Saturable Transport Research Articles

Synthesis, radiosynthesis, and in vitro characterization of [ 125I]-2- iodo-L-phenylalanine in a R1M rhabdomyosarcoma cell model as a new potential tumor tracer for SPECT

[ 125I]-2-iodo-L-phenylalanine, a new radioiodinated phenylalanine analog was evaluated as a potential specific tumor tracer for SPECT. The tracer is obtained with an overall radiochemical yield of at least 98%, a purity of >99%, and a specific activity of 11 MBq/mmol in one pot Kit conditions using the Cu 1+ assisted isotopic exchange. The tracer is evaluated in vitro using R1M rat rabdomyosarcoma cells in HEPES buffer with and without Na + ions and in MEM buffer. The uptake of [ 125I]-2-iodo-L-phenylalanine follows a reversible pseudo-first-order reaction which is the same in presence and absence of Na + ions, but the compound is not incorporated into the cell proteins. The reversible uptake is proven to occur with the same affinity as L-henylalanine by a saturable transport system which is competitively inhibited by BCH, an L transport type selective molecule. Trans-stimulation of the efflux by BCH and typical L transported amino acids shows that the transporter is of the antiport type and fulfils all the properties of the LAT1 heterodimer transport system. [ 125I]-2-iodo-L-phenylalanine is thus a phenylalanine analog that for the uptake uses for the major part the LAT1 transport system which is known to be over-expressed in tumor cells. This, together with the easy Kit preparation, makes [ 123I]-2-iodo-L-phenylalanine a promising tumor specific tracer for SPECT.

Cerebrospinal fluid leptin levels in preeclampsia: relation to maternal serum leptin levels.

To determine whether cerebrospinal fluid (CSF) and circulating levels of leptin differ between women with preeclampsia and women who had an uncomplicated pregnancy. Maternal serum and CSF leptin concentrations obtained in the third trimester of the gestation were compared in 16 women with mild preeclampsia and 23 normotensive pregnant women who underwent cesarean section. Before administering local anesthetic for spinal anesthesia, 2 mL CSF and 4 mL venous blood sample were taken and were stored at -30 degrees C until serum and CSF leptin levels were measured by enzyme-linked immunosorbent assay (ELISA). Mean CSF leptin concentrations were not significantly different between the two groups (preeclampsia 9.7 +/- 4.2 ng/mL, normotensive 13.6 +/- 4.3 ng/mL, p = 0.952). Similarly, mean serum leptin concentrations were similar between the two groups (mild preeclampsia 21.7 +/- 7.1 ng/mL, normotensive 18.3 +/- 6.7 ng/mL, p = 0.698). CSF leptin levels are inversely related to the serum leptin concentrations in preeclamptic patients (r = -0.87, p = 0.000). An inverse relationship was also detected between CSF and serum leptin levels in normotensive pregnant subjects (r = -0.66, p = 0.000). CSF and serum leptin levels were similar in patients with preeclampsia and normotensive pregnant women. However, the CSF leptin was negatively correlated with the serum leptin concentrations in preeclamptic and normotensive control subjects, suggesting that leptin enters the brain by a saturable transport system. Further work is needed to confirm our findings.

Photoaffinity labeling of the anionic sites in Caco-2 cells mediating saturable transport of hydrophilic cations ranitidine and famotidine.

The H(2) antagonists, ranitidine and famotidine, exhibit saturable absorptive transport across Caco-2 cell monolayers and human intestine via a yet unidentified mechanism. A photoreactive derivative of famotidine has been synthesized and evaluated as a photoaffinity probe for the putative transporter protein(s). The probe irreversibly inhibited ranitidine transport across Caco-2 cell monolayers and irreversibly increased the transepithelial electrical resistance (TEER) after UV activation. Photoaffinity labeling was protected by a molar excess of famotidine.

Polypeptide delivery across the blood-brain barrier.

