PubMed Health⌕ Search

Biomedical subjects

Stefan Bröer

Publications and source records attributed to Stefan Bröer.

At least 19 recordsLinked to original sources
PubMed2006

Heterologous expression of the glutamine transporter SNAT3 in Xenopus oocytes is associated with four modes of uncoupled transport.

The glutamine transporter SNAT3 (SLC38A3, former SN1) plays a major role in glutamine release from brain astrocytes and in glutamine uptake into hepatocytes and kidney epithelial cells. Here we expressed rat SNAT3 in oocytes of Xenopus laevis and reinvestigated its transport modes using two-electrode voltage clamp and pH-sensitive microelectrodes. In addition to the established coupled Na+-glutamine-cotransport/H+ antiport, we found that there are three conductances associated with SNAT3, two dependent and one independent of the amino acid substrate. The glutamine-dependent conductance is carried by cations at pH 7.4, whereas at pH 8.4 the inward currents are still dependent on the presence of external Na+ but are carried by H+. Mutation of threonine 380 to alanine abolishes the cation conductance but leaves the proton conductance intact. Under Na+-free conditions, where the substrate-dependent conductance is suppressed, a substrate-independent, outwardly rectifying current becomes apparent at pH 8.4 that is carried by K+ and H+. In addition, we identified a glutamine-dependent uncoupled Na+/H+ exchange activity that becomes apparent upon removal of Na+ in the presence of glutamine. In conclusion, our results suggest that, in addition to coupled transport, SNAT3 mediates four modes of uncoupled ion movement across the membrane.

Amino Acid Sequence↗
PubMed2006

Sodium-dependent uptake of inorganic phosphate by the intracellular malaria parasite.

As the malaria parasite, Plasmodium falciparum, grows within its host erythrocyte it induces an increase in the permeability of the erythrocyte membrane to a range of low-molecular-mass solutes, including Na+ and K+ (ref. 1). This results in a progressive increase in the concentration of Na+ in the erythrocyte cytosol. The parasite cytosol has a relatively low Na+ concentration and there is therefore a large inward Na+ gradient across the parasite plasma membrane. Here we show that the parasite exploits the Na+ electrochemical gradient to energize the uptake of inorganic phosphate (P(i)), an essential nutrient. P(i) was taken up into the intracellular parasite by a Na+-dependent transporter, with a stoichiometry of 2Na+:1P(i) and with an apparent preference for the monovalent over the divalent form of P(i). A P(i) transporter (PfPiT) belonging to the PiT family was cloned from the parasite and localized to the parasite surface. Expression of PfPiT in Xenopus oocytes resulted in Na+-dependent P(i) uptake with characteristics similar to those observed for P(i) uptake in the parasite. This study provides new insight into the significance of the malaria-parasite-induced alteration of the ionic composition of its host cell.

Animals↗
PubMed2006

The SLC6 orphans are forming a family of amino acid transporters.

Transporters in the human genome are grouped in solute carrier families (SLC). The SLC6 family is one of the biggest transporter families in the human genome comprising 20 members. It is usually referred to as the neurotransmitter transporter family because its founding members encode transporters for the neurotransmitters GABA, noradrenaline, serotonin and dopamine. The family also includes a number of 'orphan' transporters, the function of which has remained elusive until recently. Identification of the broadly specific neutral amino acid transporter SLC6A19 (also called B(0)AT1) suggested that all orphan transporters may in fact be amino acid transporters. This was subsequently confirmed by the identification of SLC6A20 as the long-sought IMINO system, a proline transporter found in kidney, intestine and brain. Very recently, SLC6A15 was identified as the neutral amino acid transporter B(0)AT2. All amino acid transporters appear to cotransport only 1Na(+) together with the amino acid substrate. Both, B(0)AT1 and B(0)AT2 are chloride independent, whereas IMINO is chloride dependent. The amino acid transporters of the SLC6 family are functionally and sequence related to the recently crystallized leucine transporter from Aquifex aeolicus. The structure elegantly explains many of the mechanistic features of the SLC6 amino acid transporters.

Amino Acid Transport Systems, Neutral↗
PubMed2006

The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2).

Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B(0)AT1 (slc6a19) and was hence named B(0)AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K(0.5) (half-saturation constant) ranging from 40 to 200 microM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K(0.5) values in the millimolar range. Transport of neutral amino acids via B(0)AT2 was Na+-dependent, Cl--independent and electrogenic. Superfusion of mouse B(0)AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na+-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na+/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K(0.5) values, suggesting that they share the same binding site. A mouse B(0)AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B(0)AT2 in transporting neurotransmitter precursors and neuromodulators is proposed.

Amino Acid Sequence↗
PubMed2006

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1.

Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.

Animals↗
PubMed2005

Characterization of mouse amino acid transporter B0AT1 (slc6a19).

The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+-amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+-substrate-transporter] complex, which is consistent with the experimental data.

Amino Acid Transport Systems↗
PubMed2005

The molecular basis of neutral aminoacidurias.

Recent success in the molecular cloning and identification of apical neutral amino acid transporters has shed a new light on inherited neutral amino acidurias, such as Hartnup disorder and Iminoglycinuria. Hartnup disorder is caused by mutations in the neutral amino acid transporter B(0) AT1 (SLC6A19). The transporter is found in kidney and intestine, where it is involved in the resorption of all neutral amino acids. The molecular defect underlying Iminoglycinuria has not yet been identified. However, two transporters, the proton amino acid transporter PAT1 (SLC36A1) and the IMINO transporter (SLC6A20) appear to play key roles in the resorption of glycine and proline. A model is presented, involving all three transporters that can explain the phenotypic variability of iminoglycinuria.

Amino Acid Transport Systems, Neutral↗
PubMed2005

Molecular cloning of the mouse IMINO system: an Na+- and Cl--dependent proline transporter.

Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of the long-sought IMINO system. The mouse has two homologues corresponding to the single human SLC6A20 gene: these have been named XT3 and XT3s1. Expression of mouse XT3s1, but not XT3, in Xenopus laevis oocytes induced an electrogenic Na+-and-Cl--dependent transporter for proline, hydroxyproline, betaine, N-methylaminoisobutyric acid and pipecolic acid. Expression of XT3s1 was found in brain, kidney, small intestine, thymus, spleen and lung, whereas XT3 prevailed in kidney and lung. Accordingly we suggest that the two homologues be termed 'XT3s1 IMINO(B)' and 'XT3 IMINO(K)' to indicate the tissue expression of the two genes.

Amino Acid Transport Systems, Neutral↗
PubMed2005

Preferred stereoselective brain uptake of d-serine--a modulator of glutamatergic neurotransmission.

Although it has long been presumed that d-amino acids are uncommon in mammalians, substantial amounts of free d-serine have been detected in the mammalian brain. d-Serine has been demonstrated to be an important modulator of glutamatergic neurotransmission and acts as an agonist at the strychnine-insensitive glycine site of N-methyl-d-aspartate receptors. The blood-to-brain transfer of d-serine is thought to be extremely low, and it is assumed that d-serine is generated by isomerization of l-serine in the brain. Stimulated by the observation of a preferred transport of the d-isomer of proline at the blood-brain barrier, we investigated the differential uptake of [3H]-d-serine and [3H]-l-serine in the rat brain 1 h after intravenous injection using quantitative autoradiography. Surprisingly, brain uptake of [3H]-d-serine was significantly higher than that of [3H]-l-serine, indicating a preferred transport of the d-enantiomer of serine at the blood-brain barrier. This finding indicates that exogenous d-serine may have a direct influence on glutamatergic neurotransmission and associated diseases.

Animals↗
PubMed2005

Preferred stereoselective transport of the D-isomer of cis-4-[18F]fluoro-proline at the blood-brain barrier.

Generally, L-amino acids are preferably transported into mammalian cells compared with their D-isomers, and only L-amino acids are incorporated into proteins. Former studies, however, indicated that D-[H]proline is accumulated in the brain of mice after injection, while L-[3H]proline is not. We investigated the differential cerebral uptake of the D- and L-isomers of the PET tracer cis-4-[18F]fluoroproline (D-/L-cis-FPro) and of D-/L-[3H]proline (D-/L-Pro) in rats by dual tracer autoradiography and the uptake of D-cis-FPro in two human subjects by PET. The standardized uptake value (SUV) of D-cis-FPro in the cerebral cortex of rats 2 h p.i. was 3.05+/-1.18 (n=9) versus 0.06+/-0.01 (n=4) for L-cis-FPro (P<0.001) and 1.29+/-0.27 (n=4) for D-Pro versus 0.30+/-0.14 (n=9) for L-Pro (P<0.001). Analysis of the rat brain tissue after injection of D-cis-FPro (n=3) revealed no radioactivity in the proteins but a relevant part in the form of L-trans-FPro. The PET studies yielded a four- to five-fold higher SUV and influx rate constant in the human cortex for D-cis-FPro than for L-cis-FPro. We conclude that D-cis-FPro and D-Pro are preferably transported at the blood-brain barrier compared with their L-isomers and isomerized to the L-form within the brain. Thus, D-Pro in the plasma might be a source of intracerebral L-proline, which has been shown to act as a modulator of excitatory neurotransmission.

