Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.     

In vitro release of cytotoxic nucleoside analogs from lactide-caprolactone and lactide-glycolide copolymers

The aims of our study were to assess the release of cytotoxic nucleoside analogs 5-fluorouracil and 2-chloro-2'-deoxyadenosine from different lactide-glycolide or lactide-caprolactone biodegradable copolymers and the effects of sterilization on this release. The polymers were sterilized either with ethylene oxide at 37 degrees C, or with gamma radiation (15 kGy, 20 kGy, or 25 kGy). The kinetics of nucleoside release from the copolymers were measured over 50 days. Four copolymers exhibited relatively constant release of nucleosides in micromolar concentrations during the entire observation period. Sterilization with either ethylene oxide or gamma radiation only slightly influenced nucleoside release. Further development of these copolymers as an intracerebral nucleoside delivery system for local treatment of brain tumors is indicated.     

Involvement of Bidirectional Adenosine Transporters in the Release of l-[3H]Adenosine from Rat Brain Synaptosomal Preparations

Abstract: Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with l -[³H]adenosine, our objectives here were to determine (1) if l -[³H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological d -adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. l -[³H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. l -[³H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 µM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with l -[³H]adenosine alone or l -[³H]adenosine plus unlabeled l -adenosine, d -adenosine, or uridine, l -[³H]-adenosine release was inhibited 42% by l -adenosine, 69% by uridine, and 81% by d -adenosine. The inhibition of l -[³H]adenosine release from the synaptosomal preparations by substrates for or inhibitors of nucleoside transporters suggests that a portion of the release was mediated by nucleoside transporters. This experimental system may prove useful for evaluating the effects of pharmacological agents on bidirectional transport of adenosine.     

Adenosine receptor on human basophils: modulation of histamine release.

Adenosine, at physiologic concentrations, inhibits in vitro IgE-mediated human basophil histamine release in a dose-dependent fashion. The inhibition dose-response curve is paralleled by an adenosine-induced increase in cAMP levels of human leukocyte preparations. Further evidence that the adenosine effect is related to changes in cAMP levels is that the nucleoside inhibits only in the first stage of antigen-induced histamine release and fails to inhibit the release caused by ionophore A23187. A poorly metabolized derivative of adenosine, 2-chloroadenosine inhibits as effectively as adenosine; dipyridamole, which blocks adenosine uptake, does not impair the inhibition caused by adenosine. Finally, theophylline, which is a competitive antagonist of adenosine in human lymphocytes also blocks the inhibition of release caused by adenosine. These data suggest that adenosine acts via a specific cell-surface receptor linked to adenylate cyclase. It appears that the human basophil has a specific receptor for adenosine and that this nucleoside may modulate the in vivo release of the mediators of immediate hypersensitivity reactions.     

Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Enhanced accumulation of pericardial fluid adenosine and inosine in patients with coronary artery disease.

Adenosine and inosine are believed to have cardioprotective effects. However, little is known about their possible role in the metabolic autoregulation of human coronaries and in pathologic conditions with supply/demand imbalance of the heart such as coronary artery disease. Since these low molecular weight nucleosides freely diffuse through the monolayer of the visceral pericardium, adenosine and inosine concentrations in pericardial fluid may well reflect the conditions in cardiac interstitium. The pericardial fluid and systemic venous blood adenosine and inosine concentrations were measured in 98 human subjects undergoing heart surgery for coronary artery disease or valvular heart disease. Adenosine and inosine concentrations were measured by HPLC with UV detection. In subjects with coronary artery disease pericardial fluid nucleoside concentrations were significantly higher than in patients with valvular heart disease (adenosine: 1545 (996-3146) nmol/L [median (25th-75th quartiles)] vs. 738 (390-2527) nmol/L, P<0.01; inosine: 658 (321-1331) nmol/L vs. 347 (159-1037) nmol/L, P<0.05), while in both patient groups pericardial fluid nucleoside concentrations were higher by an order of magnitude than in venous plasma. Our results show the enhanced release of adenosine and inosine by the ischemic myocardium as a marker of supply/demand imbalance and support the hypothesis that these cardiac nucleosides may have an important role in the adaptation of coronary blood flow in human coronary artery disease.     

