RESUMEN
This paper examines a unique hypothesis regarding an important role for taurine in renal development. Taurine-deficient neonatal kittens show renal developmental abnormalities, one of several lines of support for this speculation. Adaptive regulation of the taurine transporter gene is critical in mammalian species because maintenance of adequate tissue levels of taurine is essential to the normal development of the retina and the central nervous system. Observations of the remarkable phenotypic similarity that exists between children with deletion of bands p25-pter of chromosome 3 and taurine-deficient kits led us to hypothesize that deletion of the renal taurine transporter gene (TauT) might contribute to some features of the 3p-syndrome. Further, the renal taurine transporter gene is down-regulated by the tumor suppressor gene p53, and up-regulated by the Wilms tumor (WT-1) and early growth response-1 (EGR-1) genes. It has been demonstrated using WT-1 gene knockout mice that WT-1 is critical for normal renal development. In contrast, transgenic mice overexpressing the p53 gene have renal development defects, including hypoplasia similar to that observed in the taurine-deficient kitten. This paper reviews evidence that altered expression of the renal taurine transporter may result in reduced intracellular taurine content, which in turn may lead to abnormal cell volume regulation, cell death and, ultimately, defective renal development.
Asunto(s)
Proteínas Portadoras/fisiología , Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Riñón/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana , Taurina/fisiología , Animales , Desarrollo Embrionario y Fetal/genética , RatasAsunto(s)
Señalización del Calcio/fisiología , Proteínas Portadoras/genética , Regulación de la Expresión Génica , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Regiones Promotoras Genéticas , Taurina/metabolismo , Región de Flanqueo 5' , Adaptación Fisiológica , Animales , Línea Celular , AMP Cíclico/metabolismo , Citosol/metabolismo , Perros , Regulación de la Expresión Génica/efectos de los fármacos , Ionomicina/farmacología , Ionóforos/farmacología , Células LLC-PK1 , Elementos de Respuesta , Porcinos , Taurina/farmacocinéticaAsunto(s)
Proteínas Portadoras/genética , Deleción Cromosómica , Cromosomas Humanos Par 3 , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Taurina/deficiencia , Anomalías Múltiples/genética , Animales , Gatos , Femenino , Humanos , Riñón/lesiones , Masculino , Fenotipo , SíndromeAsunto(s)
Proteínas Portadoras/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Regiones Promotoras Genéticas , Taurina/metabolismo , Región de Flanqueo 5' , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario , Perros , Eliminación de Gen , Guanina , Datos de Secuencia Molecular , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Factor de Transcripción Sp1/metabolismo , Porcinos , Timina , Sitio de Iniciación de la TranscripciónRESUMEN
Previous studies have shown that the Madin-Darby canine kidney cell taurine transporter (pNCT) is downregulated by protein kinase C (PKC) activation. In this study, it is hypothesized that the highly conserved serine-322 (Ser-322) located in the fourth intracellular segment (S4) may play an important role in the function of taurine transporter, which is modulated by PKC phosphorylation. It is demonstrated that Ser-322 is the critical site of PKC phosphorylation, as determined by site-directed mutagenesis. When Ser-322 of pNCT was changed to alanine (S322A) and this mutant was evaluated in an oocyte expression system, taurine transport activity increased threefold compared with control (wild-type pNCT). Activation of PKC by the active phorbol ester 12-myristate 13-acetate did not influence taurine transport by mutant S322A. Kinetic analysis showed that the mutation of Ser-322 essentially changed the Vmax, rather than the Km, of the transporter. Mutation of all other PKC consensus sites did not affect transporter activity when expressed in the oocyte system. Western blot analysis showed that expression of taurine transporter protein was similar in oocytes injected with either wild-type or mutant pNCT cRNA, indicating that the enhanced taurine transport activity by mutant S322A was not caused by a greater amount of transporter expressed in the oocyte. Furthermore, this study demonstrated that the taurine transporter was phosphorylated after PKC activation, and this effect was not observed in mutant S322A. In conclusion, Ser-322 is critical in PKC regulation of taurine transporter activity. The steady-state taurine transporter activity is tightly controlled by endogenous PKC phosphorylation of Ser-322, which is located in the fourth intracellular segment of the taurine transporter.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Riñón/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteína Quinasa C/metabolismo , Taurina/metabolismo , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Línea Celular , Secuencia de Consenso , Perros , Regulación hacia Abajo , Activación Enzimática , Femenino , Técnicas In Vitro , Cinética , Glicoproteínas de Membrana/genética , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Fosforilación , Serina/química , Serina/genética , Xenopus laevisRESUMEN
Studies have shown that the renal tubular epithelium adapts to alterations in the sulfur amino acid composition of the diet. The renal adaptive response has been described in man, mouse, rat, dog, and pig. The observed phenomenon involves increased or decreased initial rate activity of the NaCl-dependent taurine transporter at the brush border membrane surface of the proximal tubule following dietary manipulation of taurine. A cDNA encoding a taurine transporter has been isolated from LLC-PK1 cells, designated pTAUT, and its functional properties have been examined in Xenopus laevis oocytes. The nucleotide sequence of the clone predicts a 621-amino acid protein with about 90% homology to other cloned taurine transporter cDNAs. When expressed in oocytes the transporter displays a Km of 25 microM and is dependent on the presence of external sodium and chloride, characteristics similar to taurine uptake by LLC-PK1 cells. The abundance of pTAUT mRNA and protein were up-regulated in cells cultured in taurine-free medium as compared with cells cultured in medium containing 500 microM taurine. Activation of PKC by PMA had no effect on adaptive regulation of pTAUT mRNA and protein, indicating that down-regulation of LLC-PK1 cell taurine transport activity by PMA occurs at the post-translational level.
