Programmable hyperbolic polaritons in van der Waals semiconductors.

Collective electronic modes or lattice vibrations usually prohibit propagation of electromagnetic radiation through the bulk of common materials over a frequency range associated with these oscillations. However, this textbook tenet does not necessarily apply to layered crystals. Highly anisotropic materials often display nonintuitive optical properties and can permit propagation of subdiffractional waveguide modes, with hyperbolic dispersion, throughout their bulk. Here, we report on the observation of optically induced electronic hyperbolicity in the layered transition metal dichalcogenide tungsten diselenide (WSe2). We used photoexcitation to inject electron-hole pairs in WSe2 and then visualized, by transient nanoimaging, the hyperbolic rays that traveled along conical trajectories inside of the crystal. We establish here the signatures of programmable hyperbolic electrodynamics and assess the role of quantum transitions of excitons within the Rydberg series in the observed polaritonic response.

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.

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 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.

Changes in hippocampal adenosine efflux, ATP levels, and synaptic transmission induced by increased temperature.

Previous studies have demonstrated that when the temperature of hippocampal brain slices is increased, there is a corresponding depression of synaptic potentials mediated by an increased activation of presynaptic adenosine A(1) receptors. The present experiments demonstrate that when the temperature of hippocampal slices is raised from 32.5 degrees C to either 38.5 degrees C or 40.0 degrees C there is a marked, temperature-dependent increase in the efflux of endogenous adenosine and a corresponding decrease in excitatory synaptic responses. The increase in efflux is rapidly reversible on lowering the slice temperature and the temperature-induced efflux is repeatable. Control experiments suggest that this increased efflux of adenosine is not the result of hypoxia or ischemia secondary to a temperature-induced increase in the metabolic rate of the slice. The increase in adenosine efflux was not accompanied by any significant change in the ATP levels in the brain slice, whereas a hypoxic stimulus sufficient to produce a comparable depression of excitatory transmission produced an approximately 75% decrease in ATP levels. These experiments indicate that changes in brain slice temperature can alter purine metabolism in such a way as to increase the adenosine concentration in the extracellular space, as well as adenosine efflux from hippocampal slices, in the absence of significant changes in ATP levels.

CHS 828, a novel pyridyl cyanoguanidine with potent antitumor activity in vitro and in vivo.

A new class of recently discovered antineoplastic agents, the pyridyl cyanoguanidines, exert a potent antitumor activity in rodents after oral administration. Optimization in vitro and in vivo has resulted in the selection of the lead candidate CHS 828 (N-(6-chlorophenoxyhexyl)-N'cyano-N"-4-pyridylguanidine). CHS 828 was found to exert potent cytotoxic effects in human breast and lung cancer cell lines, with lesser effects on normal fibroblasts and endothelial cells. In a study using a panel of cell lines with different resistance patterns, the effects of CHS 828 showed a low correlation with the activity patterns of known anticancer agents, and no sensitivity to known mechanisms of multidrug resistance was observed. In nude mice bearing human tumor xenografts, CHS 828, at doses from 20 to 50 mg/kg/day p.o., inhibited the growth of MCF-7 breast cancer tumors and caused regression of NYH small cell lung cancer tumors. Oral administration of CHS 828 once weekly improved efficacy without increasing toxicity. CHS 828 was found to compare favorably with established chemotherapeutic agents such as cyclophosphamide, etoposide, methotrexate, and paclitaxel. In mice with NYH tumors, long-term survival (>6 months) was observed after treatment with CHS 828 was stopped. In conclusion, CHS 828 is an effective new antitumor agent, with a potentially new mechanism of action. CHS 828 is presently being tested in Phase I clinical trials in collaboration with the European Organization for Research and Treatment of Cancer.

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.

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.

Down-regulation of laminin-binding integrins by 1 alpha,25-dihydroxyvitamin D3 in human melanoma cells in vitro.

