Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (K(M) = 5.3 microm), which is competitively inhibited by cyanidine 3-glucoside (Ki = 51.6 microm) and mainly noncompetitively by cyanidin (Ki = 88.3 microm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd = 1.7 microm) on the carnation carrier only, and a high-affinity bilirubin-binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabeled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.     

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

Immunolocalisation of bilitranslocase in mucosecretory and parietal cells of the rat gastric mucosa

SummaryBilitranslocase is a plasma membrane carrier localised at the vascular pole of the rat liver cell, where it mediates uptake of organic anions from the blood into the liver. This carrier is also present in the epithelium of the rat gastric mucosa, with similar molecular mass and functional properties. An immunohistochemical study reveals that both the mucus-secreting cells of the gastric pit and the H⁺-secreting parietal cells express bilitranslocase. These data point to a possible role of bilitranslocase and of its food-borne substrates (anthocyanins and nicotinic acid) in regulating the function and the permeability of the gastric mucosa.     

VEGFR1 receptor tyrosine kinase localization to the Golgi apparatus is calcium-dependent

Vascular endothelial growth factor receptor 1 (VEGFR1) is an essential receptor tyrosine kinase that regulates mammalian vascular development and embryogenesis but its function is not well understood. Herein, we present evidence whereby endothelial VEGFR1 is largely resident within the Golgi apparatus but translocates to the plasma membrane via a calcium-regulated process. Primary human endothelial cells reveal differing VEGFR1 and VEGFR2 intracellular distribution and dynamics. The major proportion of the full-length VEGFR1 membrane protein was resident within the Golgi apparatus in primary endothelial cells. Whereas VEGFR2 displayed down-regulation in response to VEGF-A, VEGFR1 was not significantly affected arguing for a significant intracellular pool that was inaccessible to extracellular VEGF-A. This intracellular VEGFR1 pool showed significant co-distribution with key Golgi residents. Brefeldin A caused VEGFR1 Golgi fragmentation consistent with redistribution to the endoplasmic reticulum. Metabolic labeling experiments and microscopy using domain-specific VEGFR1 antibodies indicated that the mature processed VEGFR1 species and an integral membrane protein was resident within Golgi apparatus. Cytosolic calcium ions play a key role in VEGFR1 trafficking as treatment with either VEGF-A, histamine, thrombin, thapsigargin or A23187 ionophore caused VEGFR1 redistribution from the Golgi apparatus to small punctate vesicles and plasma membrane. We thus propose a model whereby the balance of VEGFR1 and VEGFR2 plasma membrane levels dictate either negative or positive endothelial signaling to influence vascular physiology.     

Dietary anthocyanins protect endothelial cells against peroxynitrite-induced mitochondrial apoptosis pathway and Bax nuclear translocation: an in vitro approach

Anthocyanins have received increasing attention because of their relatively high intake in humans and wide range of potential health-promoting effects, including anti-atherogenic properties. Evidences support their vascular protective effects but the involved molecular mechanisms have not been well clarified. The endothelium seems to have a central role in atherogenesis and apoptosis is emerging as a crucial event in this disease progression. Following our previous work on the biochemical pathways underlying peroxynitrite-triggered apoptosis in endothelial cells, here we investigated potential mechanisms responsible for the cytoprotective actions of three common anthocyanins, namely cyanidin- delphinidin- and pelargonidin-3-glucoside, against this process. Beyond their antioxidant properties, all these flavonoids, possessing either catecholic or monophenolic structures, were able to counteract peroxynitrite-induced apoptotic effects in endothelial cells through the inhibition of several crucial signaling cascades. Actually, pre-incubation of cells with 25 μM anthocyanins prevented them from peroxynitrite-mediated apoptosis, which was evaluated by the loss of mitochondrial membrane potential, caspases-9 and-3 activation, the increase in cytoplasmatic Bax levels and the inactivation of the PI3 K/Akt pathway. Moreover, they counteracted the translocation of Bax into the nucleus, as observed by immunocytochemistry and immunoblot, an event shown for the first time in endothelial cells apoptotic process. Such cellular actions could not be inferred from their in vitro antioxidant properties. These results suggest a potential role of dietary anthocyanins in the modulation of several apoptotic signaling pathways triggered by peroxynitrite in endothelial cells, supporting mechanistically their health benefits in the context of prevention of endothelial dysfunction and, ultimately, of atherosclerosis.     

