Ascorbic acid transport and accumulation in human neutrophils

The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake.     

Ascorbic acid metabolism and glycolysis in the polymorphonuclear leucocyte of the guinea pig

Exudate leucocytes lost approximately 30% of their original intracellular ascorbic acid content during two hour incubation in glucose medium. The same loss was observed for cells containing initially both high and low levels of ascorbic acid. High concentrations of ascorbic acid in the incubation medium depressed lactic acid production and increased oxygen uptake by the cells. Iodoacetate and fluoride at low concentrations decreased ascorbic acid loss from cells during incubation; at high concentrations they increased loss. Ascorbic acid uptake from the medium was inhibited by iodoacetate but stimulated by fluoride.     

Effect of Escherichia coli endotoxin on ascorbic acid transport in isolated adrenocortical cells

The active uptake of ascorbic acid by isolated rat adrenocortical cells increases with ascorbic acid concentration, depends on time and calcium, and is inhibited by ACTH concentrations required for maximal steroidogenesis. Lipopolysaccharide of Escherichia coli 0111:B4 modifies the ascorbic acid uptake in a calcium-dependent manner. At low calcium concentrations, lipopolysaccharide exerts a stimulatory effect on ascorbic acid transport and at high concentrations lipopolysaccharide produces a dose-dependent inhibitory effect. This inhibition of the ascorbic acid transport by the endotoxin can alter the ascorbic acid accumulation in the adrenal gland during endotoxin shock.     

Effect of Sorbinil and Ascorbic Acid on myo-Inositol Transport in Cultured Rat Schwann Cells Exposed to Elevated Extracellular Glucose

Abstract: The effect of long-term (2 weeks) exposure to 0–50 m M glucose and 0–1 m M sorbitol on myo -inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo -inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo -Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in ≥30 m M glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 m M sorbinil. Ascorbic acid also prevented the reduction in myo -inositol uptake due to excess extracellular glucose, at 3 and 30 µ M concentrations, but not at 300 µ M . Neither sorbinil nor ascorbic acid could prevent the alterations in myo -inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo -inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo -inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.     

Osmotic stress,glucose transport capacity and consequences for glutamate overproduction in Corynebacterium glutamicum

Glucose uptake by Corynebacterium glutamicum is predominantly assured by a mannose phosphotransferase system (PTS) with a high affinity for glucose (Km=0.35 mM). Mutants selected for their resistance to 2-deoxyglucose (2DG) and lacking detectable PEP-dependent glucose-transporting activity, retained the capacity to grow on media in which glucose was the only carbon and energy source, albeit at significantly diminished rates, due to the presence of a low affinity (Ks=11 mM) non-PTS uptake system. During growth in media of different osmolarity, specific rates of glucose consumption and of growth of wild type cells were diminished. Cell samples from these cultures were shown to possess similar PTS activities when measured under standard conditions. However, when cells were resuspended in buffer solutions of different osmolarity measurable PTS activity was shown to be dependent upon osmolarity. This inhibition effect was sufficient to account for the decreased rates of both sugar uptake and growth observed in fermentation media of high osmolarity. The secondary glucose transporter was, however, not influenced by medium osmolarity. During industrial fermentation conditions with accumulation of glutamic acid and the corresponding increase in medium osmolarity, similar inhibition of the sugar transport capacity was observed. This phenomenon provokes a major process constraint since the decrease in specific rates leads to an increasing proportion of sugar catabolised for maintenance requirements with an associated decrease in product yields.     

Recycling of vitamin C by a bystander effect

Human cells transport dehydroascorbic acid through facilitative glucose transporters, in apparent contradiction with evidence indicating that vitamin C is present in human blood only as ascorbic acid. On the other hand, activated host defense cells undergoing the oxidative burst show increased vitamin C accumulation. We analyzed the role of the oxidative burst and the glucose transporters on vitamin C recycling in an in vitro system consisting of activated host-defense cells co-cultured with human cell lines and primary cells. We asked whether human cells can acquire vitamin C by a "bystander effect" by taking up dehydroascorbic acid generated from extracellular ascorbic acid by neighboring cells undergoing the oxidative burst. As activated cells, we used HL-60 neutrophils and normal human neutrophils activated with phorbol 12 myristate 13-acetate. As bystander cells, we used immortalized cell lines and primary cultures of human epithelial and endothelial cells. Activated cells produced superoxide anions that oxidized extracellular ascorbic acid to dehydroascorbic acid. At the same time, there was a marked increase in vitamin C uptake by the bystander cells that was blocked by superoxide dismutase but not by catalase and was inhibited by the glucose transporter inhibitor cytochalasin B. Only ascorbic acid was accumulated intracellularly by the bystander cells. Glucose partially blocked vitamin C uptake by the bystander cells, although it increased superoxide production by the activated cells. We conclude that the local production of superoxide anions by activated cells causes the oxidation of extracellular ascorbic acid to dehydroascorbic acid, which is then transported by neighboring cells through the glucose transporters and immediately reduced to ascorbic acid intracellularly. In addition to causing increased intracellular concentrations of ascorbic acid with likely associated enhanced antioxidant defense mechanisms, the bystander effect may allow the recycling of vitamin C in vivo, which may contribute to the low daily requirements of the vitamin in humans.     

