Adenosine as a putative transmitter in the cerebral cortex. Studies with potentiators and antagonists.

Adenosine has a potent depressant action on cerebral cortical neurons, including identified corticospinal cells. Adenosine 2′-, 3′- and 5′-phosphates, including adenosine 5′-imidodiphosphate, had comparable depressant actions and 2-chloroadenosine was an even more potent depressant. Inhibitors of adenosine uptake, hexobendine and papaverine, potentiated the actions of adenosine and adenosine 5′-monophosphate. Theophylline and caffeine antagonized the depressant actions of adenosine and adenosine 5′-monophosphate. The results are compatible with the hypothesis that adenosine depresses neurons by activating an extracellular receptor and that this effect can be blocked by theophylline and caffeine.     

Inhibition of adenosine kinase and adenosine uptake in guinea-pig CNS tissue by halogenated tubercidin analogues

Two halogenated analogues of tubercidin (7-deazaadenosine) viz. 5-iodotubercidin and 5′-deoxy-5-iodotubercidin, previously were shown to be potent inhibitors of guinea-pig brain adenosine kinase activity and adenosine uptake in guinea-pig cerebral cortex slices. A further series of halogenated tubercidin analogues have been investigated; of the 9 compounds tested, 5′-deoxy-5-iodotubericidin was the most potent adenosine kinase inhibitor while 5-iodotubercidin was the most potent in inhibiting the facilitated uptake of adenosine. These compounds may be useful for elucidating the involvement of adenosine kinase in adenosine uptake, the maintenance of intracellular adenosine levels and in the neuromodulatory actions of adenosine in the CNS.     

Stimulation of guinea-pig brain adenylate cyclase by adenosine analogues with potent pharmacological activity in vivo

1-Methylisoguanosine, a marine natural product with potent muscle-relaxant and cardiovascular actions $\text{in vivo}$, interacts directly with adenosine receptors in guinea-pig brain slices to stimulate adenylate cyclase. These effects are blocked by theophylline. Comparison of the $\text{in vivo}$ pharmacological activity of a number of synthetic analogues of 1-methylisoguanosine with $\text{in vitro}$ adenylate cyclase-stimulating ability indicates that compounds lacking the latter biochemical activity have little muscle-relaxant activity. Adenosine is a potent stimulator of adenylate cyclase but is inactive $\text{in vivo}$ because of rapid removal from the extracellular environment by uptake and deamination. Unlike adenosine, 1-methylisoguanosine is resistant to deamination and is only poorly accumulated by brain tissue slices or homogenates containing synaptosomes. Since it is an extremely weak competitive inhibitor of adenosine deaminase and only a weak inhibitor of adenosine uptake, it is unlikely to act by potentiating the effects of adenosine itself at extracellular receptors. Thus, the pharmacological effects of 1-methylisoguanosine are apparently due to its actions as a long-lasting adenosine analogue.     

The effect of various centrally active drugs on adenosine uptake by the central nervous system

1. Adenosine and its analogs depress the firing of neurons in various brain regions. The primary mode of action of adenosine in exerting this effect appears to be the depression of transmitter release from presynaptic nerve terminals. This is a result of reduced calcium mobilization. 2. Adenosine uptake inhibitors and deaminase inhibitors depress the firing of central neurons. Adenosine antagonists, caffeine and theophylline, excite central neurons. Adenosine is therefore likely to be released in sufficient quantities to exert an ongoing modulation of synaptic transmission in the intact brain. 3. A number of groups of centrally active drugs inhibit adenosine uptake by brain synaptosomal preparations. These include the benzodiazepines, phenothiazines, various other sedatives and hypnotics, tricyclic antidepressants, non-steroidal anti-inflammatory analgesics, some steroids, diphenylhydantoin, puromycin and toyocamycin. 4. It is proposed that many agents with anxiolytic, sedative, analgesic or anti-convulsant actions may achieve their effects by inhibiting adenosine uptake and thus potentiating extracellular adenosine levels. 5. Morphine also elevates extracellular adenosine levels but achieves this by enhancing adenosine release.     