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.     

Flavonoid transport by mammalian endothelial cells

Despite the ever-growing body of literature reporting the effects of flavonoids on animals at both the cellular and systemic levels, one of the most basic questions-"Are the effects of flavonoids on animal cells initiated through their interaction with extracellular targets or intracellular targets?"-has yet to be addressed. Because many effects of flavonoids on cells can be detected within minutes of flavonoid application and because flavonoids diffuse across lipid membranes slowly or not at all, intracellular mechanisms would necessitate a flavonoid transport system for rapid flavonoid uptake. The specific aims of this investigation were (1) to determine if endothelial cells contain a mechanism that mediates rapid flavonoid uptake and (2) to provide evidence for or against the hypothesis that rapid flavonoid effects on endothelial cell synthesis of prostacyclin and endothelin are initiated through the interaction of flavonoids with intracellular targets. Data show that bovine and human aortic endothelial cells possess a transport system that mediates rapid uptake of the flavonoid morin and suggest that the flavonoid uptake system utilizes a variety of oxygenated phenolic compounds as substrates. Further investigation into flavonoid transport should expedite future investigation into the mechanisms of flavonoid actions, because it may allow research to focus on the cellular locations where flavonoids are concentrated. Although endothelial cells contain a mechanism for the rapid uptake of morin, data reported herein suggest that morin initiates its rapid effects on endothelial cell synthesis of prostacyclin and endothelin through an interaction with extracellular targets.     

Lipid metabolism of myocardial endothelial cells

The vascular endothelium can be regarded as a widely distributed organ, interposed between the intravascular and extravascular spaces, with a pluripotent function in the regulation of capillary diameter, vascular homeostasis, lipoprotein metabolism and the vascular response to injury. In the basal physiological state these processes provide a non-thrombotic, non-inflammatory vascular lining preventing uncontrolled inflammation and coagulation. Endothelial cells respond to potential harmful conditions (mechanical stress, anoxia, ischemia and oxidative stress) and a variety of hormones and vasoactive mediators by inducing coagulation and production of inflammatory mediators through the production of bioactive lipids. Although the number of studies in isolated myocardial endothelial cells is limited, from the presumed metabolic analogy with endothelial cells isolated (and cultured) from other organs, one may conclude that the bioactive lipids include oxygenated arachidonate metabolites (eicosanoids) and the platelet activating factor (1--O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF). All aspects of lipid metabolism, related to the production of eicosanoids and PAF, are present within myocardial endothelial cells. There is uptake and incorporation of fatty acids by endothelial cells and liberation from endogenous triacylglycerol and (membrane) phospholipid stores by (phospho)lipases. Endothelial cells oxidize fatty acids in a carnitine-dependent, mitochondrial, pathway. Endothelial cells actively interact with high density lipoprotein (HDL) and low density lipoprotein (LDL) leading to uptake of cholesterol(esters) that undergo intracellular hydrolysis, and re-esterification to phosphoand neutral lipids, and leaving the LDL-particle modified in a way that makes them bind to the scavenger receptor on macrophages. Extravascular triacylglycerols in lipoproteins (very low density lipoprotein (VLDL), chylomicrons) are handled by endothelial cell lipoprotein lipase, providing substrate fatty acids for the underlying muscle tissue. Eicosanoid production from (membrane)phospholipids and PAF synthesis from alkylphospholipids are tightly coupled and interrelated to the flow of arachidonic acid between cellular lipid pools. (Mol Cell Biochem116: 171–179, 1992)     

Flavonoid transport across RBE4 cells: A blood-brain barrier model

There is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer’s and Parkinson’s diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.     