In the present investigation the effect of 1 alpha,25(OH)2D3 on the expression of the integrin laminin receptor on the melanoma cell line SK-MEL-28 has been examined. The SK-MEL-28 cells were shown to contain high-affinity receptors for 1 alpha,25(OH)2D3 and cell proliferation was found to be inhibited in a dose-dependent manner in response to the hormone. Using monoclonal antibodies against the alpha 6-sub-unit of the integrin laminin receptor, immunocytochemistry demonstrated that exposure of cells to 1 alpha,25(OH)2D3 for 5 days caused a reduced staining intensity. This observation was further confirmed by dot blot analysis, where a dose-dependent decline of alpha 6 expression was obtained after treatment of the cells with 1 alpha,25(OH)2D3 for 6 days. FACS-analysis was performed in order to quantify this decline, and it was found that the level of alpha 6-subunits on the cell surface was reduced by more than 40%. Additional investigations including Northern blot analyses of poly(A)+RNA extracts also showed a dose-dependent reduction of alpha 6 mRNA. Interestingly, the decrease of alpha 6 expression on the surface of SK-MEL-28 melanoma cells was accompanied by a reduced ability of the cells to adhere to an artificial basement membrane. In conclusion, the present investigation shows that besides having an antiproliferative effect on the SK-MEL-28 melanoma cells, 1 alpha,25(OH)2D3 is also able to inhibit the surface expression of the alpha 6-subunit of the integrin laminin receptor. Moreover, the results strongly indicate that 1 alpha,25(OH)2D3 exerts its regulatory effect on the alpha 6-subunit at the transcriptional level rather than at the protein level.

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.

The contribution of different types of calcium channels to electrically-evoked adenosine release from rat hippocampal slices.

The role of L-, N- and P-type voltage-dependent calcium channels (VDCCs) in the release of adenosine from rat hippocampal slices was investigated by evaluating the effect of the L-channel activator 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyr idi ne carboxylic acid methyl ester (Bay K 8644) and of three calcium channel antagonists: the L-channel antagonist nifedipine, the N-channel blocker omega-conotoxin GVIA (omega-CgTx) and the P-channel blocker omega-agatoxin IVA (omega-Aga-IVA). Adenosine and inosine release, evoked by 5 min electrical stimulation at 10 Hz of hippocampal slices, was assayed by HPLC with ultraviolet absorbance or fluorimetric detection. Nifedipine (100 nM) did not affect adenosine and inosine release evoked by electrical stimulation. Bay K 8644 (100 nM) brought about a statistically significant increase in adenosine evoked release (70%). At a higher concentration (1 microM) Bay K 8644 had no significant effect either on adenosine or inosine release evoked by electrical stimulation. The increase in adenosine release elicited by 100 nM Bay K 8644 was abolished by nifedipine (100 nM). Both omega-CgTx (10 microM) and omega-Aga-IVA (200 nM) caused a statistically significant reduction (77-78%) in evoked release of adenosine. When the previously demonstrated glutamate-dependent component of the release of adenosine was suppressed in the presence of the NMDA and non-NMDA receptor antagonists, D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7. 100 microM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 microM), the remaining release of adenosine was again significantly reduced by omega-CgTx (10 microM) (60%) and omega-Aga-IVA (200 nM) (73%). These data suggest that, while L-type VDCCs are involved in the regulation of the evoked release of adenosine only when activated by Bay K 8644, both P- and N-channels play a direct role in the calcium entry involved in the coupling process between electrical stimulation and adenosine release.

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.

Adenosine by activating A1 receptors prevents GABAA-mediated actions during hypoxia in the rat hippocampus.

The relative contribution of adenosine and gamma-aminobutyric acid (GABA) for the hypoxia-induced depression of field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices, was investigated. It is concluded that both adenosine and GABA, by activating A1 and GABAA receptors, could be responsible for the inhibition of synaptic transmission during hypoxia, but the action of endogenous GABA becomes evident only when the adenosine A1 receptor action is precluded.

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.

Purinoceptors in the Central Nervous System.