Asunto(s)
Transporte Biológico , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Taurina/metabolismo , Adaptación Fisiológica , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Clonación Molecular , Células LLC-PK1 , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Porcinos , Taurina/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The importance of taurine in the diet of pre-term and term infants has not always been clearly understood and is a topic of interest to students of infant nutrition. Recent evidence indicates that it should be considered one of the "conditionally essential" amino acids in infant nutrition. Plasma values for taurine will fall if infants are fed a taurine-free formula or do not have taurine provided in the TPN solution. Urine taurine values also fall, which is indicative of an attempt by the kidney to conserve taurine. The very-low-birth-weight infant, for a variety of reasons involving the maturation of tubular transport function, cannot maximally conserve taurine by enhancing renal reabsorption and, hence, is potentially at greater risk for taurine depletion than larger pre-term or term infants, and certainly more than older children who have taurine in their diet. Taurine has an important role in fat absorption in pre-term and possibly term infants and in children with cystic fibrosis. Because taurine-conjugated bile acids are better emulsifiers of fat than glycine-conjugated bile acids, the dietary (or TPN) intake has a direct influence on absorption of lipids. Taurine supplementation of formulas or TPN solutions could potentially serve to minimize the brain phospholipid fatty acid composition differences between formula-fed and human milk-fed infants. Taurine appears to have a role in infants, children, and even adults receiving most (> 75%) of their calories from TPN solutions in the prevention of granulation of the retina and electroencephalographic changes. Taurine has also been reported to improve maturation of auditory-evoked responses in pre-term infants, although this point is not fully established. Clearly, taurine is an important osmolyte in the brain and the renal medulla. At these locations, it is a primary factor in the cell volume regulatory process, in which brain or renal cells swell or shrink in response to osmolar changes, but return to their previous volume according to the uptake or release of taurine. While there is a dearth of clinical studies in man concerning this volume regulatory response, studies in cats, rats, and dog kidney cells indicate the protective role of taurine in hyperosmolar stress. The infant depleted of taurine may not be able to respond to hyper- or hyponatremic stress without massive changes in neuronal volume, which has obvious clinical significance. The fact that the brain content of taurine is very high at birth and falls with maturation may be a protective feature, or compensation for renal immaturity Defining an amino acid as "conditionally essential" requires that deficiency result in a clinical consequence or consequences which can be reversed by supplementation. In pre-term and term infants, taurine insufficiency results in impaired fat absorption, bile acid secretion, retinal function, and hepatic function, all of which can be reversed by taurine supplementation. Therefore, this small beta-amino acid, taurine, is indeed conditionally essential.
Asunto(s)
Fenómenos Fisiológicos Nutricionales del Lactante , Taurina/fisiología , Animales , Humanos , LactanteRESUMEN
Traditionally, bulk amino acid reabsorption in the kidney has been thought to be localized to the early portions of the proximal nephron. Adult Sprague-Dawley rats were fed diets with low, normal, and high taurine content for two weeks. Kidneys were hybridized with an 35S-radiolabeled complementary RNA probe to the rB16a subclone encoding the extracellular and transmembrane domains of the rat brain taurine transporter. Identical fragments were generated by RT-PCR from rat brain and kidneys as confirmed by DNA sequencing. Hybridization was localized to the outer zone of the medulla of all the kidneys. In the normal diet animals, taurine transporter mRNA was localized to the S3 segment of the proximal tubule, to the loop of Henle in the medulla, and to the glomerular epithelial cell layer. With taurine restriction, taurine transporter mRNA expression was up-regulated predominantly in the S3 segment and was virtually absent in this segment in animals supplemented with taurine. These experiments have precisely localized the rat kidney taurine transporter gene, demonstrating regulation that is limited to the S3 segment of the proximal tubule.