Copper-stimulated endocytosis and degradation of the human copper transporter,hCtr1

Copper uptake at the plasma membrane and subsequent delivery to copper-dependent enzymes is essential for many cellular processes, including mitochondrial oxidative phosphorylation, free radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. However, intracellular levels of this nutrient must be controlled because it is potentially toxic in excess concentrations. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. In this study, we demonstrate that levels of the hCtr1 protein at the plasma membrane of HEK293 cells were reduced when cells were exposed to elevated copper. This decrease in surface hCtr1 levels was associated with an increased rate of endocytosis, and low micromolar concentrations of copper were sufficient to stimulate this process. Inhibitors of clathrin-dependent endocytosis prevented the trafficking of hCtr1 from the plasma membrane, and newly internalized hCtr1 and transferrin were co-localized. Significantly, elevated copper concentrations also resulted in the degradation of the hCtr1 protein. Our findings suggest that hCtr1-mediated copper uptake into mammalian cells is regulated by a post-translational mechanism involving copper-stimulated endocytosis and degradation of the transporter.     

Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimer's and Parkinson's. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca(2+) influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca(2+) despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.     

Measurement of sulfobromophthalein uptake in isolated rat hepatocytes by a direct spectrophotometric method

A spectrophotometric technique is described for the continuous recording of sulfobromophthalein uptake by isolated hepatocytes. The technique is based on the principle that sulfobromophthalein behaves as a pH-indicator and may be followed photometrically when moving from the medium at pH 7.8 into the interior of the cell. Data show that upon addition of cells to a sulfobromophthalein solution, an absorbance change can be recorded. The kinetics of the process is biphasic and the initial rate is linearly related to the amount of cells added. By this technique it was confirmed that the substrate dependence of the initial velocity of transport is a compound function including a saturable portion with an apparent Km in the mu molar region. Experiments carried out either in the presence of valinomycin or of high concentrations of potassium chloride indicate that the presence of a membrane potential opposes the entry of sulfobromophthalein into isolated hepatocytes. This finding is in agreement with previous observations in isolated plasma membrane vesicles and in liposomes reconstituted with purified bilitranslocase which indicate a rheogenic type of transport for the dye. Low concentrations of nicotinate (1.6 microM) efficiently inhibit the saturable transport. It is suggested, in addition, that the sensitivity of the transport to valinomycin could be used as an early indication of the functional integrity of cell preparations.     

Structural analysis of a peptide fragment of transmembrane transporter protein bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane.     

Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress

The objective of this study was to investigate the ability of endothelial cells (EC) to incorporate anthocyanins and to examine their potential benefits against various oxidative stressors. Endothelial dysfunction has been proposed to play an important role in the initiation and development of vascular disease, with studies having shown that administration of antioxidants improves endothelial function. Elderberry extract contains 4 anthocyanins, which where incorporated into the plasma membrane and cytosol of EC following 4 h incubation at 1 mg.ml(-1). However, incorporation within the cytosol was considerably less than that in the membrane. Uptake within both regions appeared to be structure dependent, with monoglycoside concentrations higher than that of the diglucosides in both compartments. The enrichment of EC with elderberry anthocyanins conferred significant protective effects in EC against the following oxidative stressors: hydrogen peroxide (H(2)O(2)); 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH); and FeSO(4)/ascorbic acid (AA). These results show for the first time that vascular EC can incorporate anthocyanins into the membrane and cytosol, conferring significant protective effects against oxidative insult. These findings may have important implications on preserving EC function and preventing the initiation of EC changes associated with vascular diseases.     

Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver: immunochemical evidence using mono- and poly-clonal antibodies

Monoclonal antibodies raised against bilitranslocase, may display either inhibitory or enhancing activity on the electrogenic transport of sulfobromophthalein, evoked in rat liver plasma-membrane vesicles by the addition of valinomycin in the presence of K+. In both cases, the target protein is identified with a 37 kDa band in SDS-mercaptoethanol gel electrophoresis of solubilized membranes. The electrophoretically homogeneous protein isolated by ion-exchange chromatography, corresponds in all respects to the 37 kDa protein band of bilitranslocase, obtained in the past by different techniques. Using this protein as antigen, a polyclonal monospecific antibody preparation has been obtained. As expected, the antibody preparation inhibits the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver. It is concluded that the 37 kDa protein of bilitranslocase is at least a necessary component of the transport system involved in the sulfobromophthalein movement in plasma membrane.     

