RESUMEN
Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis-Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier.
Asunto(s)
Canalículos Biliares/metabolismo , Glutatión/metabolismo , Hígado/metabolismo , Animales , Proteínas de Transporte de Anión , Canalículos Biliares/enzimología , Canalículos Biliares/efectos de la radiación , Transporte Biológico/efectos de la radiación , Proteínas Portadoras/química , Glutatión/química , Glutatión/farmacología , Técnicas In Vitro , Cinética , Hígado/enzimología , Hígado/efectos de la radiación , Masculino , Proteínas de Transporte de Membrana , Ratas , Ratas Sprague-Dawley , Radioisótopos de AzufreRESUMEN
We have previously shown GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.
Asunto(s)
Encéfalo/irrigación sanguínea , Proteínas Portadoras/análisis , Endotelio Vascular/metabolismo , Glutatión/metabolismo , Sodio/farmacología , Animales , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Transformada , Membrana Celular/metabolismo , Femenino , Técnicas de Transferencia de Gen , Cinética , Proteínas de Transporte de Membrana , Ratones , Oocitos/metabolismo , ARN Mensajero/metabolismo , Xenopus laevis , gamma-Glutamiltransferasa/metabolismoRESUMEN
PURPOSE: To determine reduced glutathione (GSH) transport in cultured human lens epithelial cells (HLE-B3) and plasma membrane vesicles and to study the expression of GSH transport in Xenopus laevis oocytes injected with poly(A)+ RNA from HLE-B3 cells. METHODS: Confluent HLE-B3 cells pretreated with 10 mM DL-buthionine sulfoximine and 0.5 mM acivicin were used in GSH uptake studies. The uptake of 35S-GSH was performed for 30 minutes in either NaCl medium (Na+-containing) or choline chloride medium (Na+-free) at 37 degrees C and 4 degrees C. The molecular form of 35S uptake was determined by high-performance liquid chromatography. GSH uptake kinetics were studied in acivicin and buthionine sulfoximine-treated HLE-B3 cells in NaCl medium in the concentration range 0.01 microM to 50 mM. The transport of GSH and the effect of Na+ on uptake also were determined in mixed plasma membrane vesicles from HLE-B3 cells. In oocyte expression studies, HLE-B3 poly(A)+ RNA was injected into X. laevis oocytes and GSH uptake experiments were performed 3 days after injection. The uptake of 35S-GSH and GSH efflux rates were determined in HLE-B3 poly(A)+ RNA-injected oocytes. RESULTS: No significant difference was found in the uptake of 1 mM GSH+/-acivicin (17.7+/-4.3 versus 15.7+/-1.4 picomoles/min(-1) per 10(6) cells). However, GSH uptake was significantly lower in Na+-free medium compared with Na+-containing medium (10.3+/-0.7 versus 16.8+/-0.9 picomoles/min(-1) per 10(6) cells; P < 0.01). GSH uptake in NaCl medium was carrier mediated. GSH uptake showed partial sodium dependency from 5 microM to 5 mM GSH in mixed plasma membrane vesicles from HLE-B3 cells. Oocytes injected with HLE-B3 poly(A) RNA expressed uptake and efflux of GSH. Uptake showed partial Na+ dependency at various GSH concentrations. The efflux rates were approximately 30-fold higher than those in water-injected oocytes (0.48+/-0.03 versus 0.016+/-0.005 (nanomoles per hour(-1) per oocyte, respectively). The molecular form of uptake in cultured cells and in oocyte studies was predominantly as intact GSH. CONCLUSIONS: HLE-B3 cells and plasma membrane vesicles transported GSH by a carrier-mediated process. HLE-B3 poly(A)+ RNA injected X laevis oocytes expressed GSH transport. GSH uptake was partially Na+ dependent in all systems. HLE-B3 cells offer a useful model for characterizing GSH transport and for studying its regulatory role in the etiology of cataracts.
Asunto(s)
Células Epiteliales/metabolismo , Glutatión/farmacocinética , Cristalino/metabolismo , Oocitos/metabolismo , ARN Mensajero/farmacología , Animales , Transporte Biológico , Butionina Sulfoximina/farmacología , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Isoxazoles/farmacología , Cristalino/citología , Microinyecciones , Oocitos/efectos de los fármacos , Sodio/farmacología , Xenopus laevis , gamma-Glutamiltransferasa/antagonistas & inhibidores , gamma-Glutamiltransferasa/metabolismoRESUMEN
Transport of GSH at the canalicular pole of hepatocytes occurs by a facilitative carrier and can account for approximately 50% of total hepatocyte GSH efflux. A low-affinity unit with sigmoidal kinetics accounts for 90% of canalicular transport at physiological GSH concentrations. A low-capacity transporter with high affinity for GSH has also been reported. It is not known whether the same or different proteins mediate low- and high-affinity GSH transport, although they do differ in inhibitor specificity. The bile of rats with a mutation in the canalicular multispecific organic anion transporter (cMOAT or MRP-2, a 170-kDa protein) is deficient in GSH, implying that cMOAT may transport GSH. However, transport of GSH in canalicular membrane vesicles (CMV) from these mutant rats remains intact. We examined the functional size of the two kinetic components of GSH transport by radiation inactivation of GSH uptake in rat hepatic CMV. High-affinity transport of GSH was inactivated as a single exponential function of radiation dose, yielding a functional size of approximately 70 kDa. In contrast, low-affinity canalicular GSH transport exhibited a complex biexponential response to irradiation, characterized by an initial increase followed by a decrease in GSH transport. Inactivation analysis yielded a approximately 76-kDa size for the low-affinity transporter. The complex inactivation indicated that the low-affinity transporter is associated with a larger protein of approximately 141 kDa, which masked approximately 80% of the potential transport activity in CMV. Additional studies, using inactivation of leukotriene C4 transport, yielded a functional size of approximately 302 kDa for cMOAT, indicating that it functions as a dimer.