Asunto(s)
Proteínas Portadoras/genética , Regulación de la Expresión Génica , Túbulos Renales Proximales/fisiología , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Animales , Autorradiografía , Dieta , Histocitoquímica , Hibridación in Situ , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Taurina/administración & dosificación , Distribución TisularRESUMEN
Renal brush border taurine transport adapts to changes in the dietary intake of sulfur amino acids with increased rates after dietary restriction and reduced transport after dietary surplus. The Xenopus laevis oocyte expression system was used to define the renal adaptive response to dietary manipulation. Injection of poly(A)+ RNA isolated from rat kidney cortex resulted in a time- and dose-dependent increase in NaCl-taurine cotransport in oocytes. The Km of the expressed taurine transporter was 22.5 microM. In oocytes, injection of 40 ng of poly(A)+ RNA from kidneys of low taurine diet (LTD)-fed rats elicited 2-fold the taurine uptake of normal taurine diet (NTD)-fed rats and >3-fold the uptake of high taurine diet (HTD)-fed rats. Northern blots of rat kidneys using a riboprobe derived from an rB16a (rat brain taurine transporter) subclone revealed 6.2- and 2.4-kb transcripts, the abundance of which were increased or decreased in LTD- or HTD-fed rats, respectively, as compared with NTD-fed rats. A approximately 70-kD protein was detected by Western blot using an antibody derived from a synthetic peptide corresponding to a conserved intracellular segment of rB16a. The abundance of the approximately 70-kD protein was increased or decreased in LTD- or HTD-fed rats, respectively, as compared with NTD-fed rats. In conclusion, expression of the rat renal taurine transporter is regulated by dietary taurine at the level of mRNA accumulation and protein synthesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Renal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Oocitos/fisiología , Taurina/metabolismo , Animales , Aniones/farmacología , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/biosíntesis , Femenino , Técnicas In Vitro , Cinética , Masculino , Glicoproteínas de Membrana/biosíntesis , Oocitos/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Transcripción Genética , Xenopus laevisRESUMEN
NaCl-dependent taurine transporter (pNCT) activity of MDCK cells (Madin-Darby canine kidney) is up- or down-regulated by medium taurine manipulation. In this study we found that the abundance of pNCT mRNA was up- or down-regulated after cells were incubated in media containing 0 microM taurine or 500 microM taurine for 24 h. Down-regulation was observed after 12 h exposure to high taurine (500 microM) and mRNA abundance was appreciably reduced after 72 h exposure. Nuclear run-off assays show that the gene for pNCT is induced at the transcriptional level by taurine. Addition of cycloheximide blocked the adaptive response and reduced transcription of pNCT mRNA in MDCK cells. Cycloheximide had virtually no effect on pNCT mRNA stability, suggesting that ongoing protein synthesis is required for adaptive regulation of pNCT gene transcription.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Riñón/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Taurina/metabolismo , Animales , Proteínas Portadoras/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Cicloheximida/farmacología , Perros , Regulación de la Expresión Génica , Riñón/citología , Riñón/efectos de los fármacos , Glicoproteínas de Membrana/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/efectos de los fármacos , Proteínas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Taurina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
Activation of protein kinase C (PKC) by the active phorbol ester 12-myristate 13-acetate (PMA, 100 nM) or phorbol-12, 13-dibutyrate (100 nM) reduced taurine uptake by 80% in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine uptake by 20% and 400%, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity K(m) (from 5.6 microM to 18.1 microM) with minimal effect on the maximal velocity (25% decrease) of the transporter. A polyclonal antibody (AbS4) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of AbS4 was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of AbS4 (12 ng/ oocyte). In contrast, downregulation of the transporter by PMA could not be restored by AbS4 when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Secuencia Conservada , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana , Proteína Quinasa C/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Proteínas Portadoras/metabolismo , Línea Celular/metabolismo , Perros , Activación Enzimática , Femenino , Riñón/citología , Riñón/metabolismo , Cinética , Glicoproteínas de Membrana/metabolismo , Oocitos/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Xenopus laevisRESUMEN
