RESUMEN
Although carbon nanotubes (CNTs) are increasingly used, their biological effects are only incompletely characterized. However, experimental evidence suggests that the intratracheal instillation of CNTs causes the formation of interstitial granulomas and progressive pulmonary fibrosis in rodents. Using human epithelial Calu-3 cells as a model of airway epithelium in vitro, we have recently reported that the exposure to commercial multi-walled CNTs (MWCNTs) causes a progressive decrease of the transepithelial electrical resistance (TEER), pointing to a CNT-dependent impairment of the epithelial barrier function. To characterize better this behavior, we compared the effects of two types of MWCNTs and single-walled CNTs (SWCNTs) of different lengths on the TEER of Calu-3 monolayers. All the materials were used at a dose of 100 microg/mL corresponding to an exposure of 73 microg/cm(2) of cell monolayer. Only the longer MWCNTs and SWCNTs cause a significant decrease in TEER. To elucidate the mechanism underlying the changes in barrier function, the expression of the junction proteins occludin and ZO-1 has been also assessed. No significant decrease in the mRNA for either protein is detectable after the exposure to any type of CNTs. It is concluded that the impairment of barrier function in Calu-3 monolayers is a peculiar effect of CNTs endowed with clear cut fiber properties and is not referable to marked changes in the expression of junction proteins.
Asunto(s)
Bronquios/fisiopatología , Nanotubos de Carbono , Secuencia de Bases , Western Blotting , Bronquios/citología , Línea Celular Tumoral , Cartilla de ADN , Células Epiteliales , Humanos , Técnicas In Vitro , Potenciales de la Membrana , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: To evaluate the activity of the antitumor enzyme L: -asparaginase (ASNase) on tumor cells of mesenchymal origin and the contribution of glutamine synthetase (GS) to the adaptation to the metabolic stress caused by the anti-tumor enzyme. METHODS: We studied the effects of ASNase in six human sarcoma cell lines: HT1080 (fibrosarcoma); RD (rhabdomyosarcoma); SW872 (liposarcoma); HOS, SAOS-2, and U2OS (osteosarcoma) in the absence or in the presence of the GS inhibitor methionine L: -sulfoximine (MSO). RESULTS: HT1080 and SW872 cells were highly sensitive to ASNase-dependent cytotoxicity. In contrast, RD, SAOS-2, HOS, and U2OS cells exhibited only a partial growth suppression upon treatment with the anti-tumor enzyme. In these cell lines ASNase treatment was associated with increased levels of GS. When ASNase was used together with MSO, the proliferation of the poorly sensitive cell lines was completely blocked and a significant decrease in the IC(50) for ASNase was observed. Moreover, when ASNase treatment was carried on in the presence of MSO, HOS and U2OS osteosarcoma cells exhibited a marked cytotoxicity, with increased apoptosis. CONCLUSIONS: In human sarcoma cells (1) GS markedly contributes to the metabolic adaptation of tumor cells to ASNase and (2) the inhibition of GS activity enhances the antiproliferative and cytotoxic effects of ASNase. The two-step interference with glutamine metabolism, obtained through the combined treatment with ASNase and MSO, may provide a novel therapeutic approach that should be further investigated in human tumors of mesenchymal origin.
Asunto(s)
Asparaginasa/farmacología , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Sarcoma/tratamiento farmacológico , Sarcoma/enzimología , Antineoplásicos/farmacología , Caspasas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Metionina Sulfoximina/farmacología , Células Tumorales CultivadasRESUMEN
We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X(-) (AG) (EAAT transporters) in cultured human fibroblasts. With 30 microM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in V(max). Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X(-) (AG) since transport systems A and y(+) are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/antagonistas & inhibidores , Aminoácidos/metabolismo , Antipsicóticos/farmacología , Benzodiazepinas/farmacología , Clorpromazina/farmacología , Clozapina/farmacología , Fibroblastos/efectos de los fármacos , Transporte Biológico , Células Cultivadas , Fibroblastos/metabolismo , Humanos , OlanzapinaRESUMEN
When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Etanol/farmacología , Neoplasias Pancreáticas/metabolismo , Solventes/farmacología , Uniones Estrechas/metabolismo , Actinas/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/ultraestructura , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Manitol/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Ocludina , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/ultraestructura , Permeabilidad/efectos de los fármacos , Fosfoproteínas/metabolismo , Uniones Estrechas/efectos de los fármacos , Proteína de la Zonula Occludens-1RESUMEN
Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 microM secretin or 10 microM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.