Animals↗
PubMed2005

PET with O-(2-18F-Fluoroethyl)-L-Tyrosine in peripheral tumors: first clinical results.

UNLABELLED: O-(2-18F-Fluoroethyl)-L-Tyrosine (18F-FET) PET has shown promising results in brain tumor diagnosis. The aim of this prospective study was to evaluate 18F-FET PET in comparison with 18F-FDG PET in patients with peripheral tumors. METHODS: Forty-four consecutive patients with suspected malignant tumors underwent 18F-FET PET and 18F-FDG PET within 7 d. Whole-body PET studies were performed 1 h after intravenous injection of 370 MBq of 18F-FET or 18F-FDG. Six patients were excluded from the analysis because a malignant tumor could not be verified. In 38 patients (7 with colorectal cancer, 6 with pancreatic cancer, 9 with head-neck cancer, 4 with lymphomas, 3 with lung cancer, 3 with ovarian cancer, 4 with breast cancer, and 2 with prostatic cancer), 18F-FET PET and 18F-FDG PET were compared. RESULTS: 18F-FET was positive in only 13 of 38 patients (8 with head-neck cancer, 3 with breast cancer, and 2 with lung cancer), whereas 18F-FDG exhibited increased uptake in 37 of 38 patients. All squamous cell carcinomas were found to be 18F-FET-positive tumors (8 head-neck cancer and 2 lung cancer), whereas most adenocarcinomas were found to be 18F-FET-negative tumors. In patients with colorectal cancer, pancreatic cancer, ovarian cancer, prostatic cancer, and lymphomas, no increased 18F-FET uptake could be identified. All lesions that exhibited increased 18F-FET uptake also showed increased 18F-FDG uptake. No additional lesion was identified by 18F-FET PET but not by 18F-FDG PET. A subgroup analysis of patients with head-neck carcinomas allowed a better distinction between malignant and inflammatory tissues with 18F-FET than with 18F-FDG. CONCLUSION: 18F-FET is inferior to 18F-FDG as a PET tracer for general tumor diagnosis. Our preliminary results suggest rather selective uptake of 18F-FET in squamous cell carcinomas. Compared with 18F-FDG PET, 18F-FET PET may allow a better distinction between tumors and inflammatory tissues in patients with squamous cell carcinomas.

Adult↗
PubMed2004

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19.

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B(0). We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G --> A and 0.001 for 718C --> T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

Amino Acid Sequence↗
PubMed2004

Molecular cloning of mouse amino acid transport system B0, a neutral amino acid transporter related to Hartnup disorder.

Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B(0). Here we have characterized a novel member of the Na(+)-dependent neurotransmitter transporter family (B(0)AT1) isolated from mouse kidney, which shows all properties of system B(0). Flux experiments showed that the transporter is Na(+)-dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB(0)AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B(0)AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B(0) transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB(0)AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder.

Amino Acid Sequence↗
PubMed2004

Cystinuria-specific rBAT(R365W) mutation reveals two translocation pathways in the amino acid transporter rBAT-b0,+AT.

