RESUMO
NaCT (SLC13A5) is a Na(+)-coupled transporter for Krebs cycle intermediates and is expressed predominantly in the liver. Human NaCT is relatively specific for citrate compared with other Krebs cycle intermediates. The transport activity of human NaCT is stimulated by Li(+), whereas that of rat NaCT is inhibited by Li(+). We studied the influence of Li(+) on NaCTs cloned from eight different species. Li(+) stimulated the activity of only NaCTs from primates (human, chimpanzee, and monkey); by contrast, NaCTs from nonprimate species (mouse, rat, dog, and zebrafish) were inhibited by Li(+). Caenorhabditis elegans NaCT was not affected by Li(+). With human NaCT, the Li(+)-induced increase in transport activity was associated with the conversion of the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type. H(+) was able to substitute for Li(+) in eliciting the stimulatory effect. The amino acid Phe500 in human NaCT was critical for Li(+)/H(+)-induced stimulation. Mutation of this amino acid to tryptophan (F500W) markedly increased the basal transport activity of human NaCT in the absence of Li(+), but the ability of Li(+) to stimulate the transporter was almost completely lost with this mutant. Substitution of Phe500 with tryptophan in human NaCT converted the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type, an effect similar to that of Li(+) on the wild-type NaCT. These studies show that Li(+)-induced activation of NaCT is specific for the transporter in primates and that the region surrounding Phe500 in primate NaCTs is important for the Li(+) effect.
Assuntos
Compostos de Lítio/farmacologia , Simportadores/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans , Linhagem Celular , Citratos/metabolismo , Cães , Feminino , Humanos , Macaca mulatta , Camundongos , Mutação , Oócitos/metabolismo , Pan troglodytes , Ratos , Especificidade da Espécie , Simportadores/genética , Xenopus laevis , Peixe-ZebraRESUMO
The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2-C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole-cell patch-clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non-malignant human tissues.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Evolução Molecular , Próstata/citologia , Sequência de Aminoácidos , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico , Proteínas de Transporte/química , Linhagem Celular , Ácido Cítrico/metabolismo , Células Epiteliais/citologia , Inativação Gênica , Humanos , Imuno-Histoquímica , Íons/metabolismo , Masculino , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na(+)-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na(+)-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na(+)-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36+/-0.04mM. Na(+)-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8+/-0.4, indicating involvement of more than one Na(+) in the activation process. Expression of SLC5A8 in Xenopuslaevis oocytes induces Na(+)-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19+/-0.01mM. The Na(+)-activation kinetics is sigmoidal with a Hill coefficient of 2.3+/-0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14+/-1microM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na(+)-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na(+) gradient-driven pyroglutamate uptake was stimulated by an inside-negative K(+) diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Membrana Celular/genética , Expressão Gênica , Humanos , Ionóforos/farmacologia , Rim/metabolismo , Cinética , Camundongos , Microvilosidades/genética , Microvilosidades/metabolismo , Transportadores de Ácidos Monocarboxílicos , Oócitos , Técnicas de Patch-Clamp , Potássio/metabolismo , Coelhos , Ratos , Valinomicina/farmacologia , Xenopus laevisRESUMO
SMCT1 is a Na(+)-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na(+)-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, beta-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Transporte de Cátions/agonistas , Diclofenaco/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Proteínas de Transporte de Cátions/biossíntese , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Transportadores de Ácidos Monocarboxílicos , Oócitos , Epitélio Pigmentado da Retina/metabolismo , Xenopus laevisRESUMO
