RESUMO
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.
Assuntos
Canalículos Biliares/metabolismo , Glutationa/metabolismo , Fígado/metabolismo , Animais , Proteínas de Transporte de Ânions , Canalículos Biliares/enzimologia , Canalículos Biliares/efeitos da radiação , Transporte Biológico/efeitos da radiação , Proteínas de Transporte/química , Glutationa/química , Glutationa/farmacologia , Técnicas In Vitro , Cinética , Fígado/enzimologia , Fígado/efeitos da radiação , Masculino , Proteínas de Membrana Transportadoras , Ratos , Ratos Sprague-Dawley , Radioisótopos de EnxofreRESUMO
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.
Assuntos
Canalículos Biliares/metabolismo , Glutationa/metabolismo , 5'-Nucleotidase/metabolismo , Animais , Proteínas de Transporte de Ânions , Canalículos Biliares/efeitos da radiação , Transporte Biológico/fisiologia , Transporte Biológico/efeitos da radiação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Transporte/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Cinética , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Dissulfetos/sangue , Compostos de Sulfidrila/sangue , Animais , Transporte Biológico , Cisteína/sangue , Cistina/sangue , Glutationa/sangue , Injeções Intravenosas , Cinética , Masculino , Modelos Biológicos , Concentração Osmolar , Ratos , Ratos Sprague-Dawley , Radioisótopos de EnxofreRESUMO
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.