P2Y1 receptor modulation of Ca2+-activated K+ currents in medium-sized neurons from neonatal rat striatal slices.

ATP signaling to neurons and glia in the nervous system occurs via activation of both P2Y and P2X receptors. Here, we investigated the effects of P2Y(1) receptor stimulation in developing striatal medium-sized neurons using patch-clamp recordings from acute brain slices of 7- and 28-day-old rats. Application of the selective P2Y(1) receptor agonist 2-(Methylthio) ADP trisodium salt (2-MeSADP; 250 nM) increased outward K(+) currents evoked by a ramp depolarization protocol in voltage-clamp recordings. This effect was observed in 59 out of 82 cells (72%) and was blocked completely by the P2Y(1) antagonist, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate. The averaged 2-MeSADP-sensitive conductance was fitted by the sum of a linear conductance and a Boltzmann relation, giving one-half activation voltage of -14.2 mV and an equivalent charge of 2.91. The 2MeSADP-mediated effect was sensitive to submillimolar concentrations of tetraethylammonium (TEA; 200 µM), to 200 nM iberiotoxin and to 100 nM apamin, suggesting the involvement of both big and small potassium (BK and SK, respectively) calcium-activated channels. In current-clamp experiments, 2-MeSADP decreased depolarization-evoked action potential (AP) firing in all 26 cells investigated, and this effect was reversed by TEA and by apamin but not by iberiotoxin. We conclude that the stimulation of P2Y(1) receptors in developing striatal neurons leads to activation of calcium-activated potassium channels [I(K(Ca))] of both BK and SK subtypes, the latter responsible for decreasing the frequency of AP firing in response to current injection. Therefore, P2Y(1) signaling leading to activation of I(K(Ca)) may be important in regulating the activity of medium-sized neurons in the striatum.

Selective adenosine A(2a) receptor agonists reduce the apoptosis in an experimental model of spinal cord trauma.

Adenosine is an important regulator of inflammatory mechanisms. Functional studies indicate a protective effect of adenosine A2A receptor agonists in spinal cord injury (SCI). The basic molecular mechanisms accounting for their protective effects from spinal cord injury have to be fully elucidated. The aim of this study is to evaluate in vivo protection by two selective A2A receptor agonists, 2-[p-(2-carboxyethyl)phenylethylamino]-50-ethylcarboxamidoadenosine (CGS 21680, 100 microg/kg) and (4-[3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl)prop-2-ynyl] piperidine-1-carboxylic acid methyl ester) (ATL 313, 3 microg/kg) on the degree of apoptosis, in the experimental model of spinal cord injury. Spinal cord trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord trauma in mice was characterised by edema, neutrophilic infiltration and apoptosis. ATL 313, administered by subcutaneously implanted osmotic minipumps after SCI, clearly reduced motor deficit for up to 19 days after operation. The selective A2A receptor agonists ATL 313 and CGS 21680 administered after SCI, reduced tissue damage, TUNEL staining, cytokine (TNF-alpha) expression, Bax, Fas-L and Caspase-3 expression, Annexin-V staining, while increasing Bcl-2 expression. In conclusion, our results demonstrate that treatment with adenosine A2A receptor agonists prevents the apoptotic process that is an important step of secondary damage after SCI.

Expression of neuronal and inducible nitric oxide synthase in neuronal and glial cells after transient occlusion of the middle cerebral artery.

