key: cord-0005446-c30cdaw5 authors: Wilson, John X.; Jaworski, Ewa M.; Kulaga, Andrew; Jeffrey Dixon, S. title: Substrate regulation of ascorbate transport activity in astrocytes date: 1990 journal: Neurochem Res DOI: 10.1007/bf00965751 sha: 51f4729404500a8c5fb6ca93e6c777a9b2b492ef doc_id: 5446 cord_uid: c30cdaw5 Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[(14)C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V (max)) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[(14)C]ascorbate. The increase inV (max) was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[(3)H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels. Ascorbate is essential for nervous system function because it is a cofactor in biosynthesis of myelin (1, 2) and catecholamines (3) , facilitates release of transmitters (4) (5) (6) , modulates binding of ligands to neural receptors (7) (8) (9) , and slows rates of transmitter clearance (10) (11) (12) . Vitamin C homeostasis in the central nervous system is maintained even when plasma ascorbate levels are drastically lowered (13) or elevated (14, 15) . Experiments with cortical slices and freshly dissociated tissue have shown that brain cells actively transport ascorbate (16, 17, 18) . Cultured brain cells are usually grown in media that are virtually free of ascorbate. This is true even for serum-supplemented media because the vitamin is not detectable (<0.5 Ixg/ml) in the commercially available sera commonly used for cell culture (19) . Ascorbate is not synthesized by rodent brain cells and is not detectable in either astrocytes or neurons cultured in medium from which the vitamin is absent (20) . However, our previous studies showed that type-1 astrocytes of rat and mouse brains possess a specific, high-affinity, Na+-ascorbate cotransport system in their plasma membrane (21, 22) . Uptake of L-ascorbate by astrocytes was found to be: i) saturable, stereoselective, temperatureand Na+-dependent; ii) specific for the vitamin since it was not diminished in the presence of other organic anions including acetate, formate, lactate, malonate, oxalate, p-aminohippurate, pyruvate and succinate; iii) lacking a specific requirement for external CI-; and iv) rapidly (_< 1 min) and reversibly inhibited by furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Rapid and reversible inhibition by the impermeant antagonists, SITS and DIDSa localized the transporter to the plasma membrane. The apparent affinity of the transporter for L-ascorbate was in the micromolar range in astrocytes incubated with a physiological concentration of extracellular Na +, indicating a high-affinity L-ascorbate transporter. However, the affinity for L-ascorbate was significantly decreased when the extracellular Na + concentration was lowered, consistent with Na+-ascorbate cotransport. Efflux proceeded much more slowly than uptake under our experimental conditions. There is evidence that the transport system is subject to regulation, since prolonged (> 1 week) exposure to dibutyryl cyclic AMP increased the apparent maximal transport rate (Vm=) of L-ascorbate in astrocytes (21, 22) . Type-1 astrocytes are an abundant glial cell type comprising approximately 30% of brain volume. It is important to characterize the transport process of asttocytes in order to understand the mechanisms underlying regulation of cerebral ascorbate concentration. The function of vitamin C in astrocytes is not known, but could be investigated by experiments involving depletion and repletion of external ascorbate, as suggested for other cell types (23) . The purpose of the present study was to examine the responses of astroglial ascorbate transport activity to changes in extraceIIular ascorbate concentrations. Materials. L-[1-14C]ascorbate (10 mCi/mmol) was purchased from Dupont Canada. 