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.     

Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K⁺) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca²⁺-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca²⁺ concentration, or increase of extracellular K⁺ to a depolarizing level that opens astrocytic L-channels for Ca²⁺ and thereby also increase intra-astrocytic Ca²⁺ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K⁺ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K⁺ concentrations.     

Distribution of Equilibrative, Nitrobenzylthioinosine-Sensitive Nucleoside Transporters (ENT1) in Brain

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [3H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35S-labeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra- and extracellular levels of adenosine in brain.     

Extracellular ATP-prinoceptor signaling and AMP-activated protein kinase regulate astrocytic glucose transporter 3 in an in vitro ischemia

Astrocytes become hypertrophic reactive in response to the ischemic stress, and they contribute to either protect or exacerbate neuronal damage, depending on the depth or duration of the stress. Astrocytes have more resistance to the ischemic stress than neurons, which is apparently due to active anerobic metabolic pathway in the emergency situation. We have been focused on the functional role of astrocytic glucose transporters in the ischemic condition. Under the physiological conditions, cultured astrocytes primarily express glucose transporter1 (GLUT1), and GLUT3 is only detected at extremely low levels. But astrocytes enhance GLUT3 expression through the signaling of nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB) under mild ischemic condition. It is reasonable since GLUT3 transports extracellular glucose about seven times faster than GLUT1, so astrocytes enhance the storage of intracellular glucose during the ischemia. However, other signaling cascades that regulate GLUT3 production remain unknown. Here we demonstrate that extracellular adenosine 5′-triphosphate (ATP)-P2Y receptor signaling also regulates GLUT3 expression. Under mild ischemic condition, astrocytes positively released existing intracellular or newly synthesized ATP by AMP-activated protein kinase (AMPK) signaling. The released extracellular ATP from pore channels activated ATP-sensitive P2Y receptor signaling, resulting in an increase in c-Fos and c-Jun proteins. Newly synthesized GLUT3 was regulated by those signaling since the inhibition of P2Y receptors or c-Fos/c-Jun signaling significantly reduced GLUT3 expression. Furthermore, the inhibition of P2Y receptors during the ischemic condition sustained intracellular ATP concentration, leading to a decrease in AMPK proteins. These results suggest AMPK-regulated ATP production triggers the release of ATP to activate P2Y receptor signaling, which is another candidate that regulates GLUT3 expression under the ischemic condition.     

Amyloid pathology disrupts gliotransmitter release in astrocytes

Accumulation of amyloid-beta (Aβ) is associated with synaptic dysfunction and destabilization of astrocytic calcium homeostasis. A growing body of evidence support astrocytes as active modulators of synaptic transmission via calcium-mediated gliotransmission. However, the details of mechanisms linking Aβ signaling, astrocytic calcium dynamics, and gliotransmission are not known. We developed a biophysical model that describes calcium signaling and the ensuing gliotransmitter release from a single astrocytic process when stimulated by glutamate release from hippocampal neurons. The model accurately captures the temporal dynamics of microdomain calcium signaling and glutamate release via both kiss-and-run and full-fusion exocytosis. We investigate the roles of two crucial calcium regulating machineries affected by Aβ: plasma-membrane calcium pumps (PMCA) and metabotropic glutamate receptors (mGluRs). When we implemented these Aβ-affected molecular changes in our astrocyte model, it led to an increase in the rate and synchrony of calcium events. Our model also reproduces several previous findings of Aβ associated aberrant calcium activity, such as increased intracellular calcium level and increased spontaneous calcium activity, and synchronous calcium events. The study establishes a causal link between previous observations of hyperactive astrocytes in Alzheimer’s disease (AD) and Aβ-induced modifications in mGluR and PMCA functions. Analogous to neurotransmitter release, gliotransmitter exocytosis closely tracks calcium changes in astrocyte processes, thereby guaranteeing tight control of synaptic signaling by astrocytes. However, the downstream effects of AD-related calcium changes in astrocytes on gliotransmitter release are not known. Our results show that enhanced rate of exocytosis resulting from modified calcium signaling in astrocytes leads to a rapid depletion of docked vesicles that disrupts the crucial temporal correspondence between a calcium event and vesicular release. We propose that the loss of temporal correspondence between calcium events and gliotransmission in astrocytes pathologically alters astrocytic modulation of synaptic transmission in the presence of Aβ accumulation.     

