Transport Of Glutathione Conjugates Research Articles

Effects of the Phenacetin Metabolite 4-Nitrosophenetol on the Glutathione Status and the Transport of Glutathione S-Conjugates in Human Red Cells

The extent of ferrihemoglobin formation in human erythrocytes by 4-nitrosophenetol and its metabolisation rate strongly depended on the availability of cellular GSH. Ferrihemoglobin formation rate was increased by inhibition of the red cell glutathione reductase, and 4-nitrosophenetol disappeared more slowly. When red cells were completely depleted from SH groups, ferrihemoglobin formation was retarded, despite 4-nitrosophenetol was hardly metabolized. In turn, the glutathione status of human red cells was strongly affected by 4-nitrosophenetol. GSSG, which was produced in large amounts, was reduced, as long as the reducing system was intact. The decreased total glutathione content, however, did not recover completely, indicating formation of stable glutathione S-conjugates. The active export of the stable model glutathione thioether S-(2,4-dinitrophenyl)glutathione was strongly inhibited by 4-nitrosophenetol. A Lineweaver-Burk plot of the transport data suggested a competitive inhibition mechanism, presumably caused by glutathione adducts. The results indicate that the strong pi-donor substituent in 4-nitrosophenetol enables metabolic reactions with glutathione, producing biological effects hitherto not observed with nitrosobenzene. Bicyclic arylamines and glutathione S-conjugates may cause ferrihemoglobin formation that is not brought about by the diaphorase reaction. The latter may be responsible for transport inhibition of GSSG and other glutathione S-conjugates.

Erythrocyte membrane transport of glutathione conjugates and oxidized glutathione in the Dubin-Johnson syndrome and in rats with hereditary hyperbilirubinemia.

The Dubin-Johnson syndrome is manifested by conjugated hyperbilirubinemia and pigment accumulation in hepatocellular lysosomes. The TR-rat model is a phenotypic model of the Dubin-Johnson syndrome and is characterized by defective ATP-dependent transport of a group of nonbile acid organic anions, including glutathione-S-conjugates and oxidized glutathione, across the bile canaliculus. Similar ATP-dependent transport mechanisms have been described in erythrocytes. Intact erythrocytes and inverted erythrocyte membrane vesicles from Dubin-Johnson patients, TR-rats and appropriate controls were studied with regard to ATP-dependent transport of dinitrophenyl glutathione and oxidized glutathione. No significant differences were observed, indicating that the erythrocyte and canalicular ATP-dependent transporters for these substrates are functionally and potentially genetically distinct.

Functional reconstitution of RLIP76 catalyzing ATP-dependent transport of glutathione-conjugates

RLIP76, a stress-responsive, multi-functional protein with multi-specific transport activity towards glutathione-conjugates (GS-E) and chemotherapeutic agents is frequently overexpressed in malignant cells. Our recent studies suggest that it plays a prominent anti-apoptotic role selectively in cancer cells. The present studies were performed to compare RLIP76 activity towards glutathione-conjugates in recombinant and K562 human erythroleukemia cells. The purity and identity of recombinant and K562 RLIP76 was established by SDS-PAGE and Western blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Comparison of ATPase activity and transport kinetics for DNP-SG and GS-HNE between recombinant vs. K562 RLIP76 revealed higher specific activity of ATPase and transport for recombinant purified RLIP76, indicating that additional factors present in recombinant purified RLIP76 can modulate its transport activity.

ATP-dependent canalicular transport of cysteinyl leukotrienes

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [ 3 H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperaturedependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (M r 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and ω-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent K m values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 μM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.

ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates.

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [3H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperature-dependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (Mr 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and omega-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent Km values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 microM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.

Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes.