Tumor cells up-regulate glycolysis but convert pyruvate into lactate instead of oxidizing it. Here, we show that pyruvate, but not lactate, is an inhibitor of histone deacetylases (HDAC) and an inducer of apoptosis in tumor cells and that SLC5A8, a Na(+)/monocarboxylate cotransporter, is obligatory for this process. We found that SLC5A8 is expressed in nontransformed breast epithelial cell lines but silenced by DNA methylation in tumor cell lines. The down-regulation of the gene is also evident in primary breast tumors. When MCF7 breast tumor cells are transfected with SLC5A8 cDNA, the cells undergo pyruvate-dependent apoptosis. Butyrate and propionate also induce apoptosis in SLC5A8-expressing cells, whereas lactate does not. The differential ability of these monocarboxylates to cause apoptosis in SLC5A8-expressing MCF7 cells correlates with their ability to inhibit HDACs. Apoptosis induced by SLC5A8/pyruvate in MCF7 cells is associated with up-regulation of p53, Bax, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptor (TRAILR) 1, and TRAILR2 and down-regulation of Bcl2 and survivin. Lactate dehydrogenase isozymes are differentially expressed in nontransformed cells and tumor cells such that the latter convert pyruvate into lactate. Silencing of SLC5A8 coupled with conversion of pyruvate into lactate in tumor cells correlates with increased HDAC activity in these cells compared with nontransformed cells. Our studies thus identify pyruvate as a HDAC inhibitor and indicate that the Na(+)-coupled pyruvate transport underlies the tumor-suppressive role of SLC5A8. We propose that tumor cells silence SLC5A8 and convert pyruvate into lactate as complementary mechanisms to avoid pyruvate-induced cell death.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/patologia , Proteínas de Transporte de Cátions/metabolismo , Inibidores de Histona Desacetilases , Piruvatos/farmacologia , Mama/citologia , Neoplasias da Mama/genética , Proteínas de Transporte de Cátions/genética , Morte Celular , Linhagem Celular Tumoral , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/fisiologia , Feminino , Citometria de Fluxo , Genes Supressores de Tumor , Humanos , Transportadores de Ácidos Monocarboxílicos , Transporte Proteico , RNA/genética , RNA/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
SMCT (sodium-coupled monocarboxylate transporter; slc5a8) is a Na+-coupled transporter for lactate, pyruvate and short-chain fatty acids. Similar to these already known substrates of SMCT, the water-soluble B-complex vitamin nicotinic acid also exists as a monocarboxylate anion (nicotinate) under physiological conditions. Therefore we evaluated the ability of SMCT to mediate the uptake of nicotinate. In mammalian cells, the cloned mouse SMCT (slc5a8) induced the uptake of nicotinate. The SMCT-induced uptake was Na+-dependent. The Michaelis constant for the uptake process was 296+/-88 microM. The Na+-activation kinetics indicated that at least two Na+ ions are involved in the process. Among the various structural analogues tested, nicotinate was the most effective substrate. Nicotinamide and methylnicotinate were not recognized by the transporter. 2-pyrazine carboxylate and isonicotinate interacted with the transporter to a moderate extent. SMCT-mediated uptake of nicotinate was inhibited by lactate and pyruvate. In the Xenopus laevis oocyte expression system, SMCT-mediated nicotinate transport was electrogenic, as evident from the nicotinate-induced inward currents under voltage-clamp conditions. Substrate-induced currents in this expression system corroborated the substrate specificity determined in the mammalian cell expression system. The kinetic parameters with regard to the affinity of the transporter for nicotinate and the Hill coefficient for Na+ activation, determined by using the oocyte expression system, were also similar to those obtained from the mammalian cell expression system. We conclude that SMCT functions not only as a Na+-coupled transporter for short-chain fatty acids and lactate but also as a Na+-coupled transporter for the water-soluble vitamin nicotinic acid.
