Serena Latini

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 (A1, A2A, A2B and A3), 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.

Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices

The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50–500 nM). 8‐Phenyltheophylline (10 μM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12±4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. In the time‐interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 μM. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode.

Adenosine Extracellular Brain Concentrations and Role of A2A Receptors in Ischemia

Abstract: Various experimental approaches have been used to determine the concentration of adenosine in extracellular brain fluid. The cortical cup technique or the microdialysis technique, when adenosine concentrations are evaluated 24 hours after implantation of the microdialysis probe, are able to measure adenosine in the nM range under normoxic conditions and in the μM range under ischemia. In vitro estimation of adenosine show that it can reach 30 μM at the receptor level during ischemia, a concentration able to stimulate all adenosine receptor subtypes so far identified. Although the protective role of A1 receptors in ischemia seems consistent, the protective role of A2A receptors appears to be controversial. Both A2A agonists and antagonists have been shown to be neuroprotective in various in vivo ischemia models. Although A2A agonists may be protective, mainly through peripherally mediated effects, A2A antagonists may be protective through local brain mediated effects. It is possible that A2A receptors are tonically activated following a prolonged increase of adenosine concentration, such as occurs during ischemia. A2A receptor activation desensitizes A1 receptors and reduces A1 mediated effects. Under these conditions A2A receptor antagonists may be protective by potentiating all the neuroprotective A1 mediated effects, including decreased neurotoxicity due to reduced ischemia induced glutamate outflow.

Investigations into the Adenosine Outflow from Hippocampal Slices Evoked by Ischemia‐Like Conditions

Abstract: The characteristics of adenosine and inosine outflow evoked by 5 min of ischemia‐like conditions in vitro (superfusion with glucose‐free Krebs solution gassed with 95% N2/5% CO2) were investigated on rat hippocampal slices. The viability of the slices after “ischemia” was evaluated by extracellular recording of the evoked synaptic responses in the CA1 region. The evoked dendritic field potentials were abolished after 5 min of superfusion under “ischemia” but a complete recovery occurred after 5 min of reperfusion with normal oxygenated Krebs solution. No recovery took place after 10 min of “ischemia.” The addition of the adenosine A, receptor antagonist 8‐phenylthe‐ ophylline to the superfusate antagonized the depression of the evoked field potentials caused by 5 min of “ischemia.” Five minutes of “ischemia” brought about a six‐ and fivefold increase in adenosine and inosine outflow, respectively, within 10 min. Tetrodotoxin reduced the outflow of adenosine and inosine by 42 and 33%, respectively, whereas the removal of Ca2+ caused a further increase. The NMDA receptor antagonist d(‐)‐2‐amino‐7‐ phoshonoheptanoic acid and the non‐NMDA antagonist 6,7‐dinitroquinoxaline‐2,3‐dione brought about small, not statistically significant decreases of adenosine and inosine outflow. The glutamate uptake inhibitor dihydrokainate did not affect the outflow of adenosine and inosine. Inhibition of ecto‐5′‐nucleotidase by α, β‐methylene ADP and GMP did not affect basal adenosine outflow but potentiated “ischemia”‐evoked adenosine outflow. It is concluded that ischemia‐like conditions in vitro evoke a Ca2+‐independent adenosine and inosine outflow, through a mechanism that partly depends on propagated nervous activity but does not involve excitatory amino acids. The efflux of adenosine is probably responsible for the depression of the evoked synaptic electrical activity during “ischemia” in the hippocampal slices.

Brief, repeated, oxygen-glucose deprivation episodes protect neurotransmission from a longer ischemic episode in the in vitro hippocampus: role of adenosine receptors

Ischemic preconditioning in the brain consists of reducing the sensitivity of neuronal tissue to further, more severe, ischemic insults. We recorded field epsps (fepsps) extracellularly from hippocampal slices to develop a model of in vitro ischemic preconditioning and to evaluate the role of A1, A2A and A3 adenosine receptors in this phenomenon. The application of an ischemic insult, obtained by glucose and oxygen deprivation for 7 min, produced an irreversible depression of synaptic transmission. Ischemic preconditioning was induced by four ischemic insults (2 min each) separated by 13 min of normoxic conditions. After 30 min, an ischemic insult of 7 min was applied. This protocol substantially protected the tissue from the irreversible depression of synaptic activity. The selective adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 100 nM), completely prevented the protective effect of preconditioning. The selective adenosine A2A receptor antagonist 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol (ZM 241385, 100 nM) did not modify the magnitude of fepsp recovery compared to control slices. The selective A3 adenosine receptor antagonists, 3‐propyl‐6‐ethyl‐5[ethyl(thio)carbonyl]‐2‐phenyl‐4‐propyl‐3‐pyridinecarboxylate (MRS 1523, 100 nM) significantly improved the recovery of fepsps after 7 min of ischemia. Our results show that in vitro ischemic preconditioning allows CA1 hippocampal neurons to become resistant to prolonged exposure to ischemia. Adenosine, by stimulating A1 receptors, plays a crucial role in eliciting the cell mechanisms underlying preconditioning; A2A receptors are not involved in this phenomenon, whereas A3 receptor activation is harmful to ischemic preconditioning.