2-Deoxy-D-[3H(G)]glucose (8.3 mei/mmol) was purchased from New England Nuclear and Amersham Canada. Horse serum was obtained from Gibco Canada. Modified Eagle's minimum essential medium (24) was made using tissue culture-grade chemicals purchased from Sigma. L-ascorbate, D-isoascorbate, DL-homocysteine, and N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) were also from Sigma. Cells. Primary cultures of astrocytes were prepared from the neopalium of 1-day-old Sprague-Dawley rats according to the procedure of Hertz et al. (24, 25) . The cells were plated onto 60 mm petri dishes (Falcon) and grown in modified Eagle's minimum essential medium with 20% horse serum nominally ascorbate4ree (37~ 95% air/5% CO2). The culture medium was changed twice weekly, with the serum concentration reduced to 10% after 3 days. Cultures reached confluency after 2-3 weeks. They were grown for an additional 2 weeks, in the presence or absence of 0.25 mM dibutyryl cyclic AMP, before being used for uptake experiments. Microscopic examination of cultures stained with silver showed that neurons were absent. The cultivated cells stained positively for glial fibrillary acidic protein using the procedure of Wilson et al. (26) . The morphology of living celt cultures was assessed by phase contrast microscopy. Treatment with 0.25 mM dibutyryl cyclic AMP changed the flat polygonal astrocytes to process-bearing stellate astrocytes. Preincubation of Astrocytes and Measurement of Transport Kinetics. The initial rate of cellular uptake of L-[~4C]ascorbate was measured at 37~ in serum-free incubation medium, essentially as described previously (21) . The medium consisted of (in raM): 134 NaCI, 5.4 KC1, 1.8 CaC12, 0.8 MgSO4, 10 glucose and 20 HEPES, adjusted to pH 7.3 wRh NaOH. Aliquots of medium were collected at the end of each uptake incubation. Incubations were terminated by washing cultures with ice-cold, isoosrriotic Tris-sucrose solution and harvesting the cells. An aliquot of the cell harvest was used for protein measurement (27) and the remainder was combined with scintillation cocktail. The radioactive contents of the medium and cells were measured by liquid scintillation counting. Ascorbate uptake proceeds linearly with time for at least 3 min (21), so 1 min incubations were used to measure initial uptake rates. Rates were computed based on the specific activity of L-[~4C]ascorbate in the medium and expressed as nmoi ascorbate/g cell protein/rain. To study the effect of external ascorbate supplementation on transport activity, astrocytes were preincubated for 0-18 h with various concentrations of unlabeled L-ascorbate (physiological substrate) or Disoascorbate (control compound for assessment of nonspecific effects) prior to measurement of L-[14C]ascorbate uptake. The medium was replenished with L-ascorbate at intervals of 12 h or less. The effect of external ascorbate deprivation was examined by preincubating astrocyte cultures with unlabeled L-ascorbate for 12-18 h (37~ subsequently washing and incubating (0-6 h) the cultures with ascorbatefree medium, and then measuring L-[~4C]ascorbate uptake during a final 1 rain incubation. Stock solutions of L-[~4C]ascorbate and unlabeled ascorbate analogs contained 0.4 mM homocysteine to prevent oxidation. The pH of the medium was not altered by the presence of these reductants at the concentrations employed. 2-Deoxy-D-[3H(G)]glucose transport studies were carried out as described by Mesmer et al. (28) . Cells were grown to confluence in six-well Costar plates (35 x 15 ram). Medium was aspirated and each well washed with 8 ml of phosphate buffered saline (PBS). Nine hundred microlitres of uptake buffer (PBS containing 1 mg/mI bovine serum albumin) were added to each well. Transport studies were carried out at 23~ and were initiated by the addition of 100 Ixl of radioactive 2deoxyglucose (0.6 raM). At appropriate times, uptake was terminated by washing the wells rapidly (less than 15 sec) twice with ice cold PBS. In all cases, 1 min uptake assays were performed and cell samples were taken at 15, 30, 45 and 60 sec after the addition of the radioactive substrate. Cells were solubilized with 1 ml of 0.1% Triton X-100; 0.8 ml aliquots were counted in 10 ml of scintillation fluid. Additional wells on each plate were used for protein determinations. Zero-time controls and background counts were subtracted from the raw data. Statistics. Results are presented as the mean -SEM of n experiments each with triplicate replications. In figures, error bars were omitted when the standard error was less than the size of the symbol. To determine the apparent Michaelis constant (Kin) and maximum rate of uptake (V,1,,,) from Lineweaver-Burk and Eadie-Hofstee plots, straight lines were fitted and intercepts calculated by linear