-amino acids to study transport activity.Plasma and brain amino acids were deranged in a fashion similar to the derangements seen in portasystemic encephalopathy, with a decrease in plasma branched chain amino acids and an increase in most neutral amino acids in brain. The changes were most pronounced after 24 hours. The brain uptake of several neutral amino acids was increased in the septic rats, while the uptake of lysine, a basic amino acid, was normal. In the brain capillaries isolated from septic rats, tyrosine and leucine transport was also greater than in sham-operated animals.Elevated neutral amino acids may play a role in the encephalopathy encountered in septic patients similar to its role in patients with portasystemic encephalopathy, as similar mechanisms appear to be operating.     

Normalization of tumor-induced increases in hepatic amino acid transport after surgical resection.

BACKGROUND: The liver of the host with cancer requires increased amounts of amino acids to support the synthesis of glucose and key defense proteins. To study the effect of the growing tumor on hepatic amino acid uptake, the authors measured hepatic transport activity in tumor-bearing rats and in rats at various times after tumor resection. METHODS: Fischer-344 rats were implanted subcutaneously with methylcholanthrene-induced fibrosarcoma cells (MCA sarcoma). When the tumors reached 10% of body weight, hepatic amino acid transport activity was assayed or the animals underwent surgical removal of the tumor. In animals that underwent tumor excision, livers were removed at 1, 3, or 5 days post-resection, and hepatic plasma membrane vesicles (HPMVs) were prepared. Nontumor-bearing pair-fed rats undergoing sham implantation or sham resection served as controls. System N (glutamine), System A (MeAIB), and System y+ (arginine) transport activity were assayed, which allowed the authors to compare differences in tumor-induced rates of transport and the influence of resection on transport activity. RESULTS: System A transport activity was unaltered by tumor growth. In contrast, the presence of the growing tumor increased arginine and glutamine uptake by the liver. Hepatic glutamine transport remained elevated for 5 days after tumor resection, although by postoperative day 5 there was a trend toward normalization. In contrast, arginine transport remained increased by twofold onpost-resection day 1 and had normalized by postoperative day 3. The enhanced arginine transport was a result of an increase in maximal transport velocity (Vmax) rather than a change in carrier affinity. CONCLUSIONS: Increases in hepatic amino acid transport normalize within several days of tumor resection, indicating a key role for the tumor in the induction of this response. The observation that hepatic glutamine transport activity remains augmented after tumor resection longer than any other transporter studied suggests a key role for this amino acid in overall hepatic nitrogen metabolism and may partially explain the persistent glutamine depletion that is characteristic of the tumor-bearing host.     

Characterization and growth factor stimulation ofl-arginine transport in a human colon cancer cell line

Background: Epidermal growth factor (EGF) and transforming growth factor α (TGFα) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation. Methods: The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [³H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [³H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGFα, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation. Results: The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65–70%), whereas the remainder was sodium-dependent (28–30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na⁺-independent transporter with a Km=55.8±5.8 μM and a Vmax =710.6±87.3 pM/mg protein/30 s. Na⁺-independent arginine uptake was pH-insensitive and markedly inhibited by system y⁺ substrates L-homoarginine, L-lysine, and L-ornithine. A single Na⁺-dependent transporter with a Km=19.8±2.3 μM and a Vmax=159.1±8.9 pM/mg protein/30 s was identified. Na⁺-dependent arginine uptake was inhibited by system B^O,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na⁺-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGFα had no effect on Na⁺-independent L-arginine uptake; however, Na⁺-dependent uptake was enhanced 60% by EGF (10 ng/ml, p<0.05) and 100% by TGFα (10 ng/ml, p<0.05), whereas cellular proliferation was increased 27% by EGF (10 ng/ml, p<0.05) and 37% by TGFα (10 ng/ml, p<0.01). Conclusions: L-arginine transport in the SW480 colon cancer cell line is principally mediated by the Na⁺-independent system y⁺ and to a lesser extent by the Na⁺-dependent system B^O,+. Furthermore, EGF and TGFα preferentially stimulate L-arginine uptake via the Na⁺-dependent transporter, ostensibly to accommodate for the mitogenic stimulus. Results of this study were presented at the 47th Annual Cancer Symposium of The Society of Surgical Oncology, Houston, Texas, March 17–20, 1994.     