Asunto(s)
Células Epiteliales/citología , Microscopía Confocal/métodos , Arilsulfonatos/química , Línea Celular , Células Cultivadas , Células Epiteliales/fisiología , Fluoresceínas/química , Colorantes Fluorescentes/química , Potenciales de la MembranaRESUMEN
System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Cinética , Modelos Biológicos , Ósmosis , Unión Proteica , Isoformas de Proteínas , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
In lysinuric protein intolerance (LPI), impaired transport of cationic amino acids in kidney and intestine is due to mutations of the SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino acid (CAA) transport in human fibroblasts obtained from LPI patients and a normal subject. In both cell types the bidirectional fluxes of arginine are due to the additive contributions of two Na(+)-independent, transstimulated transport systems. One of these mechanisms, inhibited by N-ethylmaleimide (NEM) and sensitive to the membrane potential, is identifiable with system y(+). The NEM- and potential-insensitive component, suppressed by L-leucine only in the presence of Na(+), is mostly due to the activity of system y(+)L. The inward and outward activities of the two systems are comparable in control and LPI fibroblasts. Both cell types express SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We conclude that LPI fibroblasts exhibit normal CAA transport through system y(+)L, probably referable to the activity of SLC7A6/y+LAT2.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Proteínas de Transporte de Anión , Antiportadores , Arginina/farmacocinética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Aminoacidurias Renales/metabolismo , Piel/citología , Piel/metabolismo , Adolescente , Sistemas de Transporte de Aminoácidos Básicos , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Cationes , Células Cultivadas , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Leucina/farmacocinética , Óxido Nítrico/metabolismo , Fenotipo , Aminoacidurias Renales/genética , Proteínas SLC4A , Sodio/farmacologíaRESUMEN
The effects of secretin, the physiological secretagogue for pancreatic ducts, were studied in CAPAN-1 pancreatic duct carcinoma cells. When grown to confluence on plastic dishes, CAPAN-1 cells form domes and exhibit marked increases in culture content of Na+ and urea distribution space (UDS). This parameter is measured as an index of both intracellular and dome compartments under the conditions adopted. Both Na increase and dome formation are inhibited by long term incubation with phorbols, DIDS, DPC, EIPA, H2DIDS, and brefeldin. Short term treatment with secretin or 8-Br-cAMP/teophylline causes dome collapse and a marked decrease in UDS and culture content of Na. Secretin-induced sodium decrease is not abolished by ion channel inhibitors, suggesting that diffusion routes other than ion channels are involved in hormone effects. This hypothesis is also in agreement with data obtained on CAPAN-1 cells cultured on permeable inserts, where no change in Na content or UDS is detected upon secretin treatment. Confluent monolayers exhibit a high transepithelial resistance (Rms) which is markedly and reversibly decreased by secretin. The hormone also decreases the transepithelial voltage (Vms) and raises the monolayer permeability to mannitol. It is concluded that secretin enhances the paracellular permeability of pancreatic duct cells. This effect of secretin, unknown thus far, may be involved in the mechanism of pancreatic secretion in vivo.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Conductos Pancreáticos/efectos de los fármacos , Secretina/farmacología , AMP Cíclico/farmacología , Humanos , Potenciales de la Membrana , Conductos Pancreáticos/citología , Conductos Pancreáticos/fisiología , Células Tumorales CultivadasRESUMEN