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.     

Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2'3'-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.     

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca(2+)-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication "Ca(2+) wave" in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant "gliotransmitter" between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a "tripartite synapse". In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS.     

Adenosine Augmentation Evoked by an ENT1 Inhibitor Improves Memory Impairment and Neuronal Plasticity in the APP/PS1 Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cognitive impairment and synaptic dysfunction. Adenosine is an important homeostatic modulator that controls the bioenergetic network in the brain through regulating receptor-evoked signaling pathways, bioenergetic machineries, and epigenetic-mediated gene regulation. Equilibrative nucleoside transporter 1 (ENT1) is a major adenosine transporter that recycles adenosine from the extracellular space. In the present study, we report that a small adenosine analogue (designated J4) that inhibited ENT1 prevented the decline in spatial memory in an AD mouse model (APP/PS1). Electrophysiological and biochemical analyses further demonstrated that chronic treatment with J4 normalized the impaired basal synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses as well as the aberrant expression of synaptic proteins (e.g., NR2A and NR2B), abnormal neuronal plasticity-related signaling pathways (e.g., PKA and GSK3β), and detrimental elevation in astrocytic A_2AR expression in the hippocampus and cortex of APP/PS1 mice. In conclusion, our findings suggest that modulation of adenosine homeostasis by J4 is beneficial in a mouse model of AD. Our study provides a potential therapeutic strategy to delay the progression of AD.     

Characterisation of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At).

Here we report on the isolation of an Arabidopsis thaliana cDNA that is able to complement a Saccharomyces cerevisiae mutant unable to synthesise adenine. This cDNA encodes a highly hydrophobic protein (ENT1,At) of 428 amino acids, showing high similarity to the human nucleoside transporter hENT1. Yeast cells expressing ENT1,At are able to grow on adenosine-containing media, adenosine import exhibited an apparent affinity (K(M)) of 3.6 microM, and led to accumulation of this nucleoside within the yeast cell. Transport is inhibited by various nucleosides. Typical inhibitors of ENT-type nucleoside transporters do not inhibit (3)H-adenosine import. The presence of protonophores abolished adenosine import, indicating that ENT1,At catalyse a proton-dependent adenosine transport. This is the first functional characterisation of a plant nucleoside transport protein.     

Zinc is released by cultured astrocytes as a gliotransmitter under hypoosmotic stress-loaded conditions and regulates microglial activity

Aim

Astrocytes contribute to the maintenance of brain homeostasis via the release of gliotransmitters such as ATP and glutamate. Here we examined whether zinc was released from astrocytes under stress-loaded conditions, and was involved in the regulation of microglial activity as a gliotransmitter.

Main methods

Hypoosmotic stress was loaded to astrocytes using balanced salt solution prepared to 214–314 mOsmol/L, and then intra- and extra-cellular zinc levels were assessed using Newport Green DCF diacetate (NG) and ICP-MS, respectively. Microglial activation by the astrocytic supernatant was assessed by their morphological changes and poly(ADP-ribose) (PAR) polymer accumulation.

Key findings

Exposure of astrocytes to hypoosmotic buffer, increased the extracellular ATP level in osmolarity-dependent manners, indicating a load of hypoosmotic stress. In hypoosmotic stress-loaded astrocytes, there were apparent increases in the intra- and extra-cellular zinc levels. Incubation of microglia in the astrocytic conditioned medium transformed them into the activated “amoeboid” form and induced PAR formation. Administration of an extracellular zinc chelator, CaEDTA, to the astrocytic conditioned medium almost completely prevented the microglial activation. Treatment of astrocytes with an intracellular zinc chelator, TPEN, suppressed the hypoosmotic stress-increased intracellular, but not the extracellular, zinc level, and the increase in the intracellular zinc level was blocked partially by a nitric oxide synthase inhibitor, but not by CaEDTA, indicating that the mechanisms underlying the increases in the intra- and extra-cellular zinc levels might be different.

Significance

These findings suggest that under hypoosmotic stress-loaded conditions, zinc is released from astrocytes and then plays a primary role in microglial activation as a gliotransmitter.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

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.     