Studies on the mechanism of adenosine transport in Trypanosoma vivax

Adenosine uptake in the presence of some metabolic inhibitors and nucleosides has been studied. The uptake of adenosine was inhibited by oubain, phlorizin, iodoacetate and coformycin. Guanosine, on the other hand stimulated adenosine uptake to a considerable extent. Neither thymidine nor inosine caused significant change in adenosine uptake. Results of the time course assay and uptake studies at various concentrations of adenosine suggest that possibly more than one mode of uptake operates in the transport of adenosine in T. Vivax.     

The Characterization of [3H] Adenosine Uptake into Rat Cerebral Cortical Synaptosomes

: Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [³H]adenosine into synaptosomes, using an adenosine concentration of 10 μ.m , was linear for 30 min at 37°C. The uptake appeared to be mediated by kinetically saturable processes with apparent K_m's of 1 μam (“high-affinity A”) and 5 μm (“high-affinity B”), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4-dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high-affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the K_m values and kinetic properties of papaverine action in the synaptosomal high-affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.     

Interactions between adenosine and nifedipine in the rat cerebral cortex

Nifedipine inhibits the uptake of [³H]adenosine into rat cerebral cortical synaptosomes with an IC₅₀ value of 1.1 μM. When applied by iontophoresis onto rat cerebral cortical neurons it potentiated the depressant effects of adenosine on spontaneous firing. Some of the calcium-antagonist actions of nifedipine may be mediated by adenosine.     

Do benzodiazepines bind at adenosine uptake sites in CNS?

Benzodiazepines inhibit adenosine uptake into rat cerebral cortical synaptosomes and their potency as inhibitors of adenosine uptake is closely correlated with therapeutic efficacy. Agents which possess “benzodiazepine like” activities such as CL218,872, zopiclone and fominoben and which displace benzodiazepine binding to brain cell membranes, are also inhibitors of adenosine uptake into brain synaptosomes. The IC₅₀ values of all these compounds as inhibitors of adenosine uptake are in close agreement with the IC₅₀ values obtained for the displacement of benzodiazepine binding to the brain receptors. Adenosine uptake inhibitors (dipyridamole, hexobendine, papaverine, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine) which competitively inhibit adenosine uptake, presumably by blocking adenosine binding to its carrier-protein, are competitive inhibitors of diazepam binding to the brain membrane receptors. The finding of a pronounced correlation between inhibition of benzodiazepine binding and inhibition of adenosine uptake further supports the proposal that benzodiazepines may exert part of their pharmacological action through the inhibition of adenosine uptake.     

Detection and characterization of a nucleoside transport system in human fibroblast lysosomes

Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.     

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.     

Uptake to adenosine in a marine bacterium is not an active transport process

The uptake and intracellular interconversions of [8-¹⁴C]adenosine in a marine bacterium Vibrio harveyi were investigated under varying physiological conditions. The results indicated that in contrast with the current views, translocation of adenosine across the cytoplasmic membrane in Vibrio harveyi was not driven by respiration. The uptake of adenosine was dependent upon its intracellular utilization and was inhibited under conditions preventing its metabolic conversions.     

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-³H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [³H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions1. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.     

Activation of adenosine A2A receptors alters postsynaptic currents and depolarizes neurons of the supraoptic nucleus