UNLABELLED: The renal tubular epithelium adapts to changes in the sulfur amino acid composition of the diet, particularly in terms of reabsorption of taurine. The adaptive response is expressed by enhanced or decreased NaCl-dependent taurine transport by rat renal brush border membrane vesicles (BBMV). Taurine transport activity in two cultured renal epithelial cell lines (MDCK and LLC-PK1) is up- or down-regulated by extracellular taurine concentration as the result of reciprocal changes in the Vmax of the transporter. In MDCK cells, abundance of taurine transporter mRNA (pNCT mRNA) was up- or down-regulated after incubation in media containing 0, 50, or 500 microM taurine. Decreased mRNA was observed in both cell lines after 12 h, and it was appreciably reduced after 72 h exposure to 500 microM taurine. Northern blot analysis of mRNA from LLC-PK1 cells using pNCT cDNA as a riboprobe showed that two transcripts, 9.6 kb and 7.2 kb, were expressed; the abundance of mRNA was increased or decreased after incubation in taurine-free or high taurine medium, respectively. Down-regulation was observed primarily in the 7.2 kb transcript after 24 h incubation. Rapid up-regulation occurred in the 9.6 kb transcript within 12 h of transfer from high to low taurine. Nuclear run-off assays showed that the gene for pNCT is induced at the transcriptional level by taurine. Regulation of expression of the taurine transporter was also studied by injection of pNCT cRNA into Xenopus laevis oocytes. Expression of transport activity was significantly reduced (64%) when oocytes were incubated in 50 microM taurine as compared to 0 microM taurine. Transport activity was totally blocked when pNCT cRNA-injected oocytes were exposed to an active phorbol ester, PMA (10(-6) M). Inhibition of uptake was reversed by staurosporine, an inhibitor of protein kinase C activity. An inactive phorbol ester, 4 alpha-phorbol, had no effect on taurine transport. A polyclonal antibody directed a highly conserved intracellular segment between homologous transmembrane domains VI and VII inhibited taurine transport activity in both pNCT cRNA-injected oocytes and BBMV. Incubation of oocytes with 10 micrograms/ml antibody (Ab) reduced taurine uptake to 46% of control, and 20-80 micrograms/ml Ab reduced uptake to 20% of control. In BBMV, active taurine uptake (10 microM) was inhibited approximately 30% by 10 pg Ab/mg protein, whereas none specific IgG had no significant effect. Proline uptake (20 microM) by BBMV was not inhibited by the Ab, nor was GABA uptake (50 microM). Two pNCT proteins, approximately 70 kD and approximately 30 kD, were detected by Western blot, and the abundance of both was regulated by medium taurine. IN CONCLUSION: (i) regulation of taurine transport activity in LLC-PK1 cells by medium taurine occurs at a level of mRNA transcription; (ii) regulation of pNCT occurs at both transcriptional and translational levels; (iii) pNCT expression is regulated by protein kinase C-dependent phosphorylation; and (iv) the intracellular segment between domains VI and VII may be required for activation of the taurine transporter; this segment may function as a gate in taurine transport.
Asunto(s)
Anticuerpos/farmacología , Proteínas Portadoras/biosíntesis , Regulación de la Expresión Génica , Corteza Renal/metabolismo , Riñón/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de Transporte de Membrana , Taurina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión de Anticuerpos , Proteínas Portadoras/fisiología , Línea Celular , Cartilla de ADN , Perros , Epitelio/metabolismo , Masculino , Glicoproteínas de Membrana/fisiología , Microvellosidades/metabolismo , Oocitos/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Taurina/farmacología , Transcripción Genética/efectos de los fármacos , Xenopus laevisRESUMEN
Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutralα-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.