The blood-brain barrier (BBB) used to be considered impermeable to polypeptides. However, this view has evolved rapidly over the past two decades. Not only do polypeptides have the potential to serve as carriers for selective therapeutic agents, but they themselves may directly cross the BBB after delivery into the bloodstream to become potential treatments for a variety of CNS disorders, including neurodegeneration, autoimmune diseases, stroke, depression, and obesity. The interactions of polypeptides with the BBB can take many forms, such as simple diffusion, saturable transport, or facilitation of entry of another peptide or protein. In some instances, interactions in the blood compartment (outside the BBB) or within the endothelial cells (at the BBB level) can significantly impede the passage of polypeptides across the BBB. We shall review the different aspects of interactions between peptides/proteins and the BBB that affect their delivery as potential drugs in their natural form, and discuss recent advances in the cell biology of polypeptide transport across the BBB. Better understanding of the BBB will provide insight and direction for future research in the treatment of CNS disorders.

The source of cerebral insulin

Insulin and its receptor are found throughout the central nervous system (CNS). Insulin administered into the CNS can exert powerful effects, yet the consensus is that little or no insulin is produced in the CNS. Therefore, CNS insulin is essentially dependent on the ability of peripheral insulin to cross the blood–brain barrier (BBB). Insulin is known to cross the BBB by a saturable transport mechanism. This transporter shows some thematic similarities to other transporters for peptides or regulatory proteins. It is unevenly distributed throughout the CNS with the olfactory bulbs having the fastest transport rate of any brain region. It is partially saturated at euglycemic levels, suggesting that its main signaling function occurs at physiological blood levels, rather than as a brake to hypoglycemic events. One probable function of the BBB transporter is to allow CNS insulin to act as a counter-regulatory hormone to peripheral insulin. The transporter is regulated, with the transport rate of insulin being altered during development and by fasting, obesity, hibernation, diabetes mellitus and Alzheimer's disease. Enhancement of insulin transport by lipopolysaccharide could be the basis for the insulin resistance seen with bacterial infections. Inhibition of insulin transport across the BBB by dexamethasone could be the basis for the enhanced appetite seen with glucocorticoid treatments. Insulin itself also has effects on the BBB, altering enzymatic and transporter functions. Overall, BBB transport of insulin provides a mechanism for peripheral insulin to act within the CNS as a regulatory peptide.

Apparent absolute oral bioavailability in excess of 100% for a vitronectin receptor antagonist (SB-265123) in rat. II. Studies implicating transporter-mediated intestinal secretion

Transporters have been increasingly identified as a factor in limiting the oralbioavailability of certain drugs. Previously, the present authors investigated a compound(SB-265123) with an apparent absolute oral bioavailability (Fapp) consistently >100%, andexcluded likely artefactual causes for this observation, as well as standard considerationsof non-stationary or non-linear pharmacokinetics. The data led the authors to believethat SB-265123 might be a transporter substrate in the rat, and it was hypothesized thattransporter interactions might be responsible for the observed Fapp>100%.In the present study, a model was proposed incorporating rapid and completeabsorption and elimination by a saturable intestinal secretory pathway. Intestinalsecretion was demonstrated for SB-265123 using a rat single-pass intestinal perfusiontechnique. In addition, in a study employing both independent and simultaneousintravenous and oral administration of SB-265123, exposure to SB-265123 was greaterthan additive on joint intravenous and oral administration, lending further support to thehypothesis of a saturable transporter. Furthermore, in a study with co-administration ofGF120918A, a transporter inhibitor, the observed Fapp for SB-265123 was only 84±17%,providing additional evidence for transporter involvement in the >100% Fappphenomenon.Experience with SB-265123 illustrates a counterintuitive impact of transporterson oral bioavailability and highlights the importance of considering transporterinteractions in the systemic disposition of xenobiotics, even those not demonstratinglow oral bioavailability.

Transport of ethinylestradiol glucuronide and ethinylestradiol sulfate by the multidrug resistance proteins MRP1, MRP2, and MRP3.