Confocal laser scanning microscopy (CLSM) was employed to visualize and measure membrane potential changes in several types of cultured adherent cells, such as human fibroblasts, mouse mammary tumor C127 cells, and human saphenous vein endothelial cells, preloaded with the anionic dye bis-1, 3,-diethylthiobarbituratetrimethineoxonol (bis-oxonol). The fluorescence of cell-associated bis-oxonol was detected in a single confocal plane. An original flow-chamber apparatus was employed to replace the extracellular medium, avoiding alterations of the plane selected for observation. In all the cell types and the experimental situations tested the intracellular distribution of the dye was typical; perinuclear zones accumulated the dye which, conversely, was excluded by the nucleus. Fluorescence was calibrated versus the membrane potential by varying the extracellular concentration of sodium in the presence of gramicidin. With this approach membrane potential was measured (i) in cultured human fibroblasts incubated under anisotonic conditions, (ii) in heterogeneous cell populations which respond unevenly to potential perturbing conditions, and (iii) in human macrovascular endothelial cells maintained in high-serum, complete growth medium. The results obtained indicate that CLSM can be successfully employed to measure changes of membrane potential in single, bis-oxonol-loaded adherent cells under experimental conditions which severely hinder conventional spectrofluorimetric approaches.
Asunto(s)
Medios de Cultivo/farmacología , Colorantes Fluorescentes/análisis , Potenciales de la Membrana , Microscopía Confocal , Tiobarbitúricos/análisis , Animales , Células Cultivadas , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Endotelio Vascular/fisiología , Endotelio Vascular/ultraestructura , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Humanos , Soluciones Hipertónicas/farmacología , Soluciones Hipotónicas/farmacología , Soluciones Isotónicas/farmacología , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/fisiopatología , Ratones , Microscopía Fluorescente , Proteínas Recombinantes/metabolismo , Vena Safena/citología , TransfecciónRESUMEN
In this study we have employed three lines of C127 murine cells. C127 CFTR w/t, C127 CFTR delta F508 and C127 mock, transfected with, respectively, wild type, delta F508 mutant human CFTR cDNA or the vector only. In the first 10 minutes of a Cl(-)-free incubation the three cell lines exhibit a significant shrinkage due to a loss of K+ and Cl-. However, C127 CFTR w/t cells shrink more than C127 CFTR delta F508 and the mock cells. The supplementation of Cl(-)-free medium with ATP causes a marked decrease in the cell volume of C127 CFTR delta F508 and of the mock cells but not of C127 CFTR w/t cells. ATP effect is mimicked by adenosine 5'-O-(3-thiotriphosphate), but neither by adenosine nor by UTP. Measurements of extracellular ATP indicate that during the Cl(-)-free incubation C127 CFTR w/t cells extrude more ATP than the other two cell lines. The results are consistent with the hypothesis that CFTR enhances K+ and Cl- permeabilities by promoting the extrusion of ATP.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Tamaño de la Célula , Clonación Molecular , Medios de Cultivo , ADN Complementario , Humanos , Mutación , Concentración Osmolar , Células Tumorales CultivadasAsunto(s)
Aminoácidos/metabolismo , Adaptación Fisiológica , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico Activo , Proteínas Portadoras/metabolismo , Células Cultivadas , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Humanos , Mesodermo/metabolismo , Modelos Biológicos , Presión Osmótica , Regulación hacia Arriba , Equilibrio HidroelectrolíticoRESUMEN
The functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X(-) AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.