A2 adenosine receptors: their presence and neuromodulatory role in the central nervous system

1. Adenosine is an endogenous neuromodulator that exerts its depressant effect on neurons by acting on the A1 adenosine receptor subtype. Excitatory actions of adenosine, mediated by the activation of the A2 adenosine receptor subtype, have also been shown in the central nervous system. 2. Adenosine A2a receptors are highly localized in the striatum, as demonstrated by the binding assay of the A2a selective agonist, CGS2680, and by analysis of the A2 receptor mRNA localization with in situ hybridization histochemistry. However, adenosine A2a, receptors, albeit at lower levels, are also localized in other brain regions, such as the cortex and the hippocampus. 3. In the striatum, adenosine A2a, receptors are implicated in the control of motor activity. Evidences exists of an antagonistic interaction between adenosine A2a and dopamine D2 receptors. 4. Utilizing selective agonists and antagonists for adenosine A2a receptors, their role in the modulation of the release of several neurotransmitters (acetylcholine, dopamine, glutamate, GABA) has been extensively studied in the brain (striatum, cortex, hippocampus). Controversial results have been obtained and, because the overall effect of endogenous adenosine in the brain is that of an inhibitory tonus, the physiological meaning of the excitatory A2 receptor remains to be clarified.

The source of brain adenosine outflow during ischemia and electrical stimulation

Adenosine outflow and adenosine and adenine nucleotide content of hippocampal slices were evaluated under two different experimental conditions: ischemia-like conditions and electrical stimulation (10 Hz). Five minutes of ischemia-like conditions brought about an 8-fold increase in adenosine outflow in the following 5 min during reperfusion, and a 2-fold increase in adenosine content, a 43% decrease in ATP, a 72% increase in AMP and a 30% decrease in energy charge (EC) at the end of the ischemic period. After 10 min of reperfusion ATP, AMP and EC returned to control values, while the adenosine content was further increased. Five minutes of electrical stimulation brought about an 8-fold increase in adenosine outflow that peaked 5 min after the end of stimulation, a 4-fold increase in adenosine content and an 18% decrease in tissue EC at the end of stimulation. After 10 min of rest conditions the adenosine content and EC returned to basal values. The origin of extracellular adenosine from S-adenosylhomocysteine (SAH) was examined under the two different experimental conditions. The SAH hydrolase inhibitor, adenosine-2,3-dialdehyde (10 microM), does not significantly modify the adenosine outflow evoked by electrical stimulation or ischemia-like conditions. This finding excludes a significant contribution by the transmethylation pathway to adenosine extracellular accumulation evoked by an electrical or ischemic stimulus, and confirms that the most likely source of adenosine is from AMP dephosphorylation.

Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices

In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia‐like conditions. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine), and of selective A2A receptor antagonists, ZM 241385 (4‐(2‐[7‐amino‐2‐(2‐furyl)‐{1,2,4}‐triazolo{2,3‐a}{1,3,5}triazin‐5‐ylamino]ethyl)phenol) and SCH 58261 (7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode. The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1,3‐dipropyl‐8‐cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia. A prolonged application of either adenosine (100 μM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM). SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia‐induced depression. These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1‐mediated depression of synaptic transmission induced by in vitro ischaemia.

Temporal correlation between adenosine outflow and synaptic potential inhibition in rat hippocampal slices during ischemia-like conditions.

The temporal correlation between adenosine outflow and changes in field excitatory post synaptic potentials (fEPSP) occurring during ischemia-like conditions was investigated in rat hippocampal slices. Five-minute long ischemia-like conditions resulted in a 100% depression of fEPSP amplitude, followed by a complete recovery after 6-7 min of reperfusion. By reducing the duration of the ischemic insult to 2 min, fEPSP was depressed by 50%. During both 5 and 2 min of ischemia-like conditions, a significant increase in adenosine outflow was detected. During reperfusion, when fEPSP amplitude recovered completely, the adenosine level in the extracellular fluid returned to basal values. The strict relationship between the increase in adenosine outflow and fEPSP inhibition supports the hypothesis that adenosine is largely responsible for the synaptic transmission depression during cerebral ischemia.

In vivo regulation of extracellular adenosine levels in the cerebral cortex by NMDA and muscarinic receptors

The adenosine concentration in samples of perfusate was determined 24 h after implantation of microdialysis fibre in the cortex. High performance liquid chromatography coupled with a fluorometric detector was used. K+ (100 mM) depolarization was followed by a 2- to 4-fold increase in adenosine efflux. The addition of tetrodotoxin (1 microM) to the perfusate was followed by a decrease in spontaneous and K(+)-evoked adenosine efflux. The increase induced by high K+ was markedly inhibited by the NMDA receptor antagonist, D(-)-2-amino-7-phosphonoheptanoic acid (1 mM, D-AP7), but not by the muscarinic receptor antagonist, atropine (1.5 microM). The acetylcholine esterase inhibitor, physostigmine (7 microM), and the muscarinic receptor agonist, oxotremorine (100 microM), significantly enhanced the K(+)-evoked increase in adenosine. The spontaneous efflux of adenosine was not modified by any of the drugs tested. A neurotoxic lesion of the cholinergic pathway innervating the cortex, although inducing a marked decrease in cortical choline acetyltransferase activity, did not significantly modify the cortical adenosine efflux. It is concluded that, under K(+)-depolarizing conditions, adenosine efflux is triggered by excitatory amino acids and enhanced by muscarinic activation.