Assuntos
Transporte Biológico Ativo , Proteínas de Transporte de Cátions/metabolismo , Eletrofisiologia , Niacina/metabolismo , Sódio/fisiologia , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Estrutura Molecular , Transportadores de Ácidos Monocarboxílicos , Niacina/química , Oócitos , Epitélio Pigmentado da Retina/citologia , Especificidade por Substrato , Xenopus laevisRESUMO
We report in the present paper, on the isolation and functional characterization of slc5a12, the twelfth member of the SLC5 gene family, from mouse kidney. The slc5a12 cDNA codes for a protein of 619 amino acids. Heterologous expression of slc5a12 cDNA in mammalian cells induces Na+-dependent transport of lactate and nicotinate. Several other short-chain monocarboxylates compete with nicotinate for the cDNA-induced transport process. Expression of slc5a12 in Xenopus oocytes induces electrogenic and Na+-dependent transport of lactate, nicotinate, propionate and butyrate. The substrate specificity of slc5a12 is similar to that of slc5a8, an Na+-coupled transporter for monocarboxylates. However, the substrate affinities of slc5a12 were much lower than those of slc5a8. slc5a12 mRNA is expressed in kidney, small intestine and skeletal muscle. In situ hybridization with sagittal sections of mouse kidney showed predominant expression of slc5a12 in the outer cortex. This is in contrast with slc5a8, which is expressed in the cortex as well as in the medulla. The physiological function of slc5a12 in the kidney is likely to mediate the reabsorption of lactate. In the intestinal tract, slc5a12 is expressed in the proximal parts, whereas slc5a8 is expressed in the distal parts. The expression of slc5a12 in the proximal parts of the intestinal tract, where there is minimal bacterial colonization, suggests that the physiological function of slc5a12 is not to mediate the absorption of short-chain monocarboxylates derived from bacterial fermentation but rather to mediate the absorption of diet-derived short-chain monocarboxylates. Based on the functional and structural similarities between slc5a8 and slc5a12, we suggest that the two transporters be designated as SMCT1 (sodium-coupled monocarboxylate transporter 1) and SMCT2 respectively.
Assuntos
Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas de Transporte de Sódio-Glucose/metabolismo , Sódio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Clonagem Molecular , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Oócitos , Especificidade de Órgãos , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis-Menten constant for the transport process is 38+/-5 mM for citrate and 37+/-6 mM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis-Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.
Assuntos
Ciclo do Ácido Cítrico , Simportadores/genética , Simportadores/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Ácido Cítrico/metabolismo , Clonagem Molecular , Transportadores de Ácidos Dicarboxílicos , Éxons , Genoma , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Sódio/farmacologia , Especificidade por Substrato , Ácido Succínico/metabolismo , Simportadores/química , Xenopus laevisRESUMO
In recent studies, we and others showed that autophagy is critical to estrogen receptor positive (ER+) breast cancer cell survival and the development of antiestrogen resistance. Consequently, new approaches are warranted for targeting autophagy in breast cancer cells undergoing antiestrogen therapy. Because crosstalk has been demonstrated between the autophagy- and proteasome-mediated pathways of protein degradation, this study investigated how the proteasome inhibitor bortezomib affects autophagy and cell survival in antiestrogen-treated ER+ breast cancer cells. Bortezomib, at clinically achievable doses, induced a robust death response in ER+, antiestrogen-sensitive and antiestrogen-resistant breast cancer cells undergoing hormonal therapy. Cleavage of PARP and lamin A was detectable as a read-out of cell death, following bortezomib-induced mitochondrial dysfunction. Prior to induction of cell death, bortezomib-treated cells showed high levels of light chain 3 (LC3) and p62, two protein markers for autophagy. The accumulation of these proteins was due to bortezomib-mediated blockade of long-lived protein turnover during macroautophagy. This novel action of bortezomib was linked to its blockade of cathepsin-L activity, which is required for autolysosomal-mediated protein turnover in ER+ breast cancer cells. Further, bortezomib-treated breast cancer cells showed induction of the unfolded protein response, with upregulation of CH OP and GRP78. Bortezomib also induced high levels of the pro-apoptotic protein BNIP3. Knockdown of CH OP and/or BNIP3 expression via RNAi targeting significantly attenuated the death-promoting effects of bortezomib. Thus, bortezomib inhibits prosurvival autophagy, in addition to its known function in blocking the proteasome, and is cytotoxic to hormonally treated ER+ breast cancer cells. These findings indicate that combining a proteasome inhibitor like bortezomib with antiestrogen therapy may have therapeutic advantage in the management of early-stage breast cancer.
