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Biomedical subjects

R Estévez

Publications and source records attributed to R Estévez.

At least 19 recordsLinked to original sources
PubMed2001

Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption and inner ear K+ secretion.

Renal salt loss in Bartter's syndrome is caused by impaired transepithelial transport in the loop of Henle. Sodium chloride is taken up apically by the combined activity of NKCC2 (Na+-K--2Cl- cotransporters) and ROMK potassium channels. Chloride ions exit from the cell through basolateral ClC-Kb chloride channels. Mutations in the three corresponding genes have been identified that correspond to Bartter's syndrome types 1-3. The gene encoding the integral membrane protein barttin is mutated in a form of Bartter's syndrome that is associated with congenital deafness and renal failure. Here we show that barttin acts as an essential beta-subunit for ClC-Ka and ClC-Kb chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either ClC-Kb or barttin compromise currents through heteromeric channels. Currents can be stimulated further by mutating a proline-tyrosine (PY) motif on barttin. This work describes the first known beta-subunit for CLC chloride channels and reveals that heteromers formed by ClC-K and barttin are crucial for renal salt reabsorption and potassium recycling in the inner ear.

Absorption↗
PubMed2001

Sequential amino acid exchange across b(0,+)-like system in chicken brush border jejunum.

In the small intestine, cationic amino acids are transported by y(+)-like and b(0,+)-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b(0,+)-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b(0,+)-like system functions for L-arginine as an obligatory exchanger since its transport capacity increases 100-200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Argin efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Argout is higher than that of Argin, suggesting a possible order of binding (Argout first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed.

Amino Acid Transport Systems↗
PubMed1999

Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta >> brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.

Amino Acid Sequence↗
PubMed1999

Phase II of doxorubicin/taxol in metastatic breast cancer. Argentine Multicenter Taxol Group.

PURPOSE: To assess the response rate, survival, and toxicity of Taxol (paclitaxel) as 1-h infusion plus doxorubicin as first-line treatment for patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: Seventy-six patients with untreated MBC were recruited. All of them had measurable disease and were evaluable for toxicity. Fifty-five percent of the patients had visceral involvement. The dose of doxorubicin was fixed at 50 mg/m2 as a short intravenous infusion, followed by 200 mg/m2 of Taxol as a 1-h intravenous infusion. Doxorubicin was administered during the first seven cycles, continuing with Taxol only up to a maximum of ten cycles. RESULTS: Neutropenia was the most important toxicity: 30% grade 3 and 18% grade 4. Only 2 patients showed a decrease in the left ventricular ejection fraction (LVEF) which caused discontinuing the treatment. No clinical congestive heart failure (CHF) was observed. Seventy-four patients were eligible for response evaluation: 10 (14%) achieved complete response (CR) and 46 (62%) achieved partial response (PR). The mean duration of response was 13.47+/-1.35 months (95% confidence interval (CI): 10.82; 16.12) and the mean survival was 21.50+/-1.42 months (95% CI: 18.72; 24.29). CONCLUSION: The overall response (OR) rate was 76%. No CHF was assessed and 2 patients stopped treatment due to LVEF decrease. Although doxorubicin 50 mg/m2 followed by Taxol 200 mg/m2 in 1-h intravenous infusion presents a toxicity profile which demands a close follow-up, it represents a convenient outpatient schedule with similar activity rate compared to longer Taxol infusions.

Adult↗
PubMed1999

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Amino Acid Sequence↗
PubMed1999

Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the baso-lateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.

Adolescent↗
PubMed1998

Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L. A candidate gene for lysinuric protein intolerance.

We have identified a new human cDNA (y+L amino acid transporter-1 (y+LAT-1)) that induces system y+L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes. Human y+LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1. Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y+L, respectively. y+LAT-1 protein forms a approximately 135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of approximately 85 kDa (i.e. 4F2hc) and approximately 40 kDa (y+LAT-1). Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the co-expressed transport activity. These data suggest that y+LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters. Human y+LAT-1 mRNA is expressed in kidney >> peripheral blood leukocytes >> lung > placenta = spleen > small intestine. The human y+LAT-1 gene localizes at chromosome 14q11.2 (17cR approximately 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P. , Savontaus, M. L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P. (1997) Am. J. Hum. Genet. 60, 1479-1486). LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues. The pattern of expression of human y+LAT-1, its co-expressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y+LAT-1 as a candidate gene for LPI.