Ethinylestradiol (EE) is one of the key constituents of oral contraceptives. Major metabolites of EE in humans are the glucuronide and sulfate conjugates, EE-3-O-glucuronide (EE-G) and EE-3-O-sulfate (EE-S). In the present study, transport of EE-G and EE-S by the human multidrug resistance proteins MRP1, MRP2, and MRP3 was investigated using inside-out membrane vesicles, isolated from Sf9 cells expressing human MRP1, MRP2, or MRP3. Vesicular uptake studies showed that EE-G was not a substrate for MRP1, whereas an ATP-dependent and saturable transport of [(3)H]EE-G was observed in MRP2 (K(m) of 35.1 +/- 3.5 microM) and MRP3 (K(m) of 9.2 +/- 2.3 microM) containing vesicles. EE-S was not transported by either MRP1, MRP2, or MRP3. However, low concentrations of EE-S stimulated MRP2-mediated uptake of ethacrynic acid glutathione. EE-S also stimulated MRP2 and MRP3-mediated uptake of 17beta-estradiol-17beta-D-glucuronide. Interestingly, EE-S stimulated strongly MRP2- and MRP3-mediated uptake of EE-G by increasing its apparent transport affinity, whereas no reciprocal stimulation of EE-S uptake by EE-G was observed. These data indicate that EE-S allosterically stimulates MRP2- and MRP3-mediated transport of EE-G and is not cotransported with EE-G. Our studies demonstrate specific active transport of a pharmacologically relevant drug conjugate by human MRP2 and MRP3, involving complex interactions with other organic anions. We also suggest that caution needs to be taken when using only competition studies as screening tools to identify substrates or inhibitors of MRP-mediated transport.

Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike beta-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.

Cellular and whole-plant chloride dynamics in barley: insights into chloride-nitrogen interactions and salinity responses.

The first analysis of chloride fluxes and compartmentation in a non-excised plant system is presented, examining ten ecologically pertinent conditions. The short-lived radiotracer couple (38)Cl/(39)Cl was used as a Cl(-) tracer in intact barley ( Hordeum vulgare L. cv. Klondike) seedlings, which were cultured and investigated under four external [Cl(-)], from abundant (0.1 mM) to potentially toxic (100 mM). Chloride-nitrogen interactions were investigated by varying N source (NO(3)(-) or NH(4)(+)) and strength (0.1 or 10 mM), in order to examine, at the subcellular compartmentation level, the antagonism, previously documented at the influx level, between Cl(-) and NO(3)(-), and the potential role of Cl(-) as a counterion for NH(4)(+) under conditions in which cytosolic [NH(4)(+)] is excessive. Cytosolic [Cl(-)] increased with external [Cl(-)] from 6 mM to 360 mM. Cl(-) influx, fluxes to vacuole and shoot, and, in particular, efflux to the external medium, also increased along this gradient. Efflux reached 90% of influx at the highest external [Cl(-)]. Half-times of cytosolic Cl(-) exchange decreased between high-affinity and low-affinity influx conditions. The relationship between cytosolic [Cl(-)] and shoot flux indicated the presence of a saturable low-affinity transport system ("SLATS") responsible for xylem loading of Cl(-). N source strongly influenced Cl(-) flux to the vacuole, and moderately influenced Cl(-) influx and shoot flux, whereas efflux and half-time were insensitive to N source. Cytosolic pool sizes were not strongly or consistently influenced by N source, indicating the low potential for Cl(-) to act as a counterion to hyperaccumulating NH(4)(+). We discuss our results in relation to salinity responses in cereals.

Efflux of human and mouse amyloid β proteins 1–40 and 1–42 from brain: impairment in a mouse model of alzheimer's disease

Brain to blood transport is believed to be a major determinant of the amount of amyloid β protein (AβP) found in brain. Impaired efflux has been suggested as a mechanism by which AβP can accumulate in the CNS and so lead to Alzheimer's disease (AD). To date, however, no study of the efflux of the form of AβP most relevant to AD, AβP1–42, has been conducted, even though a single amino acid substitution in AβP can greatly alter efflux. Here, we examined the efflux of AβP mouse1–42, mouse1–40, human1–42, and human1–40 in young CD-1, young senesence accelerated mouse (SAM) P8, and aged SAMP8 mice. The SAMP8 mouse with aging spontaneously overproduces AβP and develops cognitive impairments reversed by AβP-directed antibody or phosphorothioate antisense oligonucleotide. CD-1 mice transported all forms of AβP, although mouse1–42 and human1–40 were transported faster than the other forms. There was a decrease in the saturable transport of mouse1–42 in SAMP8 mice regardless of age. Efflux of mouse1–40 and human1–42 was only by a non-saturable mechanism in young SAMP8 mice and their efflux was totally absent in aged SAMP8 mice. These differences in the efflux of the various forms of AβP among the three groups of mice supports the hypothesis that impaired efflux is an important factor in the accumulation of AβP in the CNS.

Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.

The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.