regression. Comparisons between mean values based on a single level of treatment (e.g. Vm~,, for cells preincubated 6 h with or without 100 ixM L-ascorbate) were evaluated using paired t-tests. For simultaneous comparisons of two or more treatments, differences between means were evaluated using repeated measures analysis of variance and the Tukey-Kramer test (29) . For all statistical tests, a P value of <0.05 was considered significant. External L-ascorbate deprivation increased astroglial L-[14C]ascorbate transport activity (Figure 1 ). Transferring astrocytes from medium containing ascorbate (100 txM nominal) to ascorbate-free medium increased the L-[14C]ascorbate uptake rate (P < 0.05). In contrast, adding L-ascorbate to the medium of previously ascorbatefree cultures led to decreased initial rates of L-[14C]ascorbate uptake (P < 0.05, Figure 2 ). The effects of both substrate deprivation and supplementation were rapid. Most of the change in astroglial L-ascorbate transport rate was achieved within 1 h of raising or lowering the concentration of the vitamin in the medium ( Figures 1 and 2) . These changes were specific effects on the L-ascorbate transport system, since the presence or absence of 100 ~M external L-ascorbate during the preincubation period did not change either the cellular morphology or protein content of the astroglial cultures (data not shown). Furthermore, preincubation of astrocytes with ascorbate did not inhibit 2-deoxy-D-[3H(G)]glucose uptake. In stellate astrocytes the initial rates of 2-deoxy-D-[3H(G)]glucose uptake were 1878 +_ 506 nmol/g protein/min under con- trol (aseorbate-free) conditions and 2092 __ 488 nmol/g protein/min after 6 h preincubation with 100 p,M external L-ascorbate (mean -SEM from three independent experiments). Ascorbate transport activities in both polygonal and stellate astrocytes were decreased by preincubation with high concentrations of external L-ascorbate ( Figure 3) . When compared to L-[14C]ascorbate uptake under control conditions (i.e. after preincubation in nominally ascorbate-free medium), uptake rates after 6 h preincubation with 300 bLM L-ascorbate were inhibited by 38_ 2% in polygonal astrocytes and 42 + 3% in stellate astrocytes. A significant (P < 0.05) decrease was achieved by 6 h preincubation with external L-ascorbate concentrations as low as 30 ~M in stellate cells and 100 I~M in polygonal cells (Figure 3) . Decreased L-ascorbate transport activity was observed following preincubation with L-ascorbate but not D-isoascorbate (Figure 3 ). Even 300 ~zM external oisoascorbate, which was the highest concentration tested, did not have the effect induced by 30 IxM L-ascorbate during the 6 h preincubation period (Figure 3) . The effects of external ascorbate deprivation on the kinetics of L-[14C]ascorbate transport were investigated. Astroglial cultures preincubated either with or without extracellular L-ascorbate demonstrated initial velocities of L-[t4C]ascorbate uptake that were concentration-de- pendent and saturable following Michaelis-Menten kinetics (Figure 4) . For stellate astrocytes preincubated in medium containing L-ascorbate (100 p.M for 6 h) or maintained in ascorbate-free medium, Lineweaver-Burk plots of the data indicated the apparent Vm= were 407 _+ 29 and 605_ 54 nmol ascorbate/g protein/min (P < 0.05), respectively, and the apparent Km were 20 _ 1 and 21 _ 2 txM ascorbate, respectively (n = 3; Figure 4 ). Eadie-Hofstee plots of the data for stellate astrocytes preincubated in medium containing L-ascorbate (100 IxM for 6 h) or maintained in ascorbate-free medium, indicated the apparent V,~= were 390___ 50 and 554_ 95 nmol ascorbate/g protein/min (P < 0.05), respectively, and the apparent Km were 17_ 1 and 17_2 p,M ascorbate, respectively (n = 3; Figure 4) . Therefore, both analyses showed that the increase in transport activity associated with substrate deprivation involved a significant increase in the apparent maximal uptake rate with no change in the affinity of the astroglial transport system for external L-ascorbate. Altered metabolic states and levels of secretory activity can result in gross changes in the availability of L-ascorbate to various cells. Regulation of ascorbate transport rates is a plausible mechanism for controlling intracellular ascorbate