RESUMEN
Urinary enzymes N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyl transpeptidase (gamma-GT) are sensitive markers of specific renal cell damage. Excessive urinary amino acid excretion may also be an indicator of renal tubular damage. We have evaluated urinary excretion of NAG, gamma-GT and 37 amino acids, phospholipids and dipeptides in 30 children (aged 2.3-18.1 years) with nephrotic syndrome (NS), 23 with minimal change nephrotic syndrome (MCNS), 7 with focal segmental glomerulosclerosis (FSGS) and 16 healthy age-matched controls. Nine MCNS patients were in relapse and 14 in remission. Enzyme activity is expressed as micromoles per milligram urinary creatinine. In FSGS, NAG excretion correlated with the following: blood urea nitrogen (BUN) (r = 0.8), serum protein (r = 0.57), serum cholesterol (r = 0.85), serum albumin (r = -0.68) and proteinuria (r = 0.56). In FSGS the gamma-GT excretion was not significantly different from MCNS in remission or in relapse. In FSGS, gamma-GT excretion correlated with the following: BUN (r = 0.48), serum creatinine (r = -0.66), serum protein (r = -0.54), serum albumin (r = -0.68) and serum cholesterol (r = 0.87). Compared with controls, the urinary excretion of 5 amino acids was increased in FSGS patients as a possible indicator of tubular damage. The value for 7 amino acids was reduced in MCNS patients. Urinary amino acid excretion was not different from controls for the other amino acids in either FSGS or MCNS.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acetilglucosaminidasa/orina , Aminoácidos/orina , Síndrome Nefrótico/diagnóstico , gamma-Glutamiltransferasa/orina , Adolescente , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Pruebas Enzimáticas Clínicas , Creatinina/orina , Diagnóstico Diferencial , Femenino , Glomeruloesclerosis Focal y Segmentaria/diagnóstico , Glomeruloesclerosis Focal y Segmentaria/enzimología , Glomeruloesclerosis Focal y Segmentaria/orina , Humanos , Masculino , Síndrome Nefrótico/enzimología , Síndrome Nefrótico/orinaRESUMEN
The erythrocyte anion exchanger, or band 3 protein, catalyzes the exchange of chloride and bicarbonate in many cells. Anion exchanger activity is predominantly found in the basolateral membrane of the cortical collecting duct. Because proximal tubular renal taurine uptake across the brush border surface is dependent upon sodium and chloride, the effect of inhibitors of anion exchanger activity on renal taurine accumulation by rat renal brush border membrane vesicles was examined. The anion exchanger probes, DIDS (4,4-'diisothiosulfonic acid), and NBD-taurine (N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]-aminoethane sulfonic acid), and NBD-taurine (N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]-aminoethane sulfonic acid), were used as inhibitors of anion exchanger activity. DIDS, NAP-, and NBD-taurine all markedly inhibit the initial rate of NaCl-dependent accumulation of taurine by brush border membrane vesicles. NAP- and NBD-taurine accumulation is chloride dependent because inhibition was not found when uptake was performed in the presence of NaNO3 in place of chloride. In the presence of maximal inhibition of taurine uptake by NAP- or NBD-taurine, no additional inhibition was evident after incubation with DIDS. On the other hand, when a competitive inhibitor of taurine uptake, beta-alanine, was used, additional inhibition of taurine accumulation was found in the presence of NAP- or NBD-taurine (p < .01 respectively). These results suggest some interaction of the anion exchanger and the taurine accumulation process at the apical surface of the proximal tubule. Because NBD-taurine is a fluorescent probe, it may be possible to isolate membrane peptides that bind NBD-taurine and demonstrate fluorescence. Preliminary isoelectric focusing experiments of brush border protein incubated with NBD-taurine show fluorescence localized to several particular fractions. Hence this observation that inhibitors of the anion exchanger system block NaCl-dependent taurine uptake can potentially serve as a means of isolating the taurine transporter protein.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/antagonistas & inhibidores , Microvellosidades/metabolismo , Cloruro de Sodio/farmacología , Taurina/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Corteza Renal/ultraestructura , Liposomas/metabolismo , Masculino , Oxadiazoles/metabolismo , Oxadiazoles/farmacología , Ratas , Ratas Sprague-Dawley , Taurina/análogos & derivados , Taurina/farmacologíaRESUMEN
The uptake of the beta-amino acid taurine by rat renal brush border membrane vesicles (BBMV) adapts to changes in dietary sulfur amino acid intake. Initial rate Vmax "upregulates" after ingestion of a low methionine and taurine diet (LTD) and "downregulates" after a high taurine diet (HTD). This is reflected in vivo by hypotaurinuria after a LTD (90% reduction in excretion) and an 18-fold increase in urine taurine after a HTD. This study was performed to determine whether taurine efflux from BBMV is adaptively regulated by external taurine concentration or by diet. Vesicles were preloaded with varying concentrations of radiolabelled and unlabelled taurine and a 150 mM concentration of various salts. Efflux conditions were: taurine and 150 mM salt inside and 150 mM salt outside. The efflux of five concentrations of taurine (10-500 microM) was linear over 6 min, reached equilibrium by 21 min, and was dependent upon intravesicular taurine content. The kinetic characteristics of efflux (E) were significantly different from influx (I): Km = 109.8 +/- 5.8 (E) versus 23.6 +/- 4.2 (I), P < 0.001 [time of linearity = 360 s (E) vs. 20 s (I)]. Efflux of taurine was dependent on the presence of both sodium and chloride in the system, but neither external taurine content (0.100 microM, 1,000 microM) nor external beta-alanine altered initial efflux. Feeding rats a normal diet, LTD, or fasting altered taurine uptake but not efflux. Efflux does not appear to play a role in the adaptive regulation of taurine transport found in all mammalian species.