L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.     

橄榄果实中PPO和POD活性抑制研究

本文研究温度和几种抑制剂对橄榄果实多酚氧化酶(PPO)和过氧化物酶(POD)活性的影响。结果表明,橄榄果实PPO活性最强的温度为27℃,POD活性最强的温度为45℃;低温对PPO活性的抑制较POD显著;抗坏血酸和亚硫酸钠可有效地抑制PPO活性,而抗坏血酸和氯化镁则对POD活性有明显的抑制作用。     

Ascorbic acid-dependent GLUT3 inhibition is a critical step for switching neuronal metabolism

Intracellular ascorbic acid is able to modulate neuronal glucose utilization between resting and activity periods. We have previously demonstrated that intracellular ascorbic acid inhibits deoxyglucose transport in primary cultures of cortical and hippocampal neurons and in HEK293 cells. The same effect was not seen in astrocytes. Since this observation was valid only for cells expressing glucose transporter 3 (GLUT3), we evaluated the importance of this transporter on the inhibitory effect of ascorbic acid on glucose transport. Intracellular ascorbic acid was able to inhibit (3)H-deoxyglucose transport only in astrocytes expressing GLUT3-EGFP. In C6 glioma cells and primary cultures of cortical neurons, which natively express GLUT3, the same inhibitory effect on (3)H-deoxyglucose transport and fluorescent hexose 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was observed. Finally, knocking down the native expression of GLUT3 in primary cultured neurons and C6 cells using shRNA was sufficient to abolish the ascorbic acid-dependent inhibitory effect on uptake of glucose analogs. Uptake assays using real-time confocal microscopy demonstrated that ascorbic acid effect abrogation on 2-NBDG uptake in cultured neurons. Therefore, ascorbic acid would seem to function as a metabolic switch inhibiting glucose transport in neurons under glutamatergic synaptic activity through direct or indirect inhibition of GLUT3.     

A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K⁺]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD⁺/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.     

Age-dependent changes in uptake and recycling of ascorbic acid in erythrocytes of Beagle dogs

Uptake and recycling of ascorbic acid (AA) were studied in erythrocytes of 1- to 12-month-old Beagle dogs. At 1 month, both AA uptake and recycling capacity were high. Ascorbic acid entered erythrocytes mainly in the oxidized form with elevated activity of Glut 1 glucose transporter. However, this trait of erythrocytes was rapidly lost in the course of postnatal growth. At 3 months, ascorbic acid uptake and recycling capacity decreased to almost adult levels. Thereafter, AA was transported mainly in the reduced form, and its uptake and recycling capacity became one-third the levels of 1-month-old dogs. Postnatal anemia and recovery were indicated by changes in hemoglobin and packed cell volume levels at 1 and 3 months. Glutathione reductase (GR) activity was twice as high as in adults in 1-month-old dogs, allowing efficient reduction of oxidized ascorbic acid, which enters cells in large amounts due to elevated activity of the Glut 1 glucose transporter. One-month-old dogs need high levels of AA for antioxidant protection and skeletal development. The high AA recycling capacity of erythrocytes is considered to balance the expenditure of AA in young Beagle dogs.     

Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B.

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.     

Reduced viability of vascular endothelial cells by high concentration of ascorbic acid in vitreous humor

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Specificity of ascorbate analogs for ascorbate transport. Synthesis and detection of [(125)I]6-deoxy-6-iodo-L-ascorbic acid and characterization of its ascorbate-specific transport properties.

Cellular ascorbic acid accumulation occurs in vitro by two distinct mechanisms: transport of ascorbate itself or transport and subsequent intracellular reduction of its oxidized product, dehydroascorbic acid. It is unclear which mechanism predominates in vivo. An easily detectable compound resembling ascorbate but not dehydroascorbic acid could be a powerful tool to distinguish the two transport activities. To identify compounds, 21 ascorbate analogs were tested for inhibition of ascorbate or dehydroascorbic acid transport in human fibroblasts. The most effective analogs, competitive inhibitors of ascorbate transport with K(i) values of 3 microM, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate. No analog inhibited dehydroascorbic acid transport. Using substitution chemistry, [(125)I]6-deoxy-6-iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection methods were developed for radiolabeled and nonradiolabeled compounds, and transport kinetics of both compounds were characterized. Transport was sodium-dependent, inhibited by excess ascorbate, and similar to that of ascorbate. Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate was investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3. Oxidation of ascorbate or its analog in media increased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorbate, although an effective inhibitor of ascorbate transport, either in its reduced or oxidized form was not a substrate for dehydroascorbic acid transport. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate provide a new means for discriminating dehydroascorbic acid and ascorbate transport in ascorbate recycling.     