Assuntos
Autofagia/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Neoplasias da Mama/patologia , Caspases/fisiologia , Catepsinas/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Pirazinas/farmacologia , Antineoplásicos/farmacologia , Autofagia/genética , Autofagia/fisiologia , Bortezomib , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Moduladores de Receptor Estrogênico/uso terapêutico , Feminino , Humanos , Metabolismo/efeitos dos fármacos , Metabolismo/genética , Receptores de Estrogênio/genética , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
BACKGROUND: 3-bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3-bromopyruvate. METHODS: The transport of 3-bromopyruvate by sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na+)-coupled, electrogenic transporter for short-chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3-bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence-activated cell-sorting analysis and colony-formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis. RESULTS: 3-Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na+-coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3-bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3-bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3-bromopyruvate. CONCLUSIONS: 3-Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na+ gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilases/metabolismo , Piruvatos/farmacologia , Animais , Apoptose , Transporte Biológico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Transportadores de Ácidos Monocarboxílicos , Ratos , Proteínas Recombinantes , Epitélio Pigmentado da Retina/metabolismo , Transfecção , Xenopus laevisRESUMO
SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters (SMCTs), the former being a high-affinity type and the latter a low-affinity type. Both transport a variety of monocarboxylates in a Na(+)-coupled manner. They are expressed in the gastrointestinal tract, kidney, thyroid, brain, and retina. SLC5A8 is localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to neurons and the retinal pigment epithelium. The physiologic functions of SLC5A8 include absorption of short-chain fatty acids in the colon and small intestine, reabsorption of lactate and pyruvate in the kidney, and cellular uptake of lactate and ketone bodies in neurons. It also transports the B-complex vitamin nicotinate. SLC5A12 is also localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to astrocytes and Müller cells. SLC5A8 also functions as a tumor suppressor; its expression is silenced in tumors of colon, thyroid, stomach, kidney, and brain. The tumor-suppressive function is related to its ability to mediate concentrative uptake of butyrate, propionate, and pyruvate, all of which are inhibitors of histone deacetylases. SLC5A8 can also transport a variety of pharmacologically relevant monocarboxylates, including salicylates, benzoate, and gamma-hydroxybutyrate. Non-steroidal anti-inflammatory drugs such as ibuprofen, ketoprofen, and fenoprofen, also interact with SLC5A8. These drugs are not transportable substrates for SLC5A8, but instead function as blockers of the transporter. Relatively less is known on the role of SLC5A12 in drug transport.
Assuntos
Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias/metabolismo , Proteínas de Transporte de Sódio-Glucose/metabolismo , Animais , Humanos , Sódio/metabolismo , Canais de Sódio/metabolismo , Distribuição TecidualRESUMO
We recently identified a novel opioid peptide transport system in the retinal pigment epithelium that transports opioid peptides by a Na+/Cl--dependent process. Here we describe a similar transport system expressed in SK-N-SH cells (a human neuronal cell line) and show for the first time that the activity of the transport system is modulated differentially by lysine and small nonopioid peptides. The transport process in SK-N-SH cells, monitored with deltorphin II as the substrate, is Na+/Cl--dependent and interacts with several opioid peptides, consisting of 5 to 13 amino acids. The activity of this transport system is markedly stimulated by specific dipeptides and tripeptides, with significant stimulation observable at low micromolar concentrations. The ion dependence, Na+/Cl--activation kinetics, and opioid peptide selectivity of the transport system, however, remain unchanged. The stimulation by the modulatory peptides is associated with an increase in maximal velocity with no change in substrate affinity of the system. Amino acids have no or little effect on the transport system, with the exception of lysine. This cationic amino acid inhibits the transport system, with significant inhibition occurring at physiologic concentrations of the amino acid. The inhibitory effect is primarily associated with a decrease in the maximal velocity of the transport system with little change in substrate affinity. Methyl and ethyl esters of lysine retain the inhibitory potency, but most other structural analogs have no effect. The differential modulation of the transport system by lysine and specific small peptides has important implications in the biology and pharmacology of opioid peptides.