RESUMEN
The expression of wild type CFTR facilitates the efflux of neutral amino acids (Rotoli et al., Biochem. Biophys. Res. Commun. 204: 653-658, 1994); as a result, after an extensive depletion of intracellular amino acid pool obtained through an incubation in saline solution, the intracellular leucine levels were lower in murine C127 cells transfected with the wild type CF gene (C127 CFTRw/t) than in cells transfected with either mutant CF (C127 CFTRΔF508 cells) or mock vector only. No change in amino acid efflux was detected when C127 CFTRw/t and C127 CFTRw/t and C127 CFTRΔF508 cells were studied under conditions known to activate protein kinase A. Upon an incubation in Cl(-) free medium, a permeant analogue of cAMP caused a marked cell depolarization of C127 CFTRw/t cells but not of C127 CFTRΔF508 cells, thus showing a functional expression of CFTR protein in the former cell line. However, we found that, upon a Cl(-) free incubation and in the absence of exogenous cAMP, C127 CFTRw/t cells developed a marked hyperpolarization that was not detected in C127 CFTRΔF508 cells. It is concluded that the expression of normal CFTR accelerates amino acid efflux and enhances cell hyperpolarization in Cl(-) free media; both these effects appear to be independent from PKA stimulation of CFTR.
RESUMEN
Trans-membrane fluxes of leucine were measured in mouse C127i cells transfected with the wild type (C127 CFTRw/t) or the delta F508 CF gene (C127 CFTR delta F508). Leucine efflux was significantly faster in C127 CFTRw/t cells. On the contrary, leucine influx was comparable in the two cell lines and referable to a "L-type" transport system. No significant differences in leucine content were detected among the two cell lines when maintained in complete growth medium; in contrast, after prolonged incubation in amino-acid-free saline solution, the amount of intracellular leucine was significantly smaller in C127 CFTRw/t than in C127 CFTR delta F508 cells. Leucine behavior was shared by other neutral amino acids with non polar side chains. These results suggest that the expression of normal CFTR increases the efflux of a subgroup of neutral amino acids.
Asunto(s)
Fibrosis Quística/metabolismo , Leucina/metabolismo , Proteínas de la Membrana/fisiología , Animales , Transporte Biológico , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Cinética , RatonesRESUMEN
The transport of amino acids has been studied in human umbilical vein endothelial cells. Neutral amino acids enter human umbilical vein endothelial cells through three distinct agencies endowed with the characteristics of systems A, ASC, and L. Each system has been studied by evaluating the influx of preferential substrates. The influx of L-proline and 2-methylaminoisobutyric acid occurs through an Na(+)-dependent adaptively regulated trans-inhibited agency identifiable with system A. L-Threonine influx occurs mainly through a distinct Na(+)-dependent trans-stimulated pathway corresponding to system ASC. System L accounts for Na(+)-independent influx of L-leucine. These systems cooperate for the transport of L-glutamine, which is due mainly to system ASC, whereas the component due to the operation of system A increases upon amino acid starvation. No clear evidence was found for a glutamine-specific system ("system N"). Two systems, one Na+ dependent (system XAG-) and the other Na+ independent (system xc-), transport anionic amino acids. L-Arginine influx exhibits a poor dependence on extracellular Na+, whereas it is sensitive to conditions known to change membrane potential and to trans-stimulation by intracellular amino acids. These features are consistent with a process mediated by system y+ and may be of significance for the regulation of the intracellular concentration of L-arginine.
Asunto(s)
Aminoácidos/metabolismo , Endotelio Vascular/metabolismo , Aniones/metabolismo , Transporte Biológico , Cationes/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Glutamina/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
The relationship between the transport of anionic amino acids and the proliferative status of the cell population has been studied in NIH-3T3 cells. Proliferative quiescence, verified by determinations of growth-rate quotient and incorporation of thymidine, is associated with a marked increase of the influx of L-aspartate. After 7-10 days of serum starvation, the initial influx of L-aspartate increases by 8-10-times with respect to the transport activity determined in growing cells. The operational properties of the influx of L-aspartate are similar in growing and quiescent cells; in particular, the influx of the anionic amino acid is mostly Na(+)-dependent and completely suppressed by an excess of L-glutamate and D-aspartate, but not of D-glutamate. These features suggest that, in both cases, aspartate uptake occurs through system X(-)AG. The quiescence-related increase in aspartate transport is gradual, sensitive to the inhibition of protein synthesis and referable to the enhanced maximal capacity of transport system X(-)AG. Restoration of serum concentration in the culture medium of serum-starved cells causes a decrease in aspartate transport that is maximal in correspondence to late G1/S phases. It is concluded that the X(-)AG system for anionic amino-acid uptake is sensitive to the proliferative status of the cell population and that, in particular, its transport activity is stimulated by the establishment of proliferative quiescence.