Transport of S-(2,4-dinitrophenyl)glutathione (DNP-SG), a model compound of glutathione S-conjugates, was studied in purified canalicular and basolateral (i.e. sinusoidal and lateral) rat liver plasma membrane (LPM) vesicles. Incubation of canalicular LPM vesicles with DNP-SG in the presence of ATP resulted in an uptake of DNP-SG into the vesicles. In contrast, in basolateral LPM vesicles the initial rate of ATP-dependent DNP-SG uptake was 3-4-fold lower than that of the canalicular LPM. This suggests that the ATP-dependent glutathione S-conjugates transport is localized in the canalicular membrane of the hepatocytes. The rate of DNP-SG uptake into canalicular LPM vesicles exhibited saturation kinetics with apparent Km values of 4 microM for DNP-SG and 260 microM for ATP. The DNP-SG uptake was inhibited by various glutathione S-conjugates and GSSG, not GSH. The bulkiness of S-substituent alkyl groups in the conjugates and cysteinylglycine of glutathione are essential for the inhibition. In contrast to the ATP-dependent process, the uptake of DNP-SG to basolateral LPM in the presence of KCl was 4-fold higher than that of canalicular LPM. The rate of DNP-SG uptake was enhanced by a valinomycin-induced K+ diffusion potential, and was inhibited by GSH. These results suggest that electrogenic transport of glutathione S-conjugates is localized in the basolateral membrane of the hepatocytes. These studies provide evidence that the ATP-dependent canalicular transport system functions in biliary secretion of glutathione S-conjugates.

ATP/Mg2+-dependent Cardiac Transport System for Glutathione S-Conjugates: A study using rat heart sarcolemma vesicles

Abstract In the present study, the transport of glutathione S-conjugate across rat heart sarcolemma has directly been proved to be an ATP-dependent process. Incubation of sarcolemma vesicles with S-(2,4-dinitrophenyl)glutathione (DNP-SG) in the presence of ATP resulted in a substantial uptake of DNP-SG into the vesicles; Mg2+ was required for ATP-stimulated transport. The rate of glutathione S-conjugate uptake was saturated with respect to ATP and DNP-SG concentrations with apparent Km values of 30 microM for ATP and 20 microM for DNP-SG. However, other nucleoside triphosphates, viz. GTP, UTP, CTP, and TTP, did not stimulate the transport effectively. The ATP-stimulated DNP-SG uptake was not affected by ouabain, EGTA, or by valinomycin-induced K+-diffusion potential, suggesting that Na+,K+-and Ca2+-ATPase activities as well as the membrane potential are not involved in the transport mechanism. ATP could not be replaced by ADP, AMP, or by ATP analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(beta,gamma-imino)triphosphate. From these observations, it is proposed that hydrolysis of gamma-phosphate of ATP is essential for the transport mechanism. The transport of DNP-SG by the sarcolemma vesicles, on the other hand, was inhibited by several different types of glutathione S-conjugates including 4-hydroxynonenal glutathione S-conjugate and leukotriene C4, and not by GSH. The transport system is suggested to have high affinities toward glutathione S-conjugates carrying a long aliphatic carbon chain (n greater than 6) and may play an important role in elimination of naturally occurring glutathione S-conjugates, such as leukotriene C4.

Leukotriene C 4 inhibits ATP-dependent transport of glutathione S-conjugate across rat heart sarcolemma Implication for leukotriene C 4 translocation mediated by glutathione S-conjugate carrier

Sarcolemmal vesicles prepared from rat heart exhibited ATP-dependent uptake of S-(2,4-dinitrophenyl)glutathione (DNP-SG), which obeyed Michaelis-Menten kinetics with an apparent K m of 21 μM for DNP-SG and a V max of 0.27 nmol · 10 min −1 · mg protein −1. Several model glutathione S-conjugates inhibited DNP-SG uptake, but leukotriene C 4 inhibited uptake much more significantly even at lower concentrations (competitive inhibition, K i = 1.5 μM). However, leukotrienes D 4 and E 4, which lack the γ-glutamyl moiety, were less effective. The results suggest that the ATP-dependent transport system has a high affinity for leukotriene C 4, and may be responsible for the translocation of this compound.

Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.

Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

Cardiac transport of glutathione disulfide and S-conjugate. Studies with isolated perfused rat heart during hydroperoxide metabolism.

