35S]S-[5-(4-benzoylphenyl)pentyl]glutathione: a versatile radioligand targeting GS-X pumps with the ability of photoaffinity labeling.

[35S]S-[5-(4-benzoylphenyl)pentyl]glutathione (GIF-0017) as a biochemical probe targeting the ATP-dependent organic anion transporters GS-X pumps was synthesized by the reaction of [35S]glutathione and excess 4-(5-bromo)pentylbenzophenone under alkaline conditions, with the radiochemical yield of 24-33% after HPLC purification. Photolysis of the mixture of [35S]GIF-0017 and plasma membrane vesicles prepared from the MRP1 cDNA-transfected LLC-PK1 cells resulted in radio-labeling of a 180-kDa membrane protein. Immunoprecipitation and western blotting using an anti-MRP1 monoclonal antibody confirmed that the [35S]GIF-0017-labeled protein was the MRPI/GS-X pump.     

MRP1/GS‐X pump ATPase expression: is this the explanation for the cytoprotection of the heart against oxidative stress‐induced redox imbalance in comparison to skeletal muscle cells?

Striated muscle activity is always accompanied by oxidative stress (OxStress): the more intense muscle work and/or its duration, the more a redox imbalance may be attained. In spite of cardiac muscle functioning continuously, it is well known that the heart does not suffer from OxStress-induced damage over a broad physiological range. Although the expression of antioxidant enzymes may be of importance in defending heart muscle against OxStress, a series of combined antioxidant therapeutic approaches have proved to be mostly ineffective in avoiding cellular injury. Hence, additional mechanisms may be involved in heart cytoprotection other than antioxidant enzyme activities. The strong cardiotoxic effect of doxorubicin-induced cancer chemotherapy shed light on the possible role for multidrug resistance-associated proteins (MRP) in this context. Muscle activity-induced 'physiological' OxStress enhances the production of glutathione disulfide (GSSG) thus increasing the ratio of GSSG to glutathione (GSH) content inside the cells, which, in turn, leads to redox imbalance. Since MRP1 gene product (a GS-X pump ATPase) is a physiological GSSG transporter, adult Wistar rats were tested for MRP1 expression and activity in the heart and skeletal muscle (gastrocnemius), in as much as the latter is known to be extremely sensitive to muscle activity-induced OxS. MRP1 expression was completely absent in skeletal muscle. In contrast, the heart showed an exercise training-dependent induction of MRP1 protein expression which was further augmented (2.4-fold) as trained rats were challenged with a session of acute exercise. On the other hand, inducible expression of the 70-kDa heat shock protein (HSP70), a universal marker of cellular stress, was completely absent in the heart of sedentary and acutely exercised rats, whereas skeletal muscle showed a conspicuous exercise-dependent HSP70 expression, which decreased by 45% with exercise training. This effect was paralleled by a 58% decrease in GSH content in skeletal muscle which was even higher (an 80%-fall) after training thus leading to a marked redox imbalance ([GSSG]/[GSH] raised up to 38-fold). In the heart, GSH contents and [GSSG]/[GSH] ratio remained virtually unchanged even after exercise challenges, while GS-X pump activity was found to be 20% higher in the heart related to skeletal muscle. These findings suggest that an intrinsic higher capacity to express the MRP1/GS-X pump may dictate the redox status in the heart muscle thus protecting myocardium by preventing GSSG accumulation in cardiomyocytes as compared to skeletal muscle fibres.     

Low expression of MRP1/GS-X pump ATPase in lymphocytes of Walker 256 tumour-bearing rats is associated with cyclopentenone prostaglandin accumulation and cancer immunodeficiency