A current review of fatty acid transport proteins (SLC27).

Long-chain fatty acids (LCFAs) are not only important metabolites but contribute to many cellular functions including activation of protein kinase C (PKC) isoforms and nuclear transcription factors such as peroxisome proliferator-activated receptors (PPAPs). To assert their diverse effects LCFAs have first to traverse the plasma membrane, a process that can occur either through diffusion or be mediated by proteins. Considerable evidence has accumulated to show that in addition to a diffusional component, the intestine, heart, adipose tissue, and the liver express a saturable and specific LCFA transport system. Identifying the postulated fatty acid transporters is of considerable importance, since both increased and decreased fatty acid uptake have been implicated in diseases such as type-2 diabetes and acute liver failure. Fatty acid transport proteins (FATPs/solute carrier family 27) are integral transmembrane proteins that enhance the uptake of long-chain and very long chain fatty acids into cells. In humans FATPs comprise a family of six highly homologous proteins, hsFATP1-6, which are found in all fatty acid-utilizing tissues of the body. This review will focus on a brief discussion of FATP expression patterns, regulation, structure, and mechanism of transport.

Interactions of cytokines with the blood-brain barrier: implications for feeding.

The blood-brain barrier (BBB) mediates interactions between the brain and the cytokines produced in the periphery. Some of these cytokines play significant roles in feeding behavior. This review will summarize various ways by which cytokines cross the BBB and discuss the implications of their transport systems in feeding. For simplicity of discussion, three categories of cytokines are discussed: (1). the proinflammatory cytokines TNFalpha, IFNgamma, IL1, and IL6; (2). the chemokines MIP-1, CINC-1 and IL8; and (3). other cytokines (LIF, CNTF, GM-CSF, FGF, EGF, and TGFalpha). The pharmacokinetics of barrier penetration, compartmental distribution, stability, and mechanism of passage (presence or absence of saturable transport) are summarized. Our understanding of cytokines interacting with the BBB is still growing; not only are more cytokines being studied, but also more details of the nature of the transport systems and how they affect feeding behavior are being explored.

Glial cell line-derived neurotrophic factor does not enter normal mouse brain

Glial cell line-derived neurotrophic factor (GDNF) is produced both in the central nervous system (CNS) and the periphery. Effective in ameliorating neurodegeneration in several animal models of CNS disease, its promise as a therapeutic agent would be greatly enhanced if it readily crossed the blood–brain barrier (BBB) in unmodified form. Here, we used the sensitive techniques of multiple-time regression analysis and ex-vivo perfusion in blood-free buffer to examine the entry of 125I-GDNF into mouse brain. The integrity of GDNF in blood and brain was examined by high performance liquid chromatography and the physicochemical properties determining permeability were measured by octanol/buffer partition coefficient and hydrogen bonding. The efflux of 125I-GDNF was determined to test for the presence of a bidirectional transport system. The results show that 125I-GDNF differs from other peptides and polypeptides in that it does not enter brain any faster than 99mTc-albumin, an effect that cannot be explained by degradation, rapid efflux, protein binding, or inadequate lipophilicity. Thus, GDNF shows a different type of interaction with the BBB. In normal mice, the BBB functions as a substantial physical barrier; in pathological or traumatic situations when the barrier is partially disrupted, the lack of restriction by a saturable transport system could make GDNF a suitable candidate for peripheral delivery in promoting neuroregeneration.

Cellular uptake and concentrations of tamoxifen upon administration in poly(ε-caprolactone) nanoparticles

In an attempt to increase the local concentration of tamoxifen in estrogen receptor positive breast cancer cells, we have prepared and characterized poly(epsilon-caprolactone) (PCL) nanoparticle formulation.PCL (mol wt 14,800 daltons) nanoparticles were prepared by the solvent displacement method in acetone-water system in the presence of Pluronic F- 68. PCL nanoparticles, labeled with rhodamine123, were incubated with MCF-7 estrogen receptor positive breast cancer cells to determine uptake, intracellular distribution, and localization as a function of time. Intracellular drug concentrations over a specified period of time using different initial doses were examined using tritiated [3H]-tamoxifen.A significant fraction of the administered rhodamine123-loaded PCL nanoparticles was found in the perinuclear region of the MCF-7 cells, where estrogen receptors are also localized, after 1 hour of incubation. Measurements of the intracellular concentrations revealed that most of the administered nanoparticle dose was internalized within the first 30 minutes of incubation, and the uptake followed saturable transport kinetics.Results of this study show that PCL nanoparticles were rapidly internalized in MCF-7 cells and intracellular tamoxifen concentrations followed a saturable process. This approach may provide better therapeutic benefit by delivering the drug locally, near the tumor cells, for a longer period of time.