concentrations in the face of a constantly changing extracellular supply. The results of previous in vivo studies are consistent with regulation of vitamin C transport by external ascorbate levels, i) In guinea pigs, which cannot synthesize ascorbate, ascorbate-deficient diets lead to a prolonged half-life of vitamin C in brain (13) . ii) Ingestion of excess vitamin C by guinea pigs slowly changes intestinal ascorbate transport with the result that initial uptake rates are decreased (30, 31) . For example, the apparent Vm~ of Na+-dependent ascorbate uptake in ileum was 32% lower in animals fed an ascorbate-enriched ration for 14 days, compared with controls fed a maintenance ration (31) . These data from long-term whole-animal experiments suggested that autoregulation of ascorbate transport may occur in cerebral and intestinal tissue, but did not provide information about cellular mechanisms. For example, apparent downregulation might have resulted from altered transport rates but also from altered catabolism of ascorbate, systemic compensations or nonspecific toxic effects. In particular, in vivo experiments did not determine if highaffinity, Na+-dependent ascorbate transport could be altered by changes in substrate availability. There does not appear to be any previous evidence for ascorbate-dependent regulation of ascorbate uptake in cell systems. Pheochromocytoma cells express a highaffinity ascorbate transport system, yet preincubation with millimolar concentrations of the vitamin in the culture medium did not alter subsequent uptake of L-[x4C]ascorbate (32) . Accumulation of ascorbate by brain cells is especially interesting because the half-life of the vitamin is several-fold longer in the brain than in any other organ studied (liver, heart, kidneys, adrenals, spleen; 13) and this half-life is influenced by cellular transport (33) . The present experiments used primary cultures of cerebral astrocytes that are useful brain cell models (34) . Although some cell types possess both high-and low-affinity ascorbate transporters (e.g. human neutrophils -23), virtually all ascorbate uptake by astrocytes appears to be through a high-affinity, Na+-dependent system (21) . The apparent affinity of the astroglial ascorbate transporter is an order of magnitude greater for h-ascorbate than for D-isoascorbate (21) , a stereoisomer that does not naturally occur in rodent brain (35) and has only 1/20 the antiscorbutic activity of vitamin C (13, 36) . The decrease in astroglial vitamin C transport activity induced by external ascorbate is also stereoselective. Whereas 30 p~M L-ascorbate and 300 ~M m-isoascorbate were equally effective in competitively blocking h-[14C]ascorbate uptake when either of the unlabeled analogs was added to the medium at the same time as the radiolabeled vitamin (21) , the present experiments found that preincubation with 30 ~zM L-ascorbate was significantly more effective than 300 txM D-isoascorbate in reducing subsequent L-[14C]ascorbate uptake. These data suggest that interaction of ligand with the external face of the ascorbate transporter is not, of itself, sufficient to modulate transport activity. More importantly, the data show that this effect does not arise from nonspecific damage by extracellular reductant. Furthermore, the effects of external ascorbate on L-ascorbate transport activity were apparently specific in that astroglial morphology, protein content and 2-deoxy-m-[3H(G)]glucose uptake were unaffected. Substrate-dependent regulation of membrane transport has been extensively documented for the Na+-independent glucose transport system. Maintenance of various cell types in the absence of D-glucose has been shown to increase the apparent Vm~,, for hexose transport with no change in the apparent Km (37) . Glucose deprivation of primary rat brain astroglial cells for 2-12 h gives rise to an increase in glucose transport activity as well as in the amount of glucose transporter protein and mRNA (38) . Half-maximal induction of glucose transport activity occurs by 2-3 h and the maximal increase in the glucose transport activity is observed within 12 h of transferring glucose-replete astroglial cells to a nominally glucose-free medium (38) . Longer periods of starvation may be less effective, since Hara et al. (39) reported