We presently investigated the time-course of neuronal nitric oxide synthase and inducible nitric oxide synthase expression and content in the rat striatum up to 6 days after ischemia induced by transient middle cerebral artery occlusion, a condition that potentially allows functional recovery, with the aim to identify the cell types expressing these two enzymes and to correlate neuronal nitric oxide synthase and inducible nitric oxide synthase changes in order to verify whether and how these changes are related to tissue damage, motor-sensory performances and survival. Before and after surgery, the animals underwent neurological evaluation. The results demonstrated that the rats with a score > or = 12 at the neurological evaluation 24 h after ischemia showed a significant increase in neuronal nitric oxide synthase-immunoreactive neurones and absence of inducible nitric oxide synthase-immunoreactive cells and survived up to the sixth day; conversely, the rats with a score < 12 at the neurological evaluation 24 h after ischemia showed a progressive significant decrease in neuronal nitric oxide synthase-immunoreactive neurones and appearance of inducible nitric oxide synthase-immunoreactive cells and none of the rats survived up to the sixth day. Microglia cells were activated in both groups but only in the latter did these cells express inducible nitric oxide synthase. Measurement of the infarct area demonstrated that it occupied a similar territory in both groups of rats but in those with a score < 12 the edema was more extended. In conclusion, we demonstrated that a neurotoxic insult such as ischemia can induce neuronal nitric oxide synthase expression in the neurones and that when neuronal nitric oxide synthase-immunoreactive neurones increase in number, microglia activation is less extended, inducible nitric oxide synthase-immunoreactive cells are absent, tissue damage reduced and the rats survive longer. Conversely, when there is a significant decrease of neuronal nitric oxide synthase-immunoreactive neurones, microglia cells are intensely activated, inducible nitric oxide synthase-immunoreactive cells appear and the animal survival is shortened.

The protective effect of adenosine A2A receptor antagonism in cerebral ischemia.

OBJECTIVES: We reviewed our most recent work on the protective effect of adenosine A(2A)antagonism in cerebral ischemia. METHODS: Focal ischemia was produced in rats by introducing a nylon monofilament pre-coated with silicone through the external carotid artery to occlude the right MCA at its origin. RESULTS: A(2A) antagonism was found protective in the model of permanent focal ischemia induced by the monofilament technique. This methodology provides the possibility of evaluating the protection against the outflow of excitatory amino acids and against an acute motor disturbance, i.e.contralateral turning to the ischemic side in the first hours after ischemia in awake rats. Hours later, a definite neurological deficit and necrotic neuronal damage can be evaluated. DISCUSSION: Our results suggest that A(2A) antagonism may be protective from the earliest up to several hours after the ischemic event.

Acetylcholine release from rat cortical slices during postnatal development and aging.

Acetylcholine release from cortical slices superfused with choline-enriched Krebs solution containing physostigmine was investigated at birth, at 7, 20 and 30 days, and at 3 and 24 months of age, in order to assess age influence on the functional efficiency of the cortical cholinergic network. The slices were electrically stimulated at frequencies from 1 to 10 Hz for 5 min periods, preceded and followed by rest periods. The superfusate was collected every 5 min and acetylcholine content quantified by bioassay. In the newborn and 7 day-old pups acetylcholine release was approximately 50% lower than that of the 3 month-old rats at all frequencies tested. The highest release was elicited in the 30 day-old rats. Beginning with this age the evoked ACh release underwent a decline which in the 24 month-old rats brought it back to the same level as in the newborn ones. The blockade of the muscarinic autoreceptors by atropine 1.5 X 10(-8) M caused an increase in acetylcholine release at 20 day, 3 and 24 months of age but not in the newborn and 7 day-old pups. Adenosine 3 X 10(-5) M decreased acetylcholine output in newborn and adult but had no effect in the senescent rats.

Phosphatidylserine increases acetylcholine release from cortical slices in aged rats.

Acetylcholine release was investigated in cortical slices superfused with choline-enriched Krebs solution containing physostigmine. Slices were prepared from 3 and 24 month old rats treated with either Tris buffer or sonicated suspensions of phosphatidylserine and phosphatidylcholine in Tris buffer. Slices were electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. ACh content of the superfusate was quantified by bioassay. In the 24 month old rats treated with Tris buffer, acetylcholine release, at all frequencies tested, was approximately 50% lower than that in the 3 month old rats. On the contrary, no significant decrease in ACh release was found in the 24 month old rats treated for 30 days with phosphatidylserine (15 mg/kg IP). The same treatment did not increase acetylcholine release in 3 month old rats. Acetylcholine release in 24 month old rats receiving a single administration of phosphatidylserine (15 mg/kg IP) or phosphatidylcholine (15 mg/kg IP) for 30 days was as low as in the 24 month old rats receiving the Tris buffer only. It is proposed that the chronic phosphatidylserine treatment may reduce the age-induced decrease in acetylcholine release by acting on the stimulus-secretion coupling mechanism.