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.     

Fluorometric quantitation of adenosine concentration in small samples of extracellular fluid.

Adenosine is a naturally occurring nucleoside which regulates many physiological processes by interacting with adenosine-specific receptors. Knowledge of the extracellular adenosine concentration at the site of adenosine receptors on target cells is required for an understanding of mechanisms involving the action of the nucleoside. Samples of extracellular fluid which reside in close proximity to the surface of target cells are frequently small in volume. This report describes improvements in accuracy and reliability of a fluorometric assay designed for determining the concentration of adenosine in microliter samples of extracellular fluids. The utility of the assay is demonstrated by determining adenosine concentrations in interstitial and coronary effluent samples from normoxic perfused rat hearts. The assay also clearly detects changes in the interstitial and coronary effluent adenosine levels produced by isoproterenol stimulation or hypoxia. Thus, this assay is useful for determining the adenosine concentration in microliter samples of extracellular fluid and should facilitate investigations dealing with the functions of adenosine.     

Evidence for the Action of Endogenous Adenosine in the Rabbit Retina: Modulation of the Light-Evoked Release of Acetylcholine

Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent. The cholinergic amacrine cells of the rabbit retina were labeled with [3H]choline, and the effects of enzymatic adenosine degradation or adenosine antagonists on the light-evoked efflux of acetylcholine were evaluated. When endogenous adenosine was degraded by addition of adenosine deaminase, the light-evoked release of radioactivity derived from [3H]choline was significantly increased compared with control values. A similar response was observed when rabbit eyecups were superfused with a selective adenosine A1 receptor antagonist. The effect elicited by adenosine deaminase could be almost completely reversed by addition of cyclopentyladenosine, a highly selective A1 receptor agonist. These effects were observed in either the presence or the absence of picrotoxin. The results demonstrate a modulation of retinal physiology by adenosine.     

Adenosine in the central nervous system: release mechanisms and extracellular concentrations

Adenosine has several functions within the CNS that involve an inhibitory tone of neurotransmission and neuroprotective actions in pathological conditions. The understanding of adenosine production and release in the brain is therefore of fundamental importance and has been extensively studied. Conflicting results are often obtained regarding the cellular source of adenosine, the stimulus that induces release and the mechanism for release, in relation to different experimental approaches used to study adenosine production and release. A neuronal origin of adenosine has been demonstrated through electrophysiological approaches showing that neurones can release significant quantities of adenosine, sufficient to activate adenosine receptors and to modulate synaptic functions. Specific actions of adenosine are mediated by different receptor subtypes (A(1), A(2A), A(2B) and A(3)), which are activated by various ranges of adenosine concentrations. Another important issue is the measurement of adenosine concentrations in the extracellular fluid under different conditions in order to know the degree of receptor stimulation and understand adenosine central actions. For this purpose, several experimental approaches have been used both in vivo and in vitro, which provide an estimation of basal adenosine levels in the range of 50-200 nM. The purpose of this review is to describe pathways of adenosine production and metabolism, and to summarize characteristics of adenosine release in the brain in response to different stimuli. Finally, studies performed to evaluate adenosine concentrations under physiological and hypoxic/ischemic conditions will be described to evaluate the degree of adenosine receptor activation.     

Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampus

Adenosine, a neuromodulator of the CNS, activates inhibitory-A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nm), facilitated adenosine uptake through a PKC-dependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nm) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nm), facilitated the evoked release of [(3)H]acetylcholine under low-frequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2) either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nm) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.     