Asunto(s)
Canalículos Biliares/metabolismo , Glutatión/metabolismo , 5'-Nucleotidasa/metabolismo , Animales , Proteínas de Transporte de Anión , Canalículos Biliares/efectos de la radiación , Transporte Biológico/fisiología , Transporte Biológico/efectos de la radiación , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Proteínas Portadoras/efectos de la radiación , Activación Enzimática/efectos de la radiación , Cinética , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Plasma glutathione (GSH), derived principally from the liver, has been proposed as the main endogenous source of plasma cysteine (CYSH). In an earlier study in immature (I) and mature (M) rats, with the use of tracer boluses of intravenous [35S]GSH, we found the movement of the label through plasma GSH, CYSH, and cystine (CYSS) pools to be incompatible with a series of precursor-product compartments (GSH-->CYSH-->CYSS). Thus plasma GSH did not appear to account for sole source of plasma CYSH. To delineate the quantitative interrelationships of plasma GSH, CYSH, and CYSS in I and M rats, we used tracer bolus injections of intravenous [35S]CYSH and [35S]CYSS. The data from the present and previous studies were then used to develop a comprehensive multicompartmental model that fits the data from all experiments. Our analysis indicates the following. 1) Plasma CYSH does not account for the sole intermediate, kinetically homogeneous pool for the movement of label from GSH to CYSS. 2) Only one-half of the irreversible disposal rate (IDR; nmol.min-1.ml-1) of plasma GSH in I rats, but all of it in M rats, is accounted for by hydrolysis to CYSH+CYSS. Thus I rats appear capable of taking up substantial amounts of plasma GSH intact. 3) Significant age-related declines take place in the following IDRs: GSH, from 38 to 18 (approximately 55%); CYSH, from 81 to 11 (approximately 85%); CYSS (in CYSH equivalents), from 30 to 10 (approximately 67%). 4) Hydrolysis of GSH supplies only approximately 22% of plasma IDR of CYSH in I rats vs. approximately 78% in M rats. Thus, in I rats, a sizable inflow of CYSH from other sources than GSH is required to maintain plasma CYSH. 5) In contrast, plasma CYSS appears fully supplied through circulating GSH and CYSH.
Asunto(s)
Disulfuros/sangre , Compuestos de Sulfhidrilo/sangre , Animales , Transporte Biológico , Cisteína/sangre , Cistina/sangre , Glutatión/sangre , Inyecciones Intravenosas , Cinética , Masculino , Modelos Biológicos , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Radioisótopos de AzufreRESUMEN
We have previously shown that sinusoidal reduced glutathione (GSH) efflux declines during development because of a declining maximum transport rate [Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G648-G656, 1991]. Because rat liver serves as the principal source of plasma GSH, we studied the response of plasma GSH to this declining inflow from liver. In immature (28- to 42-day) and mature (90- to 151-day) rats we injected tracer boluses of [35S]GSH intravenously and collected arterial samples over a 0.75- to 8-min interval while plasma GSH pool remained at steady state. Concentrations and radioactivities of GSH, oxidized glutathione (GSSG), cysteine (CYSH), cystine (CYSS), and cysteine-glutathione disulfides (CYSSG) and the radio-activities of proteins were measured in plasma. Our results show the following changes in plasma concentrations (microM): decreases in unbound (free) GSH (26.0 +/- 2.1 to 12.4 +/- 0.98; P < 0.001), total unbound GSH equivalents GSH + 2GSSG (29.1 +/- 2.1 to 15.3 +/- 1.2; P < 0.001), total reducible (unbound + bound) GSH (39.3 +/- 2.2 to 28.9 +/- 2.6; P < 0.025), and free CYSH (57.6 +/- 8.5 to 29.9 +/- 4.0; P < 0.05); no changes in GSSG (1.57 +/- 0.27 vs. 1.47 +/- 0.41), CYSS (36.7 +/- 12 vs. 43.4 +/- 17), and total unbound CYSH equivalents CYSH + 2CYSS (131 +/- 15 vs. 117 +/- 18); increases in total reducible (unbound + bound) CYSH (158 +/- 8.1 to 203 +/- 24; P < 0.05) and CYSSG (1.80 +/- 0.42 to 4.94 +/- 1.4 in microM GSH equivalents; P < 0.05). A concurrent decline occurred in irreversible disposal rate (IDR) of plasma GSH from 38.5 +/- 4.9 to 16.4 +/- 1.4 nmol.min-1.ml-1 (P < 0.001) as determined by compartmental analysis of tracer data. This 57% decrease in IDR parallels a decrease of 53% in the inflow of GSH estimated by perfused livers (17.0 to 8.0 nmol.min-1.ml plasma-1). However, perfused liver estimates do not match > 44-49% of plasma IDR. Thus perfused liver appears to underestimate the true rate of sinusoidal GSH efflux taking place in vivo. Some earlier arteriovenous data and our present portal vein-to-hepatic vein difference measurements appear to corroborate this view.