that glucose starvation for 40-65 h does not change subsequent 2-deoxy-D-glucose uptake activity in cultured rat astrocytes. Dissociated cells from rat brain show accelerative exchange of 2-deoxy-I>glucose (i.e. transstimulation of transport). When these cells are preincubated for 20 rain in the presence of various concentrations of unlabeled 2-deoxyglucose or 3-O-methylglucose, so as to increase intracellular sugar concentrations, the subsequent rate of 2-deoxy-m-[3H(G)]glucose uptake is enhanced (40) . No evidence for accelerative exchange was found in our experiments with astroglial ascorbate transport. Na+-dependent ascorbate transport has a much higher affinity (apparent Km = 17-21 IxM ascorbate; present experiments) than does Na+-independent hexose transport (apparent K m = 360 ~M for 2-deoxyglucose; 41) by similar rat astroglial cultures. Na+-dependent plasma membrane transport systems which are modulated by external substrate deprivation include: i) Na+-glucose cotransport activity in enterocytes which increases during glucose deprivation (42) ; ii) Na+-amino acid cotransport in fibroblasts, hepatocytes and C6 glioma cells which increases during amino acid deprivation (43) (44) (45) (46) (47) (48) ; iii) Na+-phosphate cotransport in several cell types which increases during phosphate deprivation (49) . In these three cases, Na +dependent substrate uptake responds to substrate deprivation (i.e. exposure to substrate-free medium) with an increase in Vm~,, but without change in Km. In some cases, stimulation of transport requires gene transcription and protein synthesis (48, 49) . However, amino acid deprivation of <1 h can increase cellular amino acid transport activity through kinetic regulation (release from transinhibition) that does not require protein synthesis (44) (45) (46) (47) . The mechanism underlying the stimulation of astroglial ascorbate Vm~x by vitamin C deprivation has not yet been determined, but may involve: i) changed kinetic properties of transporters, affecting Vma,~ but not Km (e.g. release from transinhibition), or ii) an increased number of functional transporters due to slowed degradation of existing transporters, activation of nonfunctional (cryptic) transporters, redistribution of transporters from an intraceIlular compartment to the plasma membrane, or synthesis of transporters de novo. However, synthesis of new transporters seems unlikely in view of the rapidity of the change in ascorbate transport rate folIowing a change in external ascorbate concentration. Under physiological conditions, ascorbate is an important reducing agent (50) . Therefore, "adaptation" to vitamin C deprivation may involve an aIteration in intracellular redox state (51) with subsequent effects on the transport process. The concentration of ascorbate in brain extracellular fluid continually fluctuates. For example, extracellular ascorbate concentration in rat striatum varies according to a circadian pattern, with a rise during the nocturnal increase in motor activity (52) . Additionally, more rapid changes in rat striatal ascorbate can be observed following tail-pinch (53) . Extracellular ascorbate levels also differ between brain regions. For example, extracellular ascorbate concentrations in the white matter of the corpus callosum are higher than in adjacent areas of grey matter (striatum and cortex; 54). Additionally, amphet-amine-induced increases in extracellular ascorbate concentration are greater in the caudate than in the nucleus accumbens of rat brain (55) . These reports indicate large temporal and regional variations in extracellular ascorbate concentration. Our data are consistent with a transport system that plays a role in regulating intracellular ascorbate levels, because changes in transport activity may compensate for fluctuations in extracellular ascorbate concentration. Schwann cell myelination in a chemically defined medium: Demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation Differentiation of axon-related Schwann cells in vitro. I. Ascorbie acid regulates basal lamina assembly and myelin formation Dopamine beta-hydroxylase Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain Synthesis and release of vasoactive intestinal polypeptide (VIP) by mouse neuroblastoma cells: modulation by cyclic