Dehydroascorbate and Ascorbate Transport in Rat Liver Microsomal Vesicles

Abstract

Ascorbate and dehydroascorbate transport was investigated in rat liver microsomal vesicles using radiolabeled compounds and a rapid filtration method. The uptake of both compounds was time-and temperaturedependent, and saturable. Ascorbate uptake did not reach complete equilibrium, it had low affinity and high capacity. Ascorbate influx could not be inhibited by glucose, dehydroascorbate, or glucose transport inhibitors (phloretin, cytochalasin B) but it was reduced by the anion transport inhibitor 4,4-diisothiocyanostilbene-2,2-disulfonic acid and by the alkylating agent N-ethylmaleimide. Ascorbate uptake could be stimulated by ferric iron and could be diminished by reducing agents (dithiothreitol, reduced glutathione). In contrast, dehydroascorbate uptake exceeded the level of passive equilibrium, it had high affinity and low capacity. Glucose cis inhibited and trans stimulated the uptake. Glucose transport inhibitors were also effective. The presence of intravesicular reducing compounds increased, while extravesicular reducing environment decreased dehydroascorbate influx. Our results suggest that dehydroascorbate transport is preferred in hepatic endoplasmic reticulum and it is mediated by a GLUTtype transporter. The intravesicular reduction of dehydroascorbate leads to the accumulation of ascorbate and contributes to the low intraluminal reduced/oxidized glutathione ratio.Ascorbate producing and utilizing pathways are connected to the endomembrane system of the cell. The final enzymatic steps of ascorbate synthesis are located in the endoplasmic reticulum of hepatocytes or kidney cells; enzymes utilizing ascorbate (prolyl-3-hydroxylase, prolyl-4-hydroxylase, and lysyl hydroxylase) or its oxidized form dehydroascorbate (protein disulfide isomerase) are characteristic proteins of the lumen (1-3). Their presence in the lumen is necessary for the posttranslational modification and folding of many proteins. Since ascorbate and dehydroascorbate are charged water-soluble compounds, transporter(s) should exist for their permeation through biological membranes. Such transporters have been thoroughly investigated in plasma membrane of different cells (4 -9) and in chromaffin granula (10), but the transport of ascorbate and dehydroascorbate in microsomes has not been described in detail. The aim of the present study was to detect and characterize the activity of the possible ascorbate and/or dehydroascorbate transporter(s) in the endoplasmic reticulum. EXPERIMENTAL PROCEDURESPreparation of Rat Liver Microsomes-Microsomes were prepared from 24-h fasted male Sprague-Dawley rats (180 -230 g) as reported (11). Microsomal fractions were resuspended in buffer A (100 mM KCl, 20 mM NaCl, 1 mM MgCl 2 , 20 mM MOPS, 1 pH 7.2). The suspensions (60 -80 mg of protein/ml) were rapidly frozen and maintained under liquid N 2 until used. Intactness of microsomal vesicles was checked by measuring the latency of mannose-6-phosphatase (12) and p-nitrophenol UDP-glucuronosyltransferase activity (13), they were greater than 95% in all the p...

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

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“…The affinity of the putative transporter for AH seemed to be low and in the millimolar range. Various cells contain a low-affinity AH transporter (K m 2-7 mM) that has been primarily described for AH influx [36,[38][39][40]. In addition, cells accumulate millimolar levels of AH intracellularly [23,38,41].…”

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

“…Paradoxically, in the ER and associated vesicular structures, millimolar levels of ascorbate have been reported (41,42). To maintain these levels the oxidized form of the vitamin, DHA, is preferentially transported into the lumen of the ER where it undergoes reduction to ascorbate (43,44). In most organisms ascorbate regeneration can occur nonenzymatically by, for example, interaction with glutathione, or by means of the activity of enzymes including protein disulfide isomerase and DHA reductase (44,45).…”

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

“…Ascorbate is easily oxidized to dehydroascorbate, and it is known that cells take up dehydroascorbate from extracellular fluid through a GLUT (Banhegyi et al, 1998), and that GLUT inhibitors such as wortmannin suppress the transport capacity of glucose. We measured the uptake of [ 14 C]ascorbate in liver slices.…”

“…During the reperfusion period, hepatocytes actively take up ascorbate to restore physiological concentrations of the vitamin. A major source of ascorbate reuptake takes the form of dehydroascorbate (DHA) transport via a glucose transporter (GLUT) (Banhegyi et al, 1998). The cytosolic dehydroascorbate is rapidly reduced to ascorbate by the action of dehdroascorbate reductase and a non-enzymatic chemical reaction.…”