New exciting developments on the occurrence and functional role of purinoceptors in mammalian brain were presented at the session "Purinoceptors in the central nervous system" chaired by Flaminio Cattabeni and Tom Dunwiddie at the Purines '96 international conference. The focus of the session were topics of recent interest, including the sources and mechanisms involved in ATP and adenosine release during physiological neurotransmission in hippocampus, the brain expression of the recently cloned P2 receptors, and the role of the various adenosine receptor subtypes in brain protection from neurodegeneration associated with trauma-, ischemia-and excessive excitatory amino acid neurotransmission. New important insights into the mechanisms responsible for the formation and release of adenosine into the extracellular space were provided by data obtained by Dunwiddie and coworkers in hippocampal pyramidal neurons. These data may have functional implications for the role of purines in modulation of synaptic plasticity and long-term potentiation in this brain area, and hence in cognitive functions. Buell provided an updated overview on the cloning, molecular characteristics and brain expression of various ligand-gated P2X purinoceptors; although the functional role of these receptors in mammalian brain still awaits elucidation, their widespread distribution in the nervous system strongly suggests that ATP-mediated events are more prevalent and important in brain than expected. Pedata presented data on the functional interrelationships between adenosine and glutamate in the brain, and also provided evidence for alterations of the reciprocal regulation between these two systems in aged brain, which may have important implications for both ischemia-and trauma-associated neurodegenerative events and senescence-associated cognitive impairment. Finally, von Lubitz provided novel data on the molecular mechanisms likely to be at the basis of the brain protective effects associated with the chronic stimulation of the adenosine A3 receptor, further confirming that this receptor represents a crucial target for the development of new antiischemic and antineurodegenerative therapeutic agents.

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.

Pharmacokinetic studies of vitamin D analogues: relationship to vitamin D binding protein (DBP).

Vitamin D(3), 25-hydroxyvitamin D(3) (25OHD(3)) and 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) bind to the vitamin D binding protein (DBP) in the serum. During the development of synthetic vitamin D analogues, it has been shown that the majority of analogues bind to DBP with a low affinity. This modifies their biological activitiesin vitro compared to 1α,25(OH)(2)D(3), since binding to DBP decreases the cellular uptake and access to the vitamin D receptor. It is therefore important to elucidate the possible role played by the binding or lack of binding to DBPin vivo. We have investigated the relationship between the binding affinity for human DBP and the serum level and serum half-life (t(1/2)) in rats of a series of new vitamin D analogues. The binding affinity for DBP was determined by displacement of(3)H-1,25(OH)(2)D(3) from DBP attached to Affi-Gel 10. The serum levels in rats following a single intravenous dose were assessed by HPLC and the serum half-life was determined for each analogue. In the group of vitamin D analogues which showed a low or no affinity for DBP, we have identified compounds with a short t(1/2) and compounds with a long t(1/2), all characterized by low initial serum levels. Compounds with a long t(1/2) were also found in the group with a high affinity for DBP, and they were easily identifiable by their high initial serum level. These results showed that the initial serum level of vitamin D analogues correlated with the affinity for DBP, but that there seemed to be no correlation with the metabolic rate as reflected by measurement of the serum half-life of the analogues.

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.

Effect of idebenone on adenosine outflow and adenine nucleotide level in hippocampal slices under ischemia-like conditions.

The effect of idebenone on the changes in adenosine and nucleotide metabolism occurring in hippocampal slices after ischemia-like conditions (superfusion with glucose-free Krebs solution gassed with 95% N2-5% CO2) and during reperfusion with normal Krebs solution was investigated by measuring adenosine and inosine outflow, and adenosine and adenine nucleotide levels by HPLC. Five minutes of ischemia-like conditions brought about an 8- and 4-fold increase in adenosine and inosine outflow 10 min after reperfusion and a 75% increase in the tissue level of adenosine, a 40% decrease in ATP, and a 50% increase in AMP at the end of the ischemic period. Ten minutes after reperfusion, ATP and AMP returned to control values. Idebenone (25-100 microM) brought about a concentration-dependent increase in adenosine and inosine outflow evoked by ischemia-like conditions. Idebenone (50 microM) also increased the adenosine content in hippocampal slices after both ischemia (+150%) and reperfusion (+320%). An 82% increase in ADP, 174% in AMP, and 56% in the total sum of nucleotides, 10 min after reperfusion were found in idebenone treated slices. These results suggest that idebenone enhances adenosine formation after ischemia-like conditions from sources other than AMP, and improves phosphorylating activity during reperfusion. Idebenone, by increasing adenosine and total nucleotide levels, may protect brain tissue from ischemic damage.

Investigations into the adenosine outflow from hippocampal slices evoked by ischemia-like conditions.

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 A1 receptor antagonist 8-phenyltheophylline 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-phosphonoheptanoic 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 alpha,beta-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 Ca(2+)-independent adenosine and inosine outflow, through a mechanism that partly depends on propagated nervous activity but does not involve excitatory amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)