In vivo amino acid release from the striatum of aging rats: adenosine modulation.

The release of glutamate, aspartate, GABA, and taurine from the striatum of young (3 months), mature (12 months), and old (22 months), freely moving male rats was investigated by using a microdialysis fiber inserted transversally in the striatum. In old rats basal extracellular glutamate and aspartate levels were decreased vs. young rats (-38 and -49%, respectively). GABA and taurine levels were unmodified by age. In the presence of the adenosine receptor antagonist 8-phenyltheophilline (8-pT) at the concentration of 50 microM, both K(+)-evoked releases of glutamate and aspartate were more than doubled in young, but not in mature and old rats. 8-pT at the concentration of 500 microM significantly decreased glutamate basal levels and K(+)-evoked aspartate release in old rats only. GABA and taurine releases were not affected by 8-pT at either dose. Our findings indicate a modified adenosine modulation on glutamate and aspartate release in aged rats, that could result from a change in the balance between A1 and A2a adenosine receptor density or an alteration of A1 and A2a receptor-effector coupling.

Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electrically-stimulated brain slices.

The effect of adenosine on release of acetylcholine (ACh) was investigated in slices of rat cortex perfused with Krebs solution, at rest and during electrical stimulation at frequencies between 0.2 and 20 Hz. Electrical stimulation brought about a linear increase in release of ACh. Adenosine, in concentrations ranging from 1 to 100 microM, reduced in a dose-dependent manner the release of ACh and was more active on the stimulated than on the resting release. However, the fractional reduction by adenosine of stimulated release of ACh did not vary with increasing stimulation rate. Adenosine triphosphate was less active than adenosine in reducing release of ACh. The inhibitory effect of adenosine was antagonized by aminophylline (0.5 mM) and did not occur when the stimulated release of ACh was enhanced by blocking muscarinic autoreceptors with atropine (15 nM). Aminophylline (0.1 and 0.5 mM) itself exerted a biphasic effect on release of ACh, increasing it at rest and during stimulation at low frequencies, and decreasing it at higher stimulation rates. The manipulation of endogenous adenosine concentrations by adding adenosine deaminase or diphyridamole, an inhibitor of adenosine uptake, had little effect on release of ACh. Dipyridamole, (4 microM), only significantly decreased release of ACh at the 20 Hz stimulation rate.

Cholinergic and noradrenergic denervations decrease labelled purine release from electrically stimulated rat cortical slices.

The origin of cortical purine release was investigated by measuring [3H]purine release from electrically stimulated cortical slices of rats after neurotoxic lesions of cholinergic, noradrenergic and serotoninergic pathways innervating the cortex. Purines were labelled by incubating the cortical slices with [3H]adenine. The 3H efflux at rest and during stimulation, analysed by high performance liquid chromatography, consisted of adenosine, inosine, hypoxanthine and a small amount of nucleotides. Twenty days after unilateral or bilateral lesion of the nucleus basalis a marked decrease in choline acetyltransferase activity was associated with a decrease in [3H]purine release. A linear relationship was found between the decrease in choline acetyltransferase activity and [3H]purine release. A partial recovery in both choline acetyltransferase activity and [3H]purine release was observed eight months after the lesion. Twenty days after intra-cerebroventricular injection of 6-hydroxydopamine a 59% decrease in cortical noradrenaline content was associated with a 44% decrease in [3H]purine release. Conversely, no change in [3H]purine release was found in rats in which a 89% decrease in cortical serotonin content was induced by intra-cerebroventricular injection of 5,7-dihydroxytryptamine. The decrease in [3H]purine release after the lesion of the cholinergic and noradrenergic pathways may depend on metabolic changes, a loss of a stimulating influence of acetylcholine and noradrenaline or may indicate a release of [3H]purine from cholinergic and noradrenergic fibres.