nucleotides and ascorbic acid Ascorbic acid enhances the release of luteinizing hormone-releasing hormone from the mediobasal hypothalamus in vitro Ascorbate decreases ligand binding to neurotransmitter receptors Ascorbic acid inhibition of alpha-adrenergic receptor binding Ascorbate modulates 5-[3H]hydroxytryptamine binding to central 5-HT sites in bovine frontal cortex Studies on the metabolism of catecholamines in the central nervous system of the mouse Cerebral monoamine metabolism in guinea-pigs with ascorbic acid deficiency Catecholamine uptake into cultured mouse astrocytes. Pages 301-305 Turnover rates of D-isoascorbic acid and Lascorbic acid in guinea pig organs. Can Effect of acute hypoxia on ascorbic acid content of plasma, cerebral cortex, and adrenal gland Effect of dietary ascorbic acid intake on tissue vitamin C in mice Transport of ascorbic acid and other watersoluble vitamins Active transport of ascorbic acid in adrenal cortex and brain cortex in vitro and the effects of ACTH and steroids Micronutrient homeostasis in mammalian brain and cerebrospinal fluid Vitamins and other metabolites in various sera commonly used for cell culturing Glutathione is present in l!igh concentrations in cultured astrocytes but not in cultured neurons Asc'orbic acid uptake by a high-affinity sodium-dependent mechanism in cultured rat astrocytes Ascorbic acid transport in mouse and rat aSi~ocytes is reversibly inhibited by furosemide, SITS and DIDS Ascorbic acid transport and accumulation in human neutrophils Methodological appendix: Astrocytes in primary cultures. Pages 175-186 Cell cultures. Pages 603-661 In vivo and in vitro models of demyelinating diseases: XV. Differentiation influences on the regulation of coronavirus infection in primary exptants of mouse CNS Protein measurement with the folin phenol reagent Use of a genetic variant to study the hexose transport properties of human skin fibroblasts Intestinal ascorbic acid transport following diets of high or low ascorbic acid content Rebound scurvy: does reduced intestinal transport of ascorbic acid play a roIe? Ascorbic acid transport by a clonal line of pheochromocytoma cells Homeostatic control of ascorbate concentration in CNS extracellular fluid Astroglia from defined brain regions as studied with primary cultures Differential determination of L-ascorbic acid and D-isoascorbic acid by reversed-phase highperformance liquid chromatography with electrochemical detection Human metabolism of L-ascorbic acid and erythorbic acid Old and new concepts of the membrane transport for glucose in cells Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells Effect of glucose starvation on glucose transport in neuronal cells in primary culture from rat brain Transport of 2-deoxy-D-glucose by dissociated brain cells Characteristics of glucose transport in neuronal cells and astmcytes from rat brain in primary culture Effects of semistarvation on transintestinaI D-glucose transport and Dglucose uptake in brush border and basolateral membranes of rat enterocytes Repression, derepression, transinhibition, and trans-stimulation of amino acid transport in rat hepatocytes and four hepatoma cell lines in culture Adaptive regulation of amino acid transport in cultured human fibroblasts. Sites and mechanism of action Simultaneous regulation of amino acid influx and efflux by system A in the hepatoma cell HTC Amino acid transport in eukaryotic cells and tissues Neutral amino acid transport in human synovial cells: substrate specificity of adaptive regulation and transinhibition Characteristics and adaptive regulation of glycine transport in cultured glial cells Adaptation to Pi deprivation of cell Na-dependent Pi uptake: a widespread process Ascorbate is an outstanding antioxidant in human blood plasma Ascorbic acid protection against free radicals Circadian changes in homovanillic acid and ascorbate levels in the rat striatum using microprocessor-controlled voltammetry Rapid changes in striatal ascorbate in respofise to tail-pinch monitored by contrast potential voltammetry Regional differences in extracellular ascorbic acid levels in the rat brain determined by high speed cyclic voltammetry Repeated administration of high doses of amphetamine increases release of ascorbie acid in caudate but not nucleus accumbens