Supraoptic nucleus (SON) neurons secrete oxytocin or vasopressin in response to various physiological stimuli (e.g., lactation/suckling, dehydration). Released near fenestrated capillaries of the neurohypophysis, these peptides enter the blood and travel to peripheral target organs. The pervasive neuromodulator adenosine, acting at A1 receptors, is an important inhibitory regulator of magnocellular neuroendocrine cell activity. Another high-affinity adenosine receptor exists in this system, however. We examined the physiological effects of adenosine A2A receptor activation and determined its localization among various cell types within the SON. In whole cell patch-clamp recordings from rat brain slices, application of the selective adenosine A2A receptor agonist CGS-21680 caused membrane depolarizations in SON neurons, often leading to increased firing activity. Membrane potential changes were persistent (>10 min) and could be blocked by the selective A2A receptor antagonist ZM-241385, or GDP-beta-S, the latter suggesting postsynaptic sites of action. However, +/--alpha-methyl-(4-carboxyphenyl)glycine or TTX also blocked CGS-21680 effects, indicating secondary actions on postsynaptic neurons. In voltage-clamp mode, application of CGS-21680 caused a slight increase (approximately 8%) in high-frequency clusters of excitatory postsynaptic currents. With the use of specific antibodies, adenosine A2A receptors were immunocytochemically localized to both the magnocellular neurons and astrocytes of the SON. Ecto-5'nucleotidase, an enzyme involved in the metabolism of ATP to adenosine, was also localized to astrocytes of the SON. These results demonstrate that adenosine acting at A2A receptors can enhance the excitability of SON neurons and modulate transmitter release from glutamatergic afferents projecting to the nucleus. We suggest that adenosine A2A receptors may function in neuroendocrine regulation through both direct neuronal mechanisms and via actions involving glia.     

Absorption and transition of [8-14C]adenosine and 2',3'-O-isopropylidene adenosine by Zaidel hepatomas and their homogenates

Transformation and uptake of [8-14C]-adenosine and its synthetic analog 2',3'-O-isopropylideneadenosine was studied in Zajdel hepatoma cells and their homogenates. Uptake and deamination of adenosine and 2',3'-O-isopropylideneadenosine by Zajdel hepatoma cells proceed differently. A small part of adenosine is phosphorylated and then it is included into biosynthesis of polymer substances. The uptake and deamination of 2',3'-O-isopropylideneadenosine by hepatoma cells occurs more intensively than uptake and deamination of adenosine. The formed 2',3'-O-isopropylideneadenosine is not splitted by purine nucleoside phosphorylase and is accumulated in cells in the incubation medium that lead to cell death. The same rate of 2',3'-O-isopropylideneadenosine deamination in cells and their homogenates indicates its high penetrability through plasma membranes. The high uptake of 2',3'-O-isopropylideneadenosine contrary to adenosine leads to deaggregation of cells and their destruction.     

Uptake of adenosine 5'-monophosphate by Escherichia coli.

Adenosine 5'-monophosphate is dephosphorylated before its uptake by cells of Escherichia coli. This is demonstrated by using a radioactive double-labeled culture, and with a 5'-nucleotidase-deficient, mutant strain. The adenosine formed is further phosphorolyzed to adenine as a prerequisite for its uptake and incorporation. The cellular localization of the enzymes involved in the catabolism of adenosine 5'-monophosphate is discussed.     

Adenosine and its receptor agonists regulate nitric oxide production and RAW 264.7 macrophages via both receptor binding and its downstream metabolites-inosine

Adenosine and its receptor agonists enhanced the production of nitric oxide (NO) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The enhancement of LPS-induced NO production by adenosine, as represented by the amount of its oxidation products, nitrite and nitrate, was inhibited by adenosine uptake inhibitors, such as dipyridamole, S(4-nitrobenzyl)-6-thioinosine (NBTI) and S(4-nitrobenzyl)-6-thioguanosine (NBTG). These indicate that the uptake of adenosine by macrophages is a prerequisite for the enhancement effects observed. A downstream metabolite of adenosine, inosine, also potentiated the LPS-induced NO production in a dose-dependent manner while its enhancement effect was also inhibited by dipyridamole. However, the degree of enhancement by inosine on NO production and nitric oxide synthase (NOS) activity in LPS-treated RAW 264.7 was weaker than the effect of adenosine. Furthermore, adenosine agonists also enhanced the NO production in a dose-dependent manner, but were not specific for A1, A2 nor A3 adenosine receptor. Adenosine uptake inhibitors had no effects on the enhancement activity of the adenosine receptor agonists. Thus, extracellular receptor/s may also play an important role in the observed enhancement responses. The results of this study indicate that the enhancement effects of adenosine on NO production in macrophages could be mediated by the extracellular adenosine receptors as well as the downstream metabolites of adenosine.     

