RESUMEN
The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHi regulation of exposure to 1 microM ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHi was 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 microM dimethylamiloride (DMA) rapidly decreased pHi by 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 microM bafilomycin A1 decreased pHi by 0.22 units. Following acid loading in NaCl Ringers with a 20 mm NH4Cl prepulse, pHi recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 microM DMA or 20 microM bafilomycin A1. Based on decreases in H+ efflux resulting from selective inhibition of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pHi recovery following acid loading. Under control conditions, ET-1 (1 microM) had no effect on pHi whereas ET-1 completely suppressed pHi recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 microM), a selective ETA receptor antagonist, pHi recovery was completely restored. Suppression of pHi recovery also occurred following stimulation of protein kinase C (PKC) with 10(-7) m phorbol myristate (PMA) whereas 10(-7) m 4 alpha phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHi recovery after inhibition of PKC with 0.5 microM calphostin C suggesting that the inhibition of pHi recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 microM) reversed the suppression of pHi recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 microM phenylarsine oxide, 10 nm okadaic acid, 10 microM cyclosporin A1 or 20 microM BAPTA, also obviated the suppression of pHi recovery by ET-1. Therefore ETA receptor mediated inhibition of pHi regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+ pump. This alteration may prevent them from becoming adequately stimulated to elicit pHi recovery in response to acid loading.
Asunto(s)
Amilorida/análogos & derivados , Diuréticos/farmacología , Endotelina-1/farmacología , Epitelio Corneal/metabolismo , Concentración de Iones de Hidrógeno , Líquido Intracelular/metabolismo , Receptores de Endotelina/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Amilorida/farmacología , Animales , Arsenicales/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Bovinos , Células Cultivadas , Quelantes/farmacología , Ciclosporina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Antagonistas de los Receptores de Endotelina , Inhibidores Enzimáticos/farmacología , Epitelio Corneal/efectos de los fármacos , Inmunosupresores/farmacología , Líquido Intracelular/efectos de los fármacos , Naftalenos/farmacología , Ácido Ocadaico/farmacología , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Espectrometría de Fluorescencia , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We have investigated the functional characteristics of the human amino acid transporter ATB degree using the Xenopus laevis oocyte expression system. When expressed in oocytes, ATB degree mediates the uptake of neutral amino acids in an Na(+)-dependent manner. In addition, this transporter is able to mediate the efflux of intracellular neutral amino acids in exchange with extracellular neutral amino acids. This homo- and hetero-exchange of amino acids is absolutely Na(+)-dependent and conforms strictly to the substrate specificity of ATB degree. Kinetic analysis indicates that the affinity of ATB degree for a given amino acid substrate is similar whether ATB degree catalyzes the influx of the amino acid or the amino acid-induced efflux of intracellular amino acids. These results demonstrate for the first time the ability of ATB degree to function as a homo- and hetero-exchanger for its substrates.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Sistemas de Transporte de Aminoácidos Neutros , Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Sodio/metabolismo , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico , Células HeLa , Humanos , Cinética , Especificidad por Sustrato , Xenopus laevisRESUMEN
We have cloned the Na(+)-dependent neutral amino acid transporter B0 (ATB0) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB0 (riATB0) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB0 mediates the transport of several neutral amino acids, including glutamine, in a Na(+)-dependent manner. Anionic amino acids, cationic amino acids, and N-methylated amino acids are excluded by riATB0. When expressed in Xenopus laevis oocytes, riATB0 increases the transport of neutral amino acids severalfold. The induced transport activity is specific for neutral amino acids, with no noticeable interaction with anionic, cationic, and N-methylated amino acids. However, riATB0 does interact with anionic amino acids at acidic pH. In oocytes expressing riATB0, the neutral amino acid threonine evokes inward currents at a holding potential of -50 mV. The amino acid-evoked current is sensitive to membrane potential. The inward current increases as the membrane potential is hyperpolarized, but the current reverses at about -30 to -40 mV. Threonine evokes outward currents if the membrane potential is depolarized beyond this value. We have also cloned the ATB0 from the human intestinal cell line Caco-2. The Caco-2 ATB0 cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB0 expressed in the human choriocarcinoma cell line JAR. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis of the RT-PCR products indicate that the human intestine and the human kidney proximal tubular cell line HKPT express an ATB0 identical to the ATB0 expressed in Caco-2 cells.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Portadoras/fisiología , Yeyuno/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Línea Celular , Coriocarcinoma , Neoplasias del Colon , Secuencia de Consenso , Biblioteca de Genes , Células HeLa , Humanos , Intestinos , Túbulos Renales Proximales , Ratones , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Reacción en Cadena de la Polimerasa , Conformación Proteica , Conejos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales Cultivadas , Xenopus laevisRESUMEN
Treatment of the JAR human placental choriocarcinoma cells with herbimycin A, an inhibitor of tyrosine kinases, led to an increase in the activity of the serotonin transporter. This effect was accompanied by an increase in the serotonin transporter density and in the steady-state levels of the serotonin transporter mRNA. A treatment time of > 4 h was necessary for herbimycin A to elicit its effect. Actinomycin D and cycloheximide blocked the effect. There was no increase in the steady-state levels of the serotonin transporter mRNA when cells were treated with herbimycin A in the presence of actinomycin D. The herbimycin A-induced increase in the transporter activity was abolished by genistein, another inhibitor of tyrosine kinases. But the increase in the transporter mRNA levels caused by herbimycin A was not affected by genistein. Treatment of cells with herbimycin A resulted in an increase in the tyrosine phosphorylation of specific cellular proteins, suggesting that herbimycin A directly or indirectly activates specific tyrosine kinases. It is concluded that tyrosine phosphorylation is an essential component in the signaling pathways participating in the regulation of the human serotonin transporter gene expression.