The relationship between the intracellular GSSG level and the rate of GSSG release was studied in the isolated perfused heart. GSSG and GSH were released from the heart into the effluent perfusion fluid at rates of 110 and 370 pmol X min-1 X g of heart-1, respectively, much lower than for similar conditions in liver. Perfusion with t-butyl hydroperoxide led to an increase in GSSG release associated with an elevated level of intracellular GSSG. Saturation kinetics was found for the rate of GSSG release with respect to the intracellular GSSG level with an apparent Km value of 30 nmol X g of heart-1 and a maximal rate of 7.5 nmol X min-1 X g of heart-1. The maximal rate of GSSG release was temperature-sensitive with a temperature coefficient of 1.8. When 1-chloro-2,4-dinitrobenzene was infused during perfusion with t-butyl hydroperoxide, mutual competition was found in the release of GSSG and the glutathione-S-conjugate, S-(2,4-dinitrophenyl)-glutathione. The maximal rate for the glutathione-S-conjugate transport was estimated to be 40 nmol X min-1 X g of heart-1. These results are consistent with the existence of a transport system for GSSG in rat heart, its capacity being substantially lower than that for the S-conjugate in heart and that for GSSG in liver. Thus, the sensitivity of heart to oxidative stress may be explained, in part, by a low capacity of GSSG export.

Glutathione S-conjugate transport using inside-out vesicles from human erythrocytes.

Previous studies [Kondo, T., Dale, G. L. and Beutler, E. (1981) Biochim. Biophys. Acta, 645, 132-136] have shown evidence for the existence of two different active-transport processes for glutathione disulphide (GSSG) in human erythrocytes (the high-Km and low-Km processes). In the present investigation adenosine-triphosphate-dependent transport of glutathione S-conjugate was characterized in comparison with active glutathione transport using inside-out vesicles from human erythrocytes. Incubation of the vesicles with glutathione S-conjugate (S-2,4-dinitrophenylglutathione) was found to inhibit competitively the high-Km process of GSSG transport but not significantly affect the low-Km process. The glutathione S-conjugate transport required ATP. A lineweaver-Burk plot of the transport rate as a function of the conjugate concentration gave an apparent Km value of 0.94 mM. The Km value of ATP-Mg was 0.76 mM. The transport of glutathione S-conjugate was dependent on temperature. Preincubation of vesicles with dithiothreitol resulted in an increase of the transport rate while thiol reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromercuribenzoate inhibited the transport. Addition of nucleotides, such as CTP, UTP or GTP had no effect on the transport. These findings suggest that glutathione S-conjugate formed by the catalytic reaction of glutathione S-transferase in erythrocytes under the exposure to electrophilic compounds, is eliminated via the same transport process for GSSG elevated under oxidative stress.

Competition between transport of glutathione disulfide (GSSG) and glutathione S-conjugates from perfused rat liver into bile

Competition between transport of glutathione disulfide (GSSG) and glutathione S-conjugates from perfused rat liver into bile

Transport of glutathione S-conjugate from human erythrocytes

Reduced glutathione (GSH) is a major product of erythrocyte metabolism and is vital for protection of red cells from oxidative damage. GSH is synthesized in erythrocytes from its constituent amino acids and has a turnover half-life of -4-5 days [ 11. The mechanism of GSH degradation in erythrocytes has been the subject of some dispute, Some reports [2-41 have suggested that GSH is broken down by y-glutamyl transpeptidase. However, while this may be the case in other tissues, more recent investigations have clearly demonstrated that erythrocytes are devoid of y-glutamyl transpeptidase [5,6]. Recently, evidence has been presented suggesting that, although GSH is not transported from erythrocytes, its turnover is due to the active transport of oxidized glutathione (GSSG) [7]. The glutathione S-transferases catalyze the formation of glutathione conjugates from GSH and a wide variety of electrophilic metabolites and zenobiotics [8]. A single form of glutathione S-transferase has been shown to be present in erythrocytes [9,10] and this indicates their capacity to form glutathione conjugates. It is generally considered that the first step in the metabolism and degradation of glutathione conjugates is the cleavage of the y-glutamyl cysteine peptide bond by the action of y-glutamyl transpeptidase [8]. Since this enzyme is not present in erythrocytes, glutathione conjugates would be expected to either accummulate, or to be transported from the cell. The experiments reported here demonstrate the transport of a glutathione S-conjugate, (S{2,4dinitrophenyl)glutathione) out of erythrocytes. The formation of glutathione conjugates by the action of glutathione S-transferase, and their transport out of the cell,may constitute a significant component of the turnover and metabolism of erythrocyte glutathione.