Assuntos
Aminoácidos/farmacologia , Cloretos/fisiologia , Oligopeptídeos/farmacologia , Peptídeos Opioides/metabolismo , Sódio/fisiologia , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Ácidos Graxos não Esterificados/farmacologia , Humanos , Cinética , Conformação Molecular , Oligopeptídeos/metabolismoRESUMO
UNLABELLED: PURPOSE. To examine the involvement of human SMCT1, a Na+-coupled transporter for short-chain fatty acids, in the transport of nicotinate/structural analogs and monocarboxylate drugs, and to analyze its expression in mouse intestinal tract. MATERIALS AND METHODS: We expressed human SMCT1 in X. laevis oocytes and monitored its function by [14C]nicotinate uptake and substrate-induced inward currents. SMCT1 expression in mouse intestinal tract was examined by immunofluorescence. RESULTS: [14C]Nicotinate uptake was several-fold higher in SMCT1-expressing oocytes than in water-injected oocytes. The uptake was inhibited by short-chain/medium-chain fatty acids and various structural analogs of nicotinate. Exposure of SMCT1-expressing oocytes to nicotinate induced Na+-dependent inward currents. Measurements of nicotinate flux and associated charge transfer into oocytes suggest a Na+:nicotinate stoichiometry of 2:1. Monocarboxylate drugs benzoate, salicylate, and 5-aminosalicylate are also transported by human SMCTI. The transporter is expressed in the small intestine as well as colon, and the expression is restricted to the lumen-facing apical membrane of intestinal and colonic epithelial cells. CONCLUSIONS: Human SMCTI transports not only nicotinate and its structural analogs but also various monocarboxylate drugs. The transporter is expressed on the luminal membrane of the epithelial cells lining the intestinal tract. SMCT1 may participate in the intestinal absorption of monocarboxylate drugs.
Assuntos
Proteínas de Transporte de Cátions/fisiologia , Trato Gastrointestinal Inferior/metabolismo , Niacina/farmacocinética , Ácidos Aminossalicílicos/química , Ácidos Aminossalicílicos/metabolismo , Ácidos Aminossalicílicos/farmacocinética , Animais , Benzoatos/química , Benzoatos/metabolismo , Benzoatos/farmacocinética , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Estrutura Molecular , Transportadores de Ácidos Monocarboxílicos/fisiologia , Niacina/química , Niacina/metabolismo , Oócitos/metabolismo , Oócitos/fisiologia , Cloreto de Sódio/farmacologia , Trítio , Xenopus laevisRESUMO
In this article, we report on the expression and function of a Na(+)-coupled transporter for citrate, NaCT, in human and rat liver cell lines and in primary hepatocytes from the rat liver. We also describe the polarized expression of this transporter in human and rat livers. Citrate uptake in human liver cell lines HepG2 and Huh-7 was obligatorily dependent on Na+. The uptake system showed a preference for citrate over other intermediates of the citric acid cycle and exhibited a Michaelis constant of approximately 6 mM for citrate. The transport activity was stimulated by Li+, and the activation was associated with a marked increase in substrate affinity. Citrate uptake in rat liver cell line MH1C1 was also Na+ dependent and showed a preference for citrate. The Michaelis constant for citrate was approximately 10 microM. The transport activity was inhibited by Li+. Primary hepatocytes from the rat liver also showed robust activity for Na+-coupled citrate uptake, with functional features similar to those described in the rat liver cell line. Immunolabeling with a specific anti-NaCT antibody showed exclusive expression of the transporter in the sinusoidal membrane of hepatocytes in human and rat livers. This constitutes the first report on the expression and function of NaCT in liver cells.