Asunto(s)
Proteínas Portadoras/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Tirosina/metabolismo , Benzoquinonas , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Coriocarcinoma/metabolismo , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica , Genisteína , Humanos , Isoflavonas/farmacología , Lactamas Macrocíclicas , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/antagonistas & inhibidores , Quinonas/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Rifabutina/análogos & derivados , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Células Tumorales Cultivadas , Neoplasias Uterinas/metabolismoRESUMEN
The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin transporter expressed in JAR human placental choriocarcinoma cells was investigated. Treatment of the cells with ATA for 16 h led to a significant stimulation of the serotonin transporter activity. This effect was not observed, however, when the treatment was done for 1-2 h. The stimulatory effect was associated with an increase in the maximal velocity of the transport process with no significant change in the Michaelis-Menten constant. Northern blot hybridization revealed that ATA treatment caused a marked increase in the steady-state levels of serotonin transporter-specific transcripts. Treatment of the cells with ATA was found to increase tyrosine phosphorylation of a 180-kDa protein. The phosphotyrosine content of a protein of a similar molecular size increased dramatically when the cells were exposed to epidermal growth factor (EGF), suggesting that this protein may be the EGF receptor. Treatment of the cells with EGF for 24 h could reproduce the stimulatory effects of ATA on the serotonin transporter activity, the maximal velocity of the transport process, and the steady-state levels of the transporter-specific mRNAs. Genistein, a tyrosine kinase inhibitor, was able to block the stimulatory effect of ATA and EGF. It is concluded that EGF increases the serotonin transporter expression in JAR cells and that the neuroprotective compound ATA produces similar effects on the transporter most likely by activating the EGF receptor through tyrosine phosphorylation.
Asunto(s)
Ácido Aurintricarboxílico/farmacología , Proteínas Portadoras/genética , Factor de Crecimiento Epidérmico/farmacología , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Fármacos Neuroprotectores/farmacología , Transporte Biológico/fisiología , Northern Blotting , Proteínas Portadoras/metabolismo , Coriocarcinoma , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Genisteína , Inhibidores de Crecimiento/farmacología , Humanos , Isoflavonas/farmacología , Cinética , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Placenta/citología , ARN Mensajero/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacos , Tirosina/metabolismoRESUMEN
We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na+-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter Bo. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter Bo, the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na+. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATBo) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATBo gene to human chromosome 19q13.3.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Secuencia de Bases , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 19/genética , Clonación Molecular , ADN Complementario/genética , Femenino , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Oocitos/metabolismo , Placenta/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sodio/metabolismo , Células Tumorales Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Xenopus laevisRESUMEN
To identify a role for a Na:H (NHE) exchanger subtype in volume regulation in bovine corneal epithelium, we determined: 1. its sensitivity to inhibition by amiloride analogues 2. the effects of either Cl removal or hypertonicity on the intracellular pH. Our results suggest that volume regulatory responses elicited by stimulation of NHE-2 may help preserve epithelial barrier function in the face of increases in tear film osmolarity.
Asunto(s)
Córnea/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Amilorida/análogos & derivados , Amilorida/metabolismo , Animales , Bovinos , Células Cultivadas , Córnea/citología , Epitelio/fisiología , Concentración de Iones de Hidrógeno , Soluciones Hipertónicas , Concentración Osmolar , Cloruro de Potasio/metabolismoRESUMEN
Amiloride analogues with N5-alkyl substitutions are specific high-affinity ligands for the Na(+)-H+ exchanger in various tissues. As a means to characterize the Na(+)-H+ exchanger in the bovine corneal epithelium, we determined the binding properties of [3H] methylisobutylamiloride (MIA) to a fraction enriched in plasma membrane from this tissue. [3H]MIA bound to these membranes in a time, -a temperature-, and -a pH-dependent manner. The binding was optimal at 4 degrees C and at pH 8.5 and it reached equilibrium at 60 min. Under these conditions, specific binding, which was inhibitable by excess unlabeled MIA, was about 85%. Scatchard analysis of this specific binding revealed a single saturable binding component with a Kd of 61 nM and a Bmax of 271 pmoles/mg protein. Inhibition of [3H]MIA specific binding by amiloride analogues showed the following order of potency: MIA > dimethylamiloride (DMA) > benzamil > amiloride. Na+ did not compete with MIA for binding. The effectiveness of clonidine, an alpha 2 agonist, and cimetidine, an H2 receptor antagonist, as inhibitors of Na(+)-H+ exchange activity was also determined because these compounds are used to distinguish between the exchanger subtypes. At concentrations higher than those needed for receptor interaction, clonidine was more effective than cimetidine in decreasing MIA binding. The activity of Na(+)-H+ exchanger, which was measured as the uptake of 22Na+ in the presence of an outwardly directly H+ gradient, was also inhibited by DMA, benzamil and amiloride with the same order of potency as obtained in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Portadoras/metabolismo , Córnea/metabolismo , Amilorida/análogos & derivados , Amilorida/antagonistas & inhibidores , Amilorida/metabolismo , Amilorida/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Proteínas Portadoras/clasificación , Bovinos , Membrana Celular/metabolismo , Cimetidina/farmacología , Clonidina/farmacología , Epitelio/metabolismo , Concentración de Iones de Hidrógeno , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno , TemperaturaRESUMEN
In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2.7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-beta-D-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H(+)-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H(+)-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H(+)-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H(+)-pump activity was: ATP >> GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg2+ with Mn2+ did not change the acidification rate but Co2+ only partly activated whereas Ca2+ and Zn2+ were ineffective as activators. The H(+)-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 microM NEM or NBD-Cl and 92% inhibited by 20 microM DCCD.(ABSTRACT TRUNCATED AT 250 WORDS)