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A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer. The endoplasmic reticulum (ER)1 of the cell is the site of the synthesis, posttranslational modification, and folding of proteins transported along the secretory pathway. The oxidizing environment in the lumen of the ER is necessary for the formation of disulfide bonds and for the proper folding of these proteins (1). The oxidative effects are reflected in and supported by the GSH redox buffer; the ratio of GSH and GSSG is around 2:1 within the lumen of ER and along the secretory pathway, whereas the cytosolic ratio ranges from 30:1 to 100:1 (2). However, the primary source(s) of the oxidative effect has not been demonstrated. Recent observations suggest two possible mechanisms. First, the preferential uptake of the oxidized member of a redox couple through the ER membrane and/or the efflux (or exocytosis) of its reduced form could ensure the oxidative environment. Alternatively, enzymes resident in the membrane or lumen of the ER could produce oxidizing compounds (e.g. reactive oxygen species) toward the lumen. Experimental evidence supports both mechanisms. Favoring the transport-based hypothesis, the preferential transport of dehydroascorbate (the oxidized form of ascorbate) has been described in rat liver microsomal vesicles (3). Similarly, the selective microsomal transport of GSSG was also reported (2, 4). On the other hand, several microsomal enzymes (cytochrome P-450s, NADPH cytochrome P-450 reductase, gulonolactone oxidase, microsomal iron protein, NADPH-dependent oxidase, sulfydryl oxidase, etc.) can produce reactive oxygen species (5-10). The recent exploration of the ER oxidase protein (ERO1) and its role in the protein folding also support the latter mechanism (11, 12). Because of the conflicting opinions, the microsomal transport of GSH and GSSG has not been unequivocally established. The presently available data are based on the detection of microsome-associated radioactivity by applying a rapid filtration method and radiolabeled compounds (2, 4); however, intraluminal GSH or GSSG contents upon transport have not been directly demonstrated. Therefore, experiments were undertaken to reinvestigate the transport of GSH and GSSG through the ER membrane.The main difficulties in the investigation of microsomal transport processes are deriving from the very small intraluminal space, the presence of (intraluminal) reactions affecting the transported c...

Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

Stress is the imbalance of homeostasis, which can be sensed even at the subcellular level. The stress-sensing capability of various organelles including the endoplasmic reticulum (ER) has been described. It has become evident that acute or prolonged ER stress plays an important role in many human diseases; especially those involving organs/tissues specialized in protein secretion. This article summarizes the emerging role of ER stress in diverse human pathophysiological conditions such as carcinogenesis and tumor progression, cerebral ischemia, plasma cell maturation and apoptosis, obesity, insulin resistance, and type 2 diabetes. Certain components of the ER stress response machinery are identified as biomarkers of the diseases or as possible targets for therapeutic intervention.

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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...

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