ADENOSINE KINASE OF MAMMALIAN BRAIN: PARTIAL PURIFICATION AND ITS ROLE FOR THE UPTAKE OF ADENOSINE

Abstract— Adenosine metabolism in the homogenate of brain mainly undergoes deamination to inosine and hypoxanthine, while uniformly labelled [¹⁴C]adenosine injected into the carotid artery or [8-¹⁴C]adenosine incubated with brain slices was mostly phosphorylated to [¹⁴C]adenine nucleotides in brain cells. Adenosine kinase has now been partially purified from homogenates of guinea pig brain. The kinase preparation was free of adenosine deaminase, almost free of adenosine triphosphatase and had a K_m of the order of 2 × 10^-5M for adenosine.
Kinetic studies with brain slices showed that adenosine reached the cells by diffusion and that the diffusion was facilitated by subsequent phosphorylation to adenine nucleotides. From the following experimental results, it is concluded that the phosphorylation is catalysed by adenosine kinase quantitatively. (1) During the uptake and phosphorylation of adenosine by brain slices, the nucleoside did not split to adenine and ribose moieties. (2) The rate of formation of adenine nucleotides in the slices was a hyperbolic function of the concentration of adenosine in the medium, showing an apparent K_m foradenosine of the order of 2 × 10^-5 M. (3) Some analogues of adenosine inhibited both the facilitated diffusion of adenosine and the kinase activity, but ouabain (0.005 mM) did not inhibit either.     

Adenosine mediates transforming growth factor-beta 1 release in kidney glomeruli of diabetic rats

Up regulation of the transforming growth factor-beta 1 (TGF-β1) axis has been recognized as a pathogenic event for progression of glomerulosclerosis in diabetic nephropathy. We demonstrate that glomeruli isolated from diabetic rats accumulate up to sixfold more extracellular adenosine than normal rats. Both decreased nucleoside uptake activity by the equilibrative nucleoside transporter 1 and increased AMP hydrolysis contribute to raise extracellular adenosine. Ex vivo assays indicate that activation of the low affinity adenosine A_2B receptor subtype (A_2BAR) mediates TGF-β1 release from glomeruli of diabetic rats, a pathogenic event that could support progression of glomerulopathy when the bioavailability of adenosine is increased.     

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5'-monophosphates from such natural metabolites as adenosine 5'-phosphosulfate and adenosine 5'-phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5'-O-phosphorofluoridate and adenosine 5'-O-(gamma-fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P-F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.     

Adenosine prevents the death of mesencephalic dopaminergic neurons by a mechanism that involves astrocytes

The purinergic nucleoside adenosine effectively prevented the death of dopaminergic neurons that occurs spontaneously and progressively in cultures of rat mesencephalon. Adenosine also significantly increased dopamine uptake, attesting to the state of differentiation and functional integrity of the neurons that were rescued. The effects of adenosine were (a) specific to the dopaminergic neurons in these cultures, (b) long-lived, (c) still observed when the treatment was delayed after plating, (d) potentiated by inhibition of adenosine deaminase, and (e) abolished when this enzyme was added in excess to the culture medium. The action of adenosine was mimicked by 5'-(N-ethylcarboxamido)adenosine and dibutyryl-cyclic AMP, but not by CGS-21680, suggesting the possible involvement of A2B subtype purinergic receptors. ATP was also neuroprotective, but its action resulted principally from conversion to adenosine by ectonucleotidases. Several anticancer drugs, including cytosine arabinoside, have been shown previously to prevent apoptosis in cultured dopaminergic neurons by a mechanism that requires the inhibition of proliferating astrocytes. In the presence of adenosine, astrocytes were more differentiated, and their proliferation rate was significantly reduced, suggesting that the neurotrophic effect of the adenine nucleoside resulted also from the repression of the astroglial cells. We did not find evidence of a trophic intermediary in adenosine-treated cultures, however, leading to the hypothesis that limitation of astrocyte replication in itself and/or ensuing changes in the glial phenotype were crucial. Our results suggest that molecules that modulate adenine nucleotide/nucleoside release may be useful for the treatment of chronic neurodegenerative conditions affecting dopaminergic neurons, such as Parkinson's disease.     