Na+ gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles.

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.

Transcapillary insulin transfer in human skeletal muscle.

Transcapillary insulin transfer is considered a rate-limiting step in insulin action at supraphysiological insulin concentrations. However, it remains unclear whether this concept also applies for physiological conditions. In the present study we set out to characterize transcapillary insulin transfer by measuring insulin concentrations in plasma and interstitial space fluid of skeletal muscle during an oral glucose tolerance test and euglycaemic hyperinsulinaemic clamp conditions, respectively. For this purpose we employed in vivo microdialysis of skeletal muscle in conjunction with an ultrasensitive insulin assay in eight healthy lean male volunteers (aged 25 +/- 1 years). Insulin concentrations at baseline were 48 +/- 8 pmol x L(-1) in plasma and 19 +/- 4 pmol x L(-1) in the interstitium (P = 0.002). The mean interstitium to plasma ratio at baseline was 0.48 +/- 0.09 pmol x L(-1). During the oral glucose tolerance test the interstitium to plasma ratio remained unchanged (0.43 +/- 0.12, P = NS vs. baseline), but was significantly reduced during euglycaemic hyperinsulinaemic clamp conditions at steady-state hyperinsulinaemia (0.12 +/- 0.01, P = 0.01 vs. baseline). In summary there is a substantial transcapillary insulin gradient in healthy human skeletal muscle under baseline and glucose-stimulated conditions. Our findings support the hypothesis of a saturable transcapillary insulin transport representing a partly rate-limiting step for insulin action.

A role for thyroid hormone transporters in transcriptional regulation by thyroid hormone receptors.

Thyroid hormones (THs) must be taken up by target cells to act at the genomic level through binding to nuclear thyroid hormone receptors (TRs). Extensive study has been made of mechanisms by which TH-bound TRs regulate transcription, yet little is known about the critical upstream step, i.e. how THs enter the cell. Growing evidence suggests that saturable transport mechanisms mediate the greater part of TH movement across the plasma membrane and have important roles in the regulation of TH bioavailability. For example, System L is a multifunctional transport system serving as a plasma membrane transporter of THs and amino acids in mammalian cells. We have used two complementary systems, the Xenopus oocyte (which has negligible basal System L activity) and the mammalian BeWo cell line (which has System L activity for TH transport), to investigate the role of this representative TH transporter in nuclear action of THs. We demonstrate that overexpression of System L in Xenopus oocytes increases both cytoplasmic and nuclear delivery of THs from external medium and also enhances transcriptional activation by TRs. Conversely, blocking endogenous System L activity in BeWo cells with specific inhibitors reduces both TH uptake and TR function. These results indicate that plasma membrane TH transporters such as System L may have important roles in gene regulation by TRs.

Incorporation of Protein-Binding Kinetics and Carrier-Mediated Membrane Transport into a Model of Chemical Kinetics in the Isolated Perfused Rat Liver

Nonlinear processes present difficult problems in extrapolating toxicological kinetics from one dose or species to another. A model that includes the details of these processes will aid in identifying those conditions under which a linear extrapolation is not valid. Previously, a kinetic model of the perfused rat liver system was presented that included the effects of equilibrium protein binding, diffusion-based and saturable membrane transport, metabolism, and biliary excretion. This model has been extended here to account for two complex processes. First, the kinetics of chemical association and dissociation from binding sites in various compartments are explicitly included to allow for the possibility that the binding of the compound is not in equilibrium. Second, mediated transport via a simple four-state carrier in the membrane has been included at both the sinusoidal and biliary membranes of the liver cell. This enables inclusion of carrier-specific transport processes (such as selective transport against a concentration gradient) in modeling the kinetics in perfused rat liver experiments. Simulations demonstrating the effects of each of these processes on observable state variables have been conducted. Physiological conditions that elicit nonlinear behavior have been identified.

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76).

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.