Apical reabsorption of dibasic amino acids and cystine in kidney is mediated by the heteromeric amino acid antiporter rBAT/b(0,+)AT (system b(0,+)). Mutations in rBAT cause cystinuria type A, whereas mutations in b(0,+)AT cause cystinuria type B. b(0,+)AT is the catalytic subunit, whereas it is believed that rBAT helps the routing of the rBAT/b(0,+)AT heterodimeric complex to the plasma membrane. In the present study, we have functionally characterized the cystinuria-specific R365W (Arg(365)-->Trp) mutation of human rBAT, which in addition to a trafficking defect, alters functional properties of the b(0,+) transporter. In oocytes, where human rBAT interacts with the endogenous b(0,+)AT subunit to form an active transporter, the rBAT(R365W) mutation caused a defect of arginine efflux without altering arginine influx or apparent affinities for intracellular or extracellular arginine. Transport of lysine or leucine remained unaffected. In HeLa cells, functional expression of rBAT(R365W)/b(0,+)AT was observed only at the permissive temperature of 33 degrees C. Under these conditions, the mutated transporter showed 50% reduction of arginine influx and a similar decreased accumulation of dibasic amino acids. Efflux of arginine through the rBAT(R365W)/b(0,+)AT holotransporter was completely abolished. This supports a two-translocation-pathway model for antiporter b(0,+), in which the efflux pathway in the rBAT(R365W)/b(0,+)AT holotransporter is defective for arginine translocation or dissociation. This is the first direct evidence that mutations in rBAT may modify transport properties of system b(0,+).

Amino Acid Substitution↗
PubMed2004

Astroglial glutamine transport by system N is upregulated by glutamate.

Release of glutamine from astrocytes is an essential step of the glutamate-glutamine cycle, and hence for the maintenance of neuronal glutamate and gamma-aminobutyric acid (GABA) pools. The glutamine transporter SNAT3 (SN1) has recently been identified as one of the major mediators of glutamine efflux from astrocytes. We investigated the regulation of SNAT3 mediated glutamine transport in cultured astrocytes. Incubation of primary astrocyte cultures with physiological concentrations of glutamate resulted in a rapid, about twofold, upregulation of SNAT3-mediated transport activity. The effect was not mediated by glutamate receptors but required uptake of glutamate into astrocytes. Both net uptake and net efflux increased after treatment of cells with glutamate, excluding an acceleration of the transport by way of an exchange mechanism. Elevated intracellular glutamate most likely reduces the K(m) of SNAT3 for its substrate glutamine. The results suggest that astrocytes respond actively to the release of glutamate by increasing glutamine release and thereby may modulate glutamatergic neurotransmission.

Amino Acid Transport Systems, Neutral↗
PubMed2004

Facilitated lactate transport by MCT1 when coexpressed with the sodium bicarbonate cotransporter (NBC) in Xenopus oocytes.

Monocarboxylate transporters (MCT) and sodium-bicarbonate cotransporters (NBC) transport acid/base equivalents and coexist in many epithelial and glial cells. In nervous systems, the electroneutral MCT1 isoform cotransports lactate and other monocarboxylates with H+, and is believed to be involved in the shuttling of energy-rich substrates between astrocytes and neurons. The NBC cotransports bicarbonate with sodium and generates a membrane current. We have expressed these transporter proteins, cloned from rat brain (MCT1) and human kidney (NBC), alone and together, by injecting the cRNA into oocytes of the frog Xenopus laevis, and measured intracellular pH changes and membrane currents under voltage-clamp with intracellular microelectrodes, and radiolabeled lactate uptake into the oocytes. We determined the cytosolic buffer capacity, the H+ and lactate fluxes as induced by 3 and 10 mM lactate in oocytes expressing MCT1 and/or NBC, and in water-injected oocytes, in salines buffered with 5 mM HEPES alone or with 5% CO2/10 mM HCO3(-) (pH 7.0). In MCT1 + NBC- but not in MCT1- or NBC-expressing oocytes, lactate activated a Na+- and HCO3(-)-dependent membrane current, indicating that lactate/H+ cotransport via MCT1, due to the induced pH change, stimulates NBC activity. Lactate/H+ cotransport by MCT1 was increased about twofold when MCT1 was expressed together with NBC. Our results suggest that the facilitation of MCT1 transport activity is mainly due to the increase in apparent buffer capacity contributed by the NBC, and thereby suppresses the build-up of intracellular H+ during the influx of lactate/H+, which would reduce MCT1 activity. Hence these membrane transporters functionally cooperate and are able to increase ion/metabolite transport activity.

Animals↗
PubMed2004

Involvement of OCTN2 and B0,+ in the transport of carnitine through an in vitro model of the blood-brain barrier.

Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood-brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B(0,+). Expression of B(0,+) in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B(0,+) could be involved in carnitine transport in both the apical and basolateral membrane.

Amino Acid Transport Systems, Neutral↗