Immunosuppression is a life-threatening complication of late cancer stages. In this regard, overproduction in the host plasma of the anti-inflammatory cyclopentenone prostaglandins (CP-PGs), which are strongly antiproliferative at high concentrations, may impair immune function. In fact, lymphoid tissues of tumour-bearing rats accumulated large amounts of CP-PGs while the tumour tissue itself did not. Expression of the CP-PG-induced 72-kDa heat shock protein (hsp70) was elevated in lymphocytes from tumour-bearing animals related to controls. As the capacity for CP-PG uptake by lymphocytes is the same as tumour cells, we investigated whether the latter could overexpress the multidrug resistance-associated protein (MRP1/GS-X pump) which extrudes CP-PGs towards the extracellular space as glutathione S-conjugates. Walker 256 tumour cells extruded 15-fold more S-conjugates than lymphocytes from the same rats (p < 0.001). This did not appear to be related to deficiency in lymphocyte glutathione (GSH) metabolism, since the major GSH metabolic routes are consistent with CP-PG conjugation in lymphocytes. This was not the case, however, for the MRP1/GS-X pump activity in lymphocyte membranes (in pmol/min/mg protein: 3.1 +/- 1.7 from normal rats, 0.2 +/- 0.2 from tumour-bearing animals vs 64.3 +/- 7.0 in tumour cells) which was confirmed by Western blot analysis for MRP1 protein. Transfection of lymphocytes with MRP1 gene completely abolished CP-PG (0-40 microM) toxicity. Taken together, these findings suggest that CP-PG accumulation in lymphocytes may be, at least partially, responsible for cancer immunodeficiency. Clinical approaches for overexpressing MRP1/GS-X pump in lymphocytes could then play a role as a tool for the management of cancer therapeutics.     

Molecular link between cholesterol,cytokines and atherosclerosis

Current investigation on the origin of atherosclerosis has initiated an intense debate over whether atherosclerosis results from hypercholesterolemia or an inappropriate immune response to vascular injury. Although the role of the immune system has been questioned, the overwhelming body of evidence clearly indicates that atherogenesis is initiated by the interplay between cholesterol and cellular secretion of cytokines (especially IL-6) and apolipoprotein E within the arterial wall. Recent studies have revealed that cells possess two cholesterol-sensors: (a) Receptor-C_k which senses the extracellular cholesterol and initiates signalling pathway responsible for the regulation of genes involved in the cell cycle, cell death, cellular cholesterol homeostasis and cytokines including IL-6; (b) LxR which senses intracellular oxysterols and controls genes involved in cell death, cellular cholesterol homeostasis and cytokine IL-8. These cholesterol sensors define the molecular mechanism responsible for cholesterol-depended regulation of cellular synthesis and secretion of cytokines (IL-6, IL-8) within arterial wall. On the basis of this mechanism, presence of cholesterol and its oxy-derivative in the modified LDL will result in transient activation/deactivation of Receptor-C_k-dependent genes which will give rise to repeated cycles of growth coupled with apoptosis leading to a situation where apoptotic-deficient cells in the arterial wall, would be selected resulting in their accumulation and formation of oligoclonal atherosclerotic plaque.     

Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.     

Oxidatively Modified GST and MRP1 in Alzheimer’s Disease Brain: Implications for Accumulation of Reactive Lipid Peroxidation Products

Alzheimer disease (AD) is a neurodegenerative disorder characterized pathologically by intracellular inclusions including neurofibrillary tangles (NFT) and senile plaques. Several lines of evidence implicate oxidative stress with the progression of AD. 4-hydroxy-2-trans-nonenal (HNE), an aldehydic product of membrane lipid peroxidation, is increased in AD brain. The alpha class of glutathione S-transferase (GST) can detoxify HNE and plays an important role in cellular protection against oxidative stress. The export of the glutathione conjugate of HNE is required to fully potentiate the GST-mediated protection. The multidrug resistance protein-1 (MRP1) and GST proteins may act in synergy to confer cellular protection. In the present study, we studied oxidative modification of GST and MRP1 in AD brain by immunoprecipitation of GST and MRP1 proteins followed by Western blot analysis using anti-HNE antibody. The results suggested that HNE is covalently bound to GST and MRP1 proteins in excess in AD brain. Collectively, the data suggest that HNE may be an important mediator of oxidative stress-induced impairment of this detoxifying system and may thereby play a role in promoting neuronal cell death. The results from this study also imply that augmenting endogenous oxidative defense capacity through dietary or pharmacological intake of antioxidants may slow down the progression of neurodegenerative processes in AD.     

Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns,expression levels and stability

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) -endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R₀ and R₁ plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.     

Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated.     

Energy-dependent export of the 13-oxooctadecadienoic acid–glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, β,γ-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K_m of 325.5 μM and a V_max of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K_m of 169 μM and a V_max of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

Glutathione transporters

Background

Glutathione (GSH) is synthesized in the cytoplasm but there is a requirement for glutathione not only in the cytoplasm, but in the other organelles and the extracellular milieu. GSH is also imported into the cytoplasm. The transports of glutathione across these different membranes in different systems have been biochemically demonstrated. However the molecular identity of the transporters has been established only in a few cases.

Scope of review

An attempt has been made to present the current state of knowledge of glutathione transporters from different organisms as well as different organelles. These include the most well characterized transporters, the yeast high-affinity, high-specificity glutathione transporters involved in import into the cytoplasm, and the mammalian MRP proteins involved in low affinity glutathione efflux from the cytoplasm. Other glutathione transporters that have been described either with direct or indirect evidences are also discussed.

Major conclusions

The molecular identity of a few glutathione transporters has been unambiguously established but there is a need to identify the transporters of other systems and organelles. There is a lack of direct evidence establishing transport by suggested transporters in many cases. Studies with the high affinity transporters have led to important structure-function insights.

General significance

An understanding of glutathione transporters is critical to our understanding of redox homeostasis in living cells. By presenting our current state of understanding and the gaps in our knowledge the review hopes to stimulate research in these fields. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Transport of organic anions by multidrug resistance-associated protein in the erythrocyte

The active transport of oxidized glutathione and glutathione S-conjugates has been demonstrated for the first time in erythrocytes and this cell remained the main subject of research on the "glutathione S-conjugate pump" for years. Further studies identifled the "glutathione S-conjugate pump" as multidrug resistance-associated protein (MRP). Even though cells overexpressing MRP and isolated MRP provide useful information on MRP structure and function, the erythrocyte remains an interesting model cell for studies of MRP1 in its natural environment, including the substrate specificity and ATPase activity of the protein.     

Lipopolysaccharide Induction of MARCKS-Related Protein and Cytokine Secretion are Differentially Impaired in Microglia from LPS-nonresponsive (C3H/HeJ) Mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (100 ng/ml) than in normal (C3H/OuJ) microglia (10 ng/ml). By contrast, TNF production was not significantly increased in C3H/HeJ microglia even at 1 g LPS/ml. Microglia expressed PKC isoforms , , , and (but not and ); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lps^d mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

Cloning and expression analyses of AtMRP4 , a novel MRP -like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated. Received: 20 February 1998 / Accepted: 9 March 1998     

Induction of MRP1 and gamma-glutamylcysteine synthetase gene expression by interleukin 1beta is mediated by nitric oxide-related signalings in human colorectal cancer cells

Treatment of human colorectal cancer cells HT29 with interleukin 1beta (IL-1beta) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP-dependent glutathione S-conjugate export (GS-X) pump and the gamma-glutamylcysteine synthetase (gamma-GCSh) gene encoding heavy (catalytic) subunit of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of glutathione (GSH). The induction can be suppressed by N(G)-methyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS). These results suggest that IL-1beta-mediated MRP1 and gamma-GCSh induction involve nitric oxide (NO) -related signaling. Further supports to the involvement of NO in the induction of MRP1 and gamma-GCSh expression are made by the following observations. (i) Expression of MRP1 and gamma-GCSh genes were induced by treating the cells with NO donors, i.e., S-nitro-N-acetyl-D,L-penicillamide (SNAP) and S-nitroso-L-glutathione, in a concentration-dependent manner. (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and gamma-GCSh. We also demonstrated that HT-29 cells treated with either 1L-1beta or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT-29 cells resulted in induction of gamma-GCSh but not MRP1 expression. Collectively, our results demonstrate that induction of MRP1 and gamma-GCSh by IL-1beta is regulated, at least in part, by an NO-related signaling, and induction of gamma-GCSh is by NO-related ceramide signaling.     

Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.