Metabolic studies in human skeletal muscle tissue. Evaluation of an in vitro preparation intended for clinical metabolic studies.

An in vitro preparation of human skeltal muscle tissue aimed to be a model for clinical metabolic studies was functionally and structurally characterized. Muscle fibres from biopsy specimens obtained at surgical operations were teased away and collected in incubation vessels for the following determinations: exchangeable water content, extracellular space, potassium content, incorporation rate of labeled leucine into proteins, uptake of labeled cycloleucine, alpha-aminoisobutyric acid (AIB), and 3-o-methy1-glucose into the intracellular space, and the incorporation rate of glucose into various metabolites. After various times of incubation, muscle fibre samples were also taken for light and electron microscopy. During the incubation of the muscle fibres a transient intracellular oedema was demonstrated by electron microscopy and by determination of the exchangeable water content. The restitution of this oedema was associated with an increasing intracellular potassium concentration. Active transport of leucine, cycloleucine and AIB showing competitive inhibition was registered. The incorporation of labeled leucine into proteins was linear during 4 h of incubation and optimal stimulation of this incorporation was demonstrated in the presence of a complete amino acids mixture at a concentration corresponding to 10 times the normal plasma concentration in man. Insulin stimulated the incorporation rate of glucose-carbon into all metabolites and ouabain stimulated its incorporation in to glycogen. Monoidoacetate inhibited the incorporation of glucose into all metabolites.     

Cloning and expression of the mouse glomerular podoplanin homologue gp38P.

BACKGROUND: Puromycin aminonucleoside nephrosis (PAN) is a rat model for human minimal change nephropathy. During PAN, severe proteinuria is induced that is paralleled by a reduced expression of a rat podocyte protein, named podoplanin. The protein probably plays a role in maintaining the unique shape of podocytes. Recently, attenuated amino acid transport has been observed in cultured mouse glomerular epithelial cells treated with puromycin aminonucleoside (PA). In the present study, gp38P, a protein homologous to rat podoplanin was cloned from mouse glomerular epithelial cells and was found to be down-regulated by PA. A role for gp38P in membrane transport in mouse podocytes has been suggested. METHODS: Based on homology to rat podoplanin, the protein gp38P was cloned from mouse glomerular epithelial cells by RT-PCR. Mouse glomerular epithelial cells, mouse cortical collecting duct cells, and Xenopus oocytes were treated with PA and the expression of gp38P was examined by RT-PCR and western blot analysis. Expression of gp38P in other mouse tissues was demonstrated by RT-PCR. The possible impact of gp38P on amino acid transport and folic acid uptake was examined in Xenopus oocytes. RESULTS: gp38P cloned from mouse glomerular epithelial cells showed strong homologies to rat podoplanin and gp38, a protein expressed in the thymus and other tissues. RT-PCR analysis demonstrated ubiquitous expression of gp38P in epithelial and non-epithelial tissues. Quantitative RT-PCR and western blot analysis indicated down-regulation of gp38P in PA-treated glomerular epithelial cells along with loss of cell shape and cell lysis, which was not observed in other cell types. When expressed in Xenopus oocytes, gp38P had no impact on folic acid uptake or transport activity of the amino acid co-transporters CAT1, EAAC1, and rBAT. CONCLUSION: Cultured mouse glomerular epithelial cells express the podoplanin homologue gp38P, which is down-regulated by PAs. gp38P is ubiquitously expressed and is likely to control specifically the unique shape of podocytes.