Interactions among adenosine deaminase, adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

Biphasic effect of methylxanthines on acetylcholine release from electrically-stimulated brain slices.

The effect of caffeine and aminophylline on the release of acetylcholine (ACh) was investigated in slices of rat cortex perfused with Krebs solution at rest and during electrical stimulation at frequencies of 0.2, 1 and 5 Hz. Both methylxanthines added to the superfusing Krebs solution at a concentration of 50 microM enhanced ACh release. Conversely, at a concentration of 0.5 mM both caffeine and aminophylline decreased ACh release. Neither caffeine nor aminophylline affected the unstimulated ACh release. Dipyridamole 10 microM potentiated the inhibitory effect of adenosine 30 microM on ACh release and antagonized both the stimulatory and inhibitory effects of caffeine on ACh release. The inhibitory effect of caffeine was antagonized by cyclohexyladenosine (CHA) 0.5 microM and N-ethylcarboxamideadenosine (NECA) 5 microM. The results indicate that methylxanthines exert both stimulatory and inhibitory effects on ACh release by acting on adenosine receptors. Methylxanthines may enhance the electrically-evoked ACh release by antagonizing the effect of endogenous adenosine on inhibitory adenosine receptors. On the other hand the mechanism through which methylxanthines decrease ACh release remains obscure.

Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices.

1. 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. 2. 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. 3. 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 microM) 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. 4. 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 microM. 5. 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.

Chronic caffeine treatment reduces caffeine but not adenosine effects on cortical acetylcholine release.

The effects of both adenosine and caffeine on the release of acetylcholine (ACh) were investigated in slices of cerebral cortex taken from rats pretreated for 30 days with caffeine (100 mg kg-1 daily, dissolved in their drinking water) at rest and during electrical stimulation at frequencies of 0.2, 1 and 5 Hz. The effect of this treatment on adenosine binding sites was also investigated in cortical membranes using N-cyclohexyl-[3H]-adenosine ([3H]-CHA) as a ligand. The chronic caffeine treatment did not change animal growth patterns. Spontaneous exploratory activity appeared to be increased at the 3rd day but was unchanged at the 30th day when compared with controls. Caffeine-treatment increased the number of high affinity binding sites for [3H]-CHA by 64% over the control values. Low affinity binding site density and affinity constants were unaffected. Adenosine 30 microM added to the superfusion fluid decreased electrically stimulated ACh release both in rats drinking tap water and rats drinking caffeine. In rats drinking tap water, caffeine added to the superfusion fluid at a concentration of 50 microM enhanced ACh release, while at 0.5 mM it decreased ACh output from the slices. Both effects were abolished by pretreatment with caffeine in vivo. The results indicate that prolonged consumption of high doses of caffeine causes changes in the responsiveness of cholinergic neurones to caffeine. The change is not shared by adenosine, through whose recognition sites caffeine is believed to act. It is therefore possible that the adaptive changes following repeated caffeine administration involve either only the coupler-transducer mechanism activated by the antagonist, or effects unrelated to receptors.

Changes in synaptosomal high affinity choline uptake following electrical stimulation of guinea-pig cortical slices: effect of atropine and physostigmine.