Asunto(s)
Ácido Aspártico/metabolismo , División Celular , Sodio/metabolismo , Células 3T3 , Animales , Sitios de Unión , Transporte Biológico , Bromodesoxiuridina , Medio de Cultivo Libre de Suero , Cicloheximida , Cinética , Ratones , TimidinaRESUMEN
The influx of L-threonine through system ASC does not influence the membrane potential in cultured human fibroblasts although comparable fluxes of amino acids through another Na(+)-dependent agency, system A, effectively depolarize the cells. The membrane potential, however, stimulates the influx of amino acids through system ASC with a maximal effect at -50 mV. The sensitivity of amino acid influx through system ASC to the membrane potential is not constant, but rather, is dependent on intracellular and extracellular concentrations of the substrates, Na+ and amino acids, of the system. Conditions which favor the loading of the ASC carrier at the external surface reduce the sensitivity of ASC-mediated amino acid influx to the membrane potential; in contrast, the sensitivity of this amino acid influx increases under conditions which favor loading of the carrier at the internal surface. Trans-stimulation, a well-known characteristic of system ASC, also varies with the concentrations of the substrates of the system and, in fact, this characteristic is not observed when external Na+ is low. These data may be accommodated by a model in which an electrically silent mode of operation of the transporter is dominant. The influence of the membrane potential on the transport system is dependent on the extent to which a charge-translocating step in the cycling of the carrier is rate limiting (relative rate limitance).
Asunto(s)
Aminoácidos/metabolismo , Potenciales de la Membrana , Sodio/metabolismo , Adolescente , Transporte Biológico , Células Cultivadas , Electricidad , Polarización de Fluorescencia , Humanos , Cinética , Masculino , Treonina/metabolismoRESUMEN
The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts.
Asunto(s)
Piel/metabolismo , Sodio/farmacología , Treonina/metabolismo , Aminoácidos/farmacología , Arginina/farmacología , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glutamatos/farmacología , Ácido Glutámico , Humanos , Cinética , Masculino , Modelos BiológicosRESUMEN
The bioenergetics of amino acid transport system A was studied in two Chinese hamster ovary (CHO) cell lines, the parent line CHO-PEOT/1 and CHY-1, a mutant of the former exhibiting a low activity of the same transport system. The steady-state transmembrane distribution ratio of the cationic amino acid L-arginine (RARG) was employed as an indicator of membrane potential (delta psi). Evidence for the reliability of RARG to measure delta psi can be summarized as follows: (1) L-arginine transmembrane distribution increased under conditions of cell hyperpolarization and decreased under conditions of cell depolarization; (2) L-arginine distribution conformed closely to that expected for a probe of delta psi in conditions in which delta psi depends largely on the transmembrane potassium gradient; and (3) the value of delta psi obtained through a valinomycin null point experiment (-72.7 mV) was very similar to the value calculated from L-arginine distribution using the Nernst equation (-73.4 mV). The transmembrane gradient of sodium electrochemical potential (delta mu Na), the driving force for the operation of system A, was slightly higher in the mutant cell line CHY-1. In the same line, the intracellular level of the specific system A substrate MeAIB at steady state was also higher. Studies of the rheogenicity of system A in the two lines indicated that the depolarization associated with the entry of substrates of system A was proportional to the amount of amino acid taken up by the cells. Kinetic analysis showed that the low activity of system A in the mutant cell line was referrable to a decrease in transport Vmax. It is concluded that neither a decrease in energy available for the operation of system A nor a decreased efficiency of coupling of the system to delta psi is responsible for the defect observed in the mutant line.