Amino Acid Metabolism, Inborn Errors↗
PubMed1998

Cystinuria calls for heteromultimeric amino acid transporters.

The proteins rBAT (related to bo,+ amino acid transporter) and 4F2hc (the heavy chain of the surface antigen 4F2) are homologous proteins that induce amino acid transport in Xenopus oocytes. The role of rBAT in amino acid transport is substantiated by the fact that mutations in the gene encoding it cause cystinuria, a heritable disease characterised by high concentrations of cystine in the urine. Structural and functional evidence supports the hypothesis that both rBAT and 4F2hc proteins form part of heterodimeric amino acid transporters. There is new evidence that the functional unit of system y+L amino acid transporter is a disulfide bridge-dependent complex of 4F2hc with a Xenopus oocyte plasma membrane protein.

Amino Acid Transport Systems↗
PubMed1998

The amino acid transport system y+L/4F2hc is a heteromultimeric complex.

4F2hc is an almost ubiquitous transmembrane protein in mammalian cells; upon expression in Xenopus laevis oocytes, it induces amino acid transport with characteristics of system y+L. Indirect evidence fostered speculation that function requires the association of 4F2hc with another protein endogenous to oocytes and native tissues. We show that expression of system y+L-like amino acid transport activity by 4F2hc in oocytes is limited by an endogenous factor and that direct covalent modification of external cysteine residue(s) of an oocyte membrane protein blocks system y+L/4F2hc transport activity, based on the following. 1) Induction of system y+L-like activity saturates at very low doses of human 4F2hc cRNA (0.1 ng/oocyte). This saturation occurs with very low expression of 4F2hc at the oocyte surface, and further increased expression of the protein at the cell surface does not result in higher induction of system y+L-like activity. 2) Human 4F2hc contains only two cysteine residues (C109 and C330). We mutated these residues, singly and in combination, to serine (C109S; CS1, C330S; CS2 and C109S-C330S, Cys-less). Mutation CS2 had no effect on the expressed system y+L-like transport activity, whereas C109S-containing mutants (CS1 and Cys-less) retained only partial y+L-like transport activity (30 to 50% of wild type). 3) Hg2+, the organic mercury compounds pCMB, and the membrane-impermeant pCMBS almost completely inactivated system y+L-like induced by human 4F2hc wild type and all the mutants studied. This was reversed by ss-mercaptoethanol, indicating that external cysteine residue(s) are the target of this inactivation. 4) Sensitivity to Hg2+ inactivation is increased by pretreatment of oocytes with ss-mercaptoethanol or in the C109S-containing mutants (CS1 and Cys-less). The increased Hg2+ reactivity of C109S-containing mutants supports the possibility that C109 may be linked by a disulfide bond to the Hg2+-targeted cysteine residue of the associated protein. These results indicate that 4F2hc is intimately associated with a membrane oocyte protein for the expression of system y+L amino acid transport activity. To our knowledge, this is the first direct evidence for a heteromultimeric protein structure of an organic solute carrier in mammals.

4-Chloromercuribenzenesulfonate↗
PubMed1998

Molecular biology of mammalian plasma membrane amino acid transporters.

Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x(c)- and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

Amino Acid Sequence↗
PubMed1997

An intracellular trafficking defect in type I cystinuria rBAT mutants M467T and M467K.

The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.

Amino Acid Transport Systems, Basic↗
PubMed1996

Obligatory amino acid exchange via systems bo,+-like and y+L-like. A tertiary active transport mechanism for renal reabsorption of cystine and dibasic amino acids.

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the "renal proximal tubular" cell line OK. System bo, +-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.

Amino Acid Transport Systems, Basic↗
PubMed1983

Improved control of chemotherapy-induced emesis by the addition of dexamethasone to metoclopramide in patients resistant to metoclopramide.

One hundred and two patients with advanced solid tumors receiving chemotherapy were treated with metoclopramide (MCP; 100 mg) to prevent emesis. Twenty-six of 102 patients who had more than five episodes of vomiting were considered MCP-resistant and were randomized to receive MCP (100 mg) plus placebo versus MCP plus dexamethasone (DM; 48 mg) iv in a double-blind, crossover trial. The median number of vomiting episodes was 18 after MCP plus placebo and five after MCP plus DM (P less than 0.001). The median duration of vomiting episodes was 40 hours after MCP plus placebo and 10 hours after MCP plus DM. Immediate toxicity of the antiemetics was not enhanced by the addition of DM to MCP.

Adult↗