Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neonatal Hypothyroidism Affects the Adenine Nucleotides Metabolism in Astrocyte Cultures from Rat Brain

Neonatal hypothyroidism is associated with multiple and severe brain alterations. We recently demonstrated a significant increase in hydrolysis of AMP to adenosine in brain of hypothyroid rats at different ages. However, the origin of this effect was unclear. Considering the effects of adenine nucleotides to brain functions and the harmful effects of neonatal hypothyroidism to normal development of the central nervous system, in this study we investigated the metabolism of adenine nucleotides in hippocampal, cortical and cerebellar astrocyte cultures from rats submitted to neonatal hypothyroidism. ATP and AMP hydrolysis were enhanced by 52 and 210%, respectively, in cerebellar astrocytes from hypothyroid rats. In hippocampus of hypothyroid rats, the 47% increase in AMP hydrolysis was significantly reverted when the astrocytes were treated with T3. Therefore, the imbalance in the ATP and adenosine levels in astrocytes, during brain development, may contribute to some of the effects described in neonatal hypothyroidism.Elizandra Braganhol and Alessandra Nejar Bruno are first authors.     

Exogenous Glutamate Is Metabolized to Glutamine and Exported by Rat Primary Astrocyte Cultures

Rat cortical astrocytes in primary culture were examined for their capacity to transport and metabolize exogenous L-[U-14C]glutamate. After incubation for time periods up to 120 min, cells and incubation media were analyzed for labelled and endogenous glutamate and its metabolic products by HPLC coupled with fluorescence detection and liquid scintillation counting. Glutamine was the major labelled metabolite after 120 min, accounted for 38% of the original glutamate label, and was found primarily in the incubation medium. A further 13.5% of the label was recovered in deaminated metabolites of glutamate, 1.2% was associated with aspartate, 23% remained in glutamate, and 10.2% was found in an acid-precipitated cell fraction. More than 84% of the label was recovered in these fraction. suggesting that the maximum possible formation and loss of 14CO2 was 16%. The rate of total glutamine synthesis was 1.1 nmol X mg protein-1 X min-1 when 9 microM exogenous glutamate was present. The total amount of glutamine synthesized greatly exceeded the consumption of glutamate, indicating that a substantial proportion of glutamine was synthesized from other carbon sources. Almost all of the newly formed glutamine was exported into the medium. These results indicate that astrocytes in primary culture, by accumulating glutamate, producing glutamine, and exporting it, are capable of carrying out the glial component of the glutamine cycle.     

Uptake and Metabolism of Iron Oxide Nanoparticles in Brain Cells

Magnetic iron oxide nanoparticles (IONPs) are used for various applications in biomedicine, for example as contrast agents in magnetic resonance imaging, for cell tracking and for anti-tumor treatment. However, IONPs are also known for their toxic effects on cells and tissues which are at least in part caused by iron-mediated radical formation and oxidative stress. The potential toxicity of IONPs is especially important concerning the use of IONPs for neurobiological applications as alterations in brain iron homeostasis are strongly connected with human neurodegenerative diseases. Since IONPs are able to enter the brain, potential adverse consequences of an exposure of brain cells to IONPs have to be considered. This article describes the pathways that allow IONPs to enter the brain and summarizes the current knowledge on the uptake, the metabolism and the toxicity of IONPs for the different types of brain cells in vitro and in vivo.     

Immunocytochemical and Biochemical Analysis of Carbonic Anhydrase in Primary Astrocyte Cultures from Rat Brain

Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein-positive, astrocytic monolayer. Smaller, process-bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2',3'-dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5- to 5-fold less than that found for cerebral homogenates from perfused 3-week-old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10(-8) M, confirming that both rat brain and the astrocyte cultures possess the high-activity type II enzyme. CA-II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA-II.     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Maturation on the Phospholipid and Phospholipid Fatty Acid Compositions in Primary Rat Cortical Astrocyte Cell Cultures

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.     

Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures

Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K⁺ (using ⁸⁶Rb), taurine, and d -aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 m M quinine, 10 µ M nimodipine, 100 µ M BAPTA-AM, 10 µ M trifluoperazine, or a calcium-free buffer significantly ( p < 0.0001) inhibited RVD. This was accompanied by inhibition of ⁸⁶Rb release but an increase in d -[³H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K⁺ channels, and inhibition of calmodulin all inhibit RVD. Because d -[³H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d -aspartate release is a main determinant of RVD. In contrast, [³H]taurine release was increased by 1 m M quinine and inhibited by 10 µ M trifluoperazine. The net release of K⁺ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K⁺ and taurine release in RVD.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures

Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na⁺/K⁺-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate–glutamine cycle, as well as glutamate and d-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

Characterization of Glycosaminoglycan Disaccharide Composition in Astrocyte Primary Cultures and the Cortex of Neonatal Rats

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

     

Uptake and Metabolism of Serotonin by Ependymal Primary Cultures

Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system.     

Handling of Iron Oxide and Silver Nanoparticles by Astrocytes

Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.     

Use of Dipeptides for the Synthesis of Glutathione by Astroglia-Rich Primary Cultures

Abstract: The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cells to use dipeptides for glutathione synthesis. For restoration of the glutathione level, after a 24-h starvation period in the absence of glucose and amino acids, glucose, glutamate, cysteine, and glycine have to be present in the incubation buffer. The dipeptides CysGly and γGluCys were able to substitute for cysteine plus glycine and glutamate plus cysteine, respectively. Half-maximal contents of glutathione were found at 20 µ M CysGly and 3 m M γGluCys. In addition, the oxidized forms of the dipeptides CysGly and GlyCys could replace cysteine plus glycine for glutathione restoration, and the glycine-containing dipeptides GlyGly, GlyLeu, GlyGlu, GlyGln, and γGluGly could partially substitute for the glycine necessary for the replenishment of glutathione. The glutathione resynthesis in the presence of CysGly plus glutamate was totally inhibited in the presence of buthionine sulfoximine, an inhibitor of γ-glutamylcysteine synthetase. In contrast, glutathione restoration from γGluCys at a concentration of 10 m M in the presence of glycine was not influenced by the inhibitor. The use of CysGly or γGluCys was not affected by the presence of the dipeptidase inhibitors cilastatin or bestatin. In addition, carnosine and several other dipeptides applied in a 50-fold excess only slightly prevented the use of CysGly, hinting at the existence in astroglial cells of a transport system specific for CysGly. The results demonstrate that astroglial cells can use dipeptides for intracellular glutathione synthesis and that the dipeptides most likely are taken up as intact molecules into astroglial cells before intracellular hydrolysis occurs.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures.

Abstract: Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-³H]alkyl- sn -glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1–20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyksn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl-, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl- compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

Inosine Mediates the Protective Effect of Adenosine in Rat Astrocyte Cultures Subjected to Combined Glucose-Oxygen Deprivation

Abstract: Preliminary evidence suggests adenosine, a neuromodulator, has neuroprotective properties during cerebral ischemia. It is unclear, however, if adenosine has glioprotective effects. We studied the effect of adenosine on cellular injury in astroglial cultures subjected to combined glucose-oxygen deprivation. Adenosine (100–1,000 µ M ) dramatically reduced astroglial injury, whereas the adenosine agonists 2-chloroadenosine (10 n M –100 µ M ), N ⁶-cyclopentyladenosine (1 n M –10 µ M ), 5'- N -ethylcarboxamidoadenosine (10 n M –100 µ M ), and N ⁶-2-(4-aminophenyl)ethyladenosine (10 n M –100 µ M ) had no effect. Furthermore, the adenosine antagonists 8-cyclopentyl-1,3-dipropylxanthine (1 n M –1 µ M ), xanthine amine congener (10 n M –10 µ M ), and 8-( p -sulfophenyl)-theophylline (10–300 µ M ) failed to reverse the protective effect of 200 µ M adenosine. Next, adenosine degradation products were studied. Inosine proved to be glioprotective at concentrations nearly identical to those of adenosine, but hypoxanthine and ribose had no effect. The protective effect of 200 µ M inosine was not reversed by 8-( p -sulfophenyl)theophylline (10–300 µ M ). Adenosine deaminase (1 unit/ml) had no effect on protection produced by adenosine, whereas erythro -9-(2-hydroxy-3-nonyl)adenine hydrochloride (10 µ M ) reversed the protective effect of adenosine. Dipyridamole (4 µ M ) inhibited the protective effect of both adenosine and inosine. We conclude that adenosine dramatically decreases astroglial injury during combined glucose-oxygen deprivation and that this protective effect appears to be mediated by inosine.