Long-term facilitation of spontaneous calcium oscillations in astrocytes with endogenous adenosine in hippocampal slice cultures

It is well established that astrocytes release gliotransmitters and moderate neuronal activity in the central nervous system via intracellular Ca(2+) dynamics. Astrocytic Ca(2+) oscillations are one type of spontaneous Ca(2+) mobilization that occurs in astrocytes. However, the modulation of spontaneous astrocytic Ca(2+) oscillations, especially in pathophysiological conditions, is not yet fully understood. Here, we demonstrate that activation of adenosine receptors induces a long-lasting increase in the frequency of astrocytic Ca(2+) oscillations in rat hippocampal slice cultures. The long-term facilitation of the frequency of Ca(2+) oscillations was mediated by endogenous adenosine generated via breakdown of extracellular ATP by ecto-ATPase. We also demonstrate that local tissue injury with ultraviolet irradiation can cause this long-term facilitation of Ca(2+) oscillations via endogenous adenosine. Our data suggest that endogenous adenosine is one of the modulators of spontaneous astrocytic Ca(2+) oscillations in the rat hippocampus, and may play a significant role in altered Ca(2+) dynamics in astrocytes observed during pathophysiological conditions.     

The adenosine transporter,ENT1, in cardiomyocytes is sensitive to inhibition by ethanol in a kinase-dependent manner: implications for ethanol-dependent cardioprotection and nucleoside analog drug cytotoxicity

The adenosine transporter 1 (ENT1) transports nucleosides, such as adenosine, and cytotoxic nucleoside analog drugs. ENT1 is well established to play a role in adenosinergic signaling in the cardiovascular system by modulating adenosine levels. Moderate ethanol consumption is cardioprotective and underlying mechanisms of action are not clear although adenosinergic signaling has been implicated. Here, we show that ethanol (5–200 mM) significantly reduces ENT1-dependent [³H] 2-chloroadenosine uptake (by up to 27 %) in the cardiomyocyte cell line, HL-1. Inhibition or absence of ENT1 is known to be cardioprotective, suggesting that the interaction of ethanol with ENT1 may promote adenosinergic cardioprotective pathways in the cardiovasculature. Ethanol sensitivity of adenosine uptake is altered by pharmacological activation of PKA and PKC. Primary cardiomyocytes from PKCε-null mice have significantly greater sensitivity to inhibition (by approximately 37 %) of adenosine uptake by ethanol than controls. These data suggest that the presence of ethanol may compromise ENT1-dependent nucleoside analog drug cytotoxicity, and indeed, ethanol (5 mM) reduces the cytotoxic effects of gemcitabine (2 nM), an anti-cancer drug, in the human cancer cell line, HTB2. Thus, the pharmacological inhibition of ENT1 by ethanol may contribute to ethanol-dependent cardioprotection but compromise gemcitabine cytotoxicity.     

Glutamate, a neurotransmitter--and so much more. A synopsis of Wierzba III

It appears almost incredible that the first indications that glutamate excites brain tissue were obtained during the second half of the 20th century, that vesicles containing glutamate were demonstrated in glutamatergic neurons less than 25 years ago, and that glutamate was not accepted as the major excitatory transmitter until about the same time. During this span of time it has also become realized that glutamate is so much more than a conventional neurotransmitter: (1) astrocytes express vesicles accumulating glutamate by vesicular transporters akin to the vesicular glutamate transporters in glutamatergic neurons, and they release glutamate by exocytosis; (2) a series of metabolic processes in astrocytes (glutamate uptake, glutamine synthetase activity, glutamine release) are involved in neuronal reutilization of transmitter glutamate; (3) glutamine may also be utilized for synthesis of GABA, the major inhibitory transmitter; (4) de novo synthesis of glutamate accounts for 20% of cerebral glucose metabolism, all of which initially occurs in astrocytes, and at steady state a corresponding amount of glutamate is oxidatively degraded, mainly or exclusively in astrocytes; (5) tissue contents of glutamate/glutamine increase during enhanced glutamatergic activity, i.e., astrocytic de novo synthesis exceeds astrocytic metabolic degradation of glutamate.