1 Superfused guinea-pig cortical slices were electrically stimulated at different frequencies and the changes in acetylcholine (ACh) content measured. Synaptosomes were prepared at the end of the stimulation period and high affinity choline uptake (HACU) rate was measured. 2 The effect of increasing KC1 concentrations was compared on ACh content of the slices and on synaptosomal HACU. 3 Electrical stimulation (2, 5, 10, 20 Hz) elicited a frequency-dependent linear increase in synaptosomal HACU rate and a decrease in ACh content of the slices. 4 The addition of atropine (1.5 x 10(-8) M) to the slices enhanced and that of physostigmine (3 x 10(-5) M) reduced the frequency-dependent increase in HACU rate. Atropine (1.5 x 10(-6) M) not only antagonized the effect of physostigmine, but the HACU rate measured after treatment with both drugs was larger than that found after atropine alone. 5 These results indicate that in the cortical cholinergic nerve endings, depolarization caused by electrical stimulation is coupled with an increase in choline transport which can be modulated by the addition of atropine or physostigmine. Furthermore, within given experimental conditions a linear relationship exists between the reciprocal of ACh content in the slices and synaptosomal HACU.

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

1. In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia-like conditions. 2. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoade nos ine ), 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. 3. 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. 4. A prolonged application of either adenosine (100 micronM) 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). 5. SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia-induced depression. 6. 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.

Purinergic modulation of cortical acetylcholine release is decreased in aging rats.

The effect of adenosine, N-ethylcarboxamide adenosine (NECA), and caffeine on acetylcholine (ACh) release was investigated in cortical slices prepared from 3 and 22-24-month-old rats. The slices were perfused with Krebs solution and electrically stimulated at 0.2, 1, and 5 Hz stimulation frequency. In old rats, ACh released by stimulation at 1 and 5 Hz was about half as large as in adult rats. In 22-24-month-old rats, the potency of adenosine was strongly reduced, and a similar significant inhibition of ACh release was obtained with concentrations of 1 microM adenosine in adult and 300 microM in old rats. Conversely, NECA, which has no effect on ACh release in adult rats, brought about a 40% decrease in old rats. Caffeine at 50 microM concentration enhanced, and at 500 microM inhibited, the evoked ACh release in adult rats, but was inactive in old rats. The possibility is envisaged that aging may modify purinergic modulation of ACh release by inducing conformational changes in purinergic receptors or changing adenosine metabolism.

Adenosine and memory storage: effect of A(1) and A(2) receptor antagonists.

RATIONALE: Caffeine is a non-selective A(1)/A(2 )adenosine receptor antagonist which is known to improve cognitive performance in humans. This effect of caffeine has been attributed to its antagonism of adenosine receptors. OBJECTIVE: The present study was devised to identify the role of A(1 )and A(2A) adenosine receptors in the facilitation of memory consolidation in mice performing a passive avoidance task. METHODS: Adult albino Swiss male mice were used. The mice were trained in a step-through inhibitory avoidance task in which they were punished by a foot-shock (0.4 mA, 5 Hz, for 3 s) delivered through the grid floor. Caffeine (0.1, 0.3, 1.0 and 3.0 mg/kg), SCH 58261 (0.1, 0.3, 1.0 and 3.0 mg/kg) and DPCPX (0.1, 0.3, 1.0 and 3.0 mg/kg) were injected IP immediately or 180 min after training. The retention test was performed 24 h after training. RESULTS: Caffeine and the selective A(2A) adenosine receptor antagonist SCH 58261 facilitated retention when administered immediately after training, but not when administered 180 min later. The dose response was a bell-shaped curve. Conversely, post-training administration of the selective A(1) adenosine receptor antagonist DPCPX did not affect retention. Caffeine and SCH 58261 had no effect in mice not given the foot-shock on the training trial, a finding indicating that the drug's effect on retention was specific. CONCLUSIONS: These results suggest that A(2A) but not A(1) adenosine receptors are involved in memory retention and consolidation.

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.

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 (E.C.) at the end of the ischemic period. After 10 min of reperfusion ATP, AMP and E.C. 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 E.C. at the end of stimulation. After 10 min of rest conditions the adenosine content and E.C. 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.

Adenosine extracellular brain concentrations and role of A2A receptors in ischemia.

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 microM range under ischemia. In vitro estimation of adenosine show that it can reach 30 microM 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.