Assuntos
Fígado/fisiologia , Simportadores/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Transportadores de Ácidos Dicarboxílicos , Hepatócitos/fisiologia , Humanos , Cinética , Lítio/farmacologia , Neoplasias Hepáticas , Ratos , Sódio/metabolismo , Simportadores/metabolismoRESUMO
PURPOSE: Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na+-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). METHODS: We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [14C]-nicotinate and [3H]-ibuprofen for direct measurements of uptake in oocytes. RESULTS: In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na+ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 +/- 16 microM. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na+ dependent, and competitive. However, there was no uptake of [3H]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [14C]-nicotinate was demonstrable under identical conditions. CONCLUSIONS: Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Fenoprofeno/farmacologia , Ibuprofeno/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Fenoprofeno/química , Humanos , Ibuprofeno/química , Potenciais da Membrana , Estrutura Molecular , Transportadores de Ácidos Monocarboxílicos , Niacina/metabolismo , Oócitos/metabolismo , Propionatos , Sódio/metabolismo , Relação Estrutura-Atividade , Transfecção , Xenopus laevisRESUMO
SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.
Assuntos
Encéfalo/citologia , Proteínas de Transporte de Cátions/fisiologia , Corpos Cetônicos/metabolismo , Lactatos/metabolismo , Neurônios/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Encéfalo/metabolismo , Isótopos de Carbono/farmacocinética , Linhagem Celular , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Hidroxibutiratos/farmacocinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Transportadores de Ácidos Monocarboxílicos , Neurônios/efeitos dos fármacos , Oócitos , Técnicas de Patch-Clamp/métodos , Fosfopiruvato Hidratase/metabolismo , Epitélio Pigmentado Ocular/citologia , Sódio/metabolismo , Transfecção/métodos , XenopusRESUMO
SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na(+)/glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known. Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na(+)-coupled transporter for these fatty acids. The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes. Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions. Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes. Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltage-clamp conditions in a Na(+)-dependent manner. These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08-2.5 mm. The substrate-induced currents decreased as the carbon chain length of the substrates increased. The Na(+)-activation kinetics indicated involvement of more than one Na(+) ion in the activation process. Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule. These studies establish the functional identity of SLC5A8 as a Na(+)-coupled transporter for short-chain fatty acids.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/fisiopatologia , Ácidos Graxos Voláteis/metabolismo , Animais , Ligação Competitiva , Proteínas de Transporte de Cátions/química , Regulação para Baixo , Eletroquímica , Regulação Neoplásica da Expressão Gênica , Humanos , Iodetos/metabolismo , Potenciais da Membrana/fisiologia , Transportadores de Ácidos Monocarboxílicos , Oócitos/fisiologia , Técnicas de Patch-Clamp , RNA Mensageiro/análise , Sódio/metabolismo , XenopusRESUMO
We report here on the expression of slc5a8 in kidney and its relevance to Na(+)-coupled reabsorption of lactate. slc5a8 is the murine ortholog of SLC5A8, a candidate tumor suppressor gene, which we recently cloned from human intestine and demonstrated its functional identity as a Na(+)-coupled transporter for short-chain fatty acids and lactate. The slc5a8 cDNA, cloned from mouse kidney, codes for a protein consisting of 611 amino acids. When expressed heterologously in mammalian cells or Xenopus oocytes, slc5a8 mediates Na(+)-coupled electrogenic transport of lactate/pyruvate as well as short-chain fatty acids (e.g. acetate, propionate, and butyrate). The Na+/fatty acid stoichiometry varies depending on the fatty acid substrate (2:1 for lactate and 4:1 for propionate). This phenomenon of variable Na+/substrate stoichiometry depending on the fatty acid substrate is also demonstrable with human SLC5A8. In situ hybridization with sagittal sections of mouse kidney demonstrates abundant expression of the transcripts in the cortex as well as the medulla. Brush border membrane vesicles prepared from rabbit kidney are able to transport lactate in a Na(+)-coupled manner. The transport process exhibits the overshoot phenomenon, indicating uphill lactate transport in response to the transmembrane Na+ gradient. The Na(+)-coupled lactate transport in these membrane vesicles is inhibitable by short-chain fatty acids. We conclude that slc5a8 is expressed abundantly in the kidney and that it plays a role in the active reabsorption of lactate. slc5a8 is the first transporter known to be expressed in mammalian kidney that has the ability to mediate the Na(+)-coupled reabsorption of lactate.