Morphological and biochemical alterations of oomycete fish pathogen <Emphasis Type="Italic">Saprolegnia parasitica</Emphasis> as affected by salinity,ascorbic acid and their synergistic action

Vegetative growth of Saprolegnia parasitica decreased by increasing the concentration of NaCl and ascorbic acid. Under these conditions, the morphological features of the vegetative hyphae were distinguishable from those used as controls. NaCl and ascorbic acid in combination improved the tolerance of S. parasitica to high levels of salinity. Sporangial formation, release and proliferation were very sensitive to even lower levels of salinity. For instance, at 0.03 M NaCl sporangia formation was rarely observed. Ascorbic acid alone had a little effect on sporangial formation and release, but when combine with NaCl the developmental processes were improved. Reduction of numbers and plasmolysis of oogonia were found at various NaCl concentrations, whereas ascorbic acid stimulated the formation of these reproductive organs at low concentrations. The synergistic effect of NaCl and ascorbic acid improved and overcomed the symptoms of oogonial plasmolysis. Protease activity of S. parasitica was significantly reduced at all NaCl concentrations, whilst ascorbic acid significantly increased and inhibited it at low concentrations and at moderate and high concentrations, respectively. The combination of these compounds reduced protease activity at all tested concentrations with significant difference at the highest concentration. The total free amino-acids content of S. parasitica mycelia was significantly reduced at all the NaCl concentrations, whereas ascorbic acid significantly increased it at low but inhibited it at higher concentrations. The combination of NaCl and ascorbic acid significantly increased the accumulation of free amino-acids at low and moderate concentrations, but decreased them at high concentrations. Total protein content was reduced at all tested concentrations of NaCl and ascorbic acid had also similar effect. However, the combined effect of NaCl and ascorbic acid significantly enhanced and reduced total protein content at low and high concentrations, respectively. Treatments with NaCl induced proline accumulation in S. parasitica, which paralleled the salt concentration.     

Ascorbic acid uptake in guinea pig intestinal mucosa

Cellular accumulation of ascorbic acid was investigated in vitro in distal intestinal mucosa of guinea pig. With ¹⁴C-ascorbic acid present at 8 μM/L in the bathing media, tissue/media (T/M) concentration ratios of at least 5 were routinely achieved. Recently absorbed ascorbic acid appeared to be free in solution in the cellular fluid in that it diffused from tissue exposed to poisons with a disappearance half-time of approximately 10 minutes. Ascorbic acid uptake was highly dependent on the presence of sodium in the bathing media; total Tris substitution resulted in a 97% decrease in uptake. Also, metabolically depleted tissue did not accumulate ascorbic acid against a concentration gradient. Uptake of ¹⁴C-ascorbic acid from a bathing solution concentration of 8 μM/L was reduced 67% in the presence of 0.8 mM/L nonlabeled ascorbic acid. Recently absorbed ¹⁴C-ascorbic acid moved more rapidly back into the lumen when the luminal solution contained nonlabeled ascorbic acid (5 mM) than when it contained mannitol (5mM). This demonstration of counter transport substantiates a carrier mechanism in the brush border.     

Stimulation of ion transport by ascorbic acid through inhibition of 3':5'-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues.

Ascorbic acid stimulates active transport of Cl-minus by the isolated intact cornea. The effect is not present in corneas previously stimulated by the theophylline, an inhibitor of 3':5"-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3':5'-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using 3-H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46%. This compares with 58% inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45% is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50%) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12%, rat brain (caudate nucleus) 48%, rabbit brain 14%, rabbit liver 16%. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3':5'-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.     

Prolonged Infusion of Fluids into Tree Seedlings Using Ascorbic Acid

A method was developed to infuse liquids into the stems of three-year-oldCercis canadensis L., Liriodendron tulipifera L., and Tiliacordata Mill. seedlings and to monitor the movement of fluidinto the stems. Ascorbic acid (14.2 mM) added to the infusionliquid, could increase the initial rate of fluid uptake 2- to3-fold compared with distilled water control treatments. Theascorbic acid treatment also prolonged fluid uptake about 4d longer than distilled water treatments. The effect of ascorbicacid on fluid uptake could not be correlated to the solutionpH, the antibiotic effect of high ascorbic acid concentrations,or to seedling height, trunk diameter or projected leaf area.Data did, however, suggest that the increased fluid uptake rateand the prolonged fluid uptake may be related to the anti-oxidantproperty of ascorbic acid. The accumulation of 1.0 g of ascorbicacid in 60 to 100 cm tall C. canadensis and L. tulipifera seedlingsdid induce defoliation and lateral bud break. The data presentedmay be useful in developing techniques for prolonged infusionof therapeutic compounds into trees and for studying, in vivo,wound response to xylem tissue. Key words: Ascorbic acid, liquid influsion, tree seedlings     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.