Dietary flavonoids attenuate tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells. Structure-function relationships and activity after first pass metabolism

Flavonoids have been suggested to exert human health benefits by anti-oxidant and anti-inflammatory mechanisms. In this study, we investigated whether and by what mechanisms dietary flavonoids inhibit expression of cellular adhesion molecules, which is relevant to inflammation and atherosclerosis. We found that the capacity of flavonoids to inhibit tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells was dependent on specific structural features of the flavonoids. The 5,7-dihydroxyl substitution of a flavonoid A-ring and 2,3-double bond and 4-keto group of the C-ring were the main structural requirements for inhibition of adhesion molecule expression. In striking contrast, hydroxyl substitutions of the B- and C-rings but not the A-ring were essential for antioxidant activity. Hence, only hydroxyl flavones, such as apigenin and chrysin, and flavonols, such as galangin, kaempferol, and quercetin, were able to inhibit endothelial adhesion molecule expression, whereas flavone, chromone, the flavanone, naringenin, and the flavanol, (-)-epicatechin, were ineffectual. At low concentrations, the active flavonoids significantly attenuated expression of E-selectin and intercellular adhesion molecule 1 but not vascular cell adhesion molecule 1. In addition, exposure of apigenin and kaempferol to cultured hepatocytes, mimicking first pass metabolism, greatly diminished the inhibitory effect of flavonoids on endothelial intercellular adhesion molecule 1 expression. We conclude that the effect of dietary flavonoids on endothelial adhesion molecule expression depends on their molecular structure, concentration, and metabolic transformation but not their antioxidant activity.     

Caveolae: a regulatory platform for nutritional modulation of inflammatory diseases

Dietary intervention strategies have proven to be an effective means of decreasing several risk factors associated with the development of atherosclerosis. Endothelial cell dysfunction influences vascular inflammation and is involved in promoting the earliest stages of lesion formation. Caveolae are lipid raft microdomains abundant within the plasma membrane of endothelial cells and are responsible for modulating receptor-mediated signal transduction, thus influencing endothelial activation. Caveolae have been implicated in the regulation of enzymes associated with several key signaling pathways capable of determining intracellular redox status. Diet and plasma-derived nutrients may modulate an inflammatory outcome by interacting with and altering caveolae-associated cellular signaling. For example, omega-3 fatty acids and several polyphenolics have been shown to improve endothelial cell function by decreasing the formation of ROS and increasing NO bioavailability, events associated with altered caveolae composition. Thus, nutritional modulation of caveolae-mediated signaling events may provide an opportunity to ameliorate inflammatory signaling pathways capable of promoting the formation of vascular diseases, including atherosclerosis.     

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.     

Hyperuricemia enhances intracellular urate accumulation via down-regulation of cell-surface BCRP/ABCG2 expression in vascular endothelial cells

Hyperuricemia has been recognized as an independent risk factor for cardiovascular disease. Urate stimulates NADPH oxidase and induces production of reactive oxygen species (ROS); consequently, intracellular urate accumulation can induce oxidative stress leading to endothelial dysfunction. Here, we studied the mechanism involved, using human umbilical vascular endothelial cells (HUVEC) as a model. Pretreatment with 15 mg/dL unlabeled uric acid (corresponding to hyperuricemia) resulted in increased uptake of [¹⁴C]uric acid at steady-state by HUVEC, whereas pretreatment with 5 mg/dL uric acid (in the normal serum concentration range) did not. However, the initial uptake rate of [¹⁴C]uric acid was not affected by uric acid at either concentration. These results suggest that efflux transport of uric acid is decreased under hyperuricemic conditions. We observed a concomitant decrease of phosphorylated endothelial nitric oxide synthase. Plasma membrane expression of breast cancer resistance protein (BCRP), a uric acid efflux transporter, was decreased under hyperuricemia, though the total cellular expression of BCRP remained constant. Uric acid did not affect expression of another uric acid efflux transporter, multidrug resistance associated protein 4 (MRP4). Moreover, phosphorylation of Akt, which regulates plasma membrane localization of BCRP, was decreased. These uric acid-induced changes of BCRP and Akt were reversed in the presence of the antioxidant N-acetylcysteine. These results suggest that in hyperuricemia, uric acid-induced ROS generation inhibits Akt phosphorylation, causing a decrease in plasma membrane localization of BCRP, and the resulting decrease of BCRP-mediated efflux leads to increased uric acid accumulation and dysregulation of endothelial function.