Role of the Intracellular Nucleoside Transporter ENT3 in Transmitter and High K+ Stimulation of Astrocytic ATP Release Investigated Using siRNA Against ENT3

This study investigates the role of the intracellular adenosine transporter equilibrative nucleoside transporter 3 (ENT3) in stimulated release of the gliotransmitter adenosine triphosphate (ATP) from astrocytes. Within the past 20 years, our understanding of the importance of astrocytic handling of adenosine, its phosphorylation to ATP, and release of astrocytic ATP as an important transmitter has become greatly expanded. A recent demonstration that the mainly intracellular nucleoside transporter ENT3 shows much higher expression in freshly isolated astrocytes than in a corresponding neuronal preparation leads to the suggestion that it was important for the synthesis of gliotransmitter ATP from adenosine. This would be consistent with a previously noted delay in transmitter release of ATP in astrocytes but not in neurons. The present study has confirmed and quantitated stimulated ATP release in response to glutamate, adenosine, or an elevated K⁺ concentration from well-differentiated astrocyte cultures, measured by a luciferin–luciferase reaction. It showed that the stimulated ATP release was abolished by downregulation of ENT3 with small interfering RNA (siRNA), regardless of the stimulus. The concept that transmitter ATP in mature astrocytes is synthesized directly from adenosine prior to release is supported by the postnatal development of the expression of the vesicular transporter SLC17A9 in astrocytes. In neurons, this transporter carries ATP into synaptic vesicles, but in astrocytes, its expression is pronounced only in immature cells and shows a rapid decline during the first 3 postnatal weeks so that it has almost disappeared at the end of the third week in well-differentiated astrocytes, where its role has probably been taken over by ENT3.     

Investigation of Possible Participation of Nucleoside Transport Systems in the Postischemic Release of Purines and Pyrimidines from Cold Stored Liver

The aim of the study was to elucidate the role of nucleoside transport systems in the postischemic release of nucleosides and nucleobases accumulated by the rat liver during cold storage. Livers were preserved for 24 h in Euro-Collins (EC) or in a lactobionate-based solution (LBS) without exogenous adenosine. The rates of release of uric acid, xanthine, hypoxanthine, inosine, adenosine, uridine, and cytidine were monitored during early reperfusion. The greater part of the purines and pyrimidines (up to 80%) was lost in the first 2 min of reperfusion. After storage in EC, uric acid and xanthine formed more than 90% of the total purines released; nucleosides did not exceed 5% of the total. After storage in LBS, hypoxanthine formed more than 80% of purine efflux and the release of inosine and uridine was increased 5-10 times. These changes were shown to be due to the presence of allopurinol in LBS. Dipyridamole (an inhibitor of equilibrative nucleoside transporters) decreased the efflux of uric acid after storage in EC but residual release remained high. Dipyridamole exerted the most pronounced effect on the release of nucleosides (inosine and uridine) from livers stored in LBS. The use of sodium-free media for liver preservation and reperfusion did not alter the rates of purine and pyrimidine release. We conclude that equilibrative nucleoside transporters mediate the postischemic release of nucleosides and also, but to a less degree, of uric acid. Simple diffusion is an important factor in the release of nucleobases. Active Na(+)/nucleoside cotransport does not play an important role in early reperfusion.     

Identification of the Adenosine Uptake Sites in Guinea Pig Brain

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.     

Differential Distribution of Purine Metabolizing Enzymes Between Glia and Neurons

Abstract: Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthlne oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 ± 61 pmol/min/10⁷ cells), nucleoside phosphorylase (187 ± B pmol/min/10⁷ cells), and adenosine deaminase (233 ± 32 pmol/min/10⁷ cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.