Uptake and metabolism of nucleosides by embryos of the sea urchin Strongylocentrotus purpuratus

Uptake and metabolism of thymidine and adenosine have been studied in embryos of the sea urchin Strongylocentrotus purpuratus. Uptake of these nucleosides is found to be mutually competitive, with the Km for uptake of thymidine similar to its Ki for inhibition of adenosine uptake and vice versa. The metabolic studies show that adenosine is rapidly and completely phosphorylated upon entry, even at high exogenous concentrations which saturate the uptake mechanism. In contrast, at concentrations which saturate nucleoside uptake, thymidine becomes appreciably catabolized (up to 60%) to thymine and beta-amino-isobutyric acid in addition to its phosphorylation to thymine nucleotides. Negligible amounts of endogenous thymidine appear to remain unmetabolized following uptake in these embryos. The data provide strong in vivo evidence for separate metabolic pathways for thymidine and adenosine which have not previously been described in this organism. The observation of mutual competition during uptake, together with different routes of metabolism for these nucleosides, would suggest that the rate-limiting step in the uptake process is transport rather than metabolism. The specificity of this transport system for its nucleoside substrate has been examined in some detail in the present report. All naturally occurring nucleosides but only a limited number of nucleoside analogs are recognized by this membrane carrier. Neither purine nor pyrimidine bases are substrates for this transport system. Previous work by this laboratory has demonstrated the strict Na+-dependence of this carrier, its high affinity for nucleoside substrate, and its activation at fertilization. These observations and the substrate specificity studies of the present work together describe a unique transport system for nucleosides in sea urchin embryos which is quite different from those previously described in mammalian cells.     

P1 receptors and cytokine secretion

Evidence has accumulated in the last three decades to suggest tissue protection and regeneration by adenosine in multiple different cell types. Adenosine produced in hypoxic or inflamed environments reduces tissue injury and promotes repair by receptor-mediated mechanisms. Among other actions, regulation of cytokine production and secretion by immune cells, astrocytes and microglia (the brain immunocytes) has emerged as a main mechanism at the basis of adenosine effects in diseases characterized by a marked inflammatory component. Many recent studies have highlighted that signalling through A₁ and A_2A adenosine receptors can powerfully prevent the release of pro-inflammatory cytokines, thus inhibiting inflammation and reperfusion injury. However, the activation of adenosine receptors is not invariably protective of tissues, as signalling through the A_2B adenosine receptor has been linked to pro-inflammatory actions which are, at least in part, mediated by increased release of pro-inflammatory cytokines from epithelial cells, astrocytes and fibroblasts. Here, we discuss the multiple actions of P1 receptors on cytokine secretion, by analyzing, in particular, the role of the various adenosine receptor subtypes, the complex reciprocal interplay between the adenosine and the cytokine systems, their pathophysiological significance and the potential of adenosine receptor ligands as new anti-inflammatory agents.     

Adenosine: a novel ulcer modulator in stomachs.

Adenosine has been demonstrated for its actions on gastric secretion and stress-induced gastric ulceration in animals. We examined the pharmacological actions of adenosine on ethanol-evoked gastric lesions and gastric mucosal blood flow (GMBF) in rats, because both of them are closely related. Adenosine pretreatment, in dose of 7.5 mg/kg increased GMBF and protected against ethanol-evoked gastric lesion formation. However, this antiulcer action was followed by an aggravation of gastric lesions and reduction in GMBF. We further investigated whether these actions could act through the adenosine A1 or A2 receptors, therefore L-phenylisopropyladenosine (L-PIA) or N-ethylcarboxamidoadenosine (NECA), the adenosine A1 or A2 receptor agonists, respectively, were used. The drugs given in doses of 10 or 50 micrograms/kg for L-PIA and 1 or 5 micrograms/kg for NECA, dose-dependently inhibited GMBF and potentiated ethanol-induced gastric damage. When the two drugs were given together to animals, they did not further aggravate the severity of ulceration and reduction of GMBF. These findings indicate that the antiulcer action of adenosine is not mediated via the adenosine A1 and A2 receptors but if acts through different adenosine receptor subtypes. It was because the lesion worsening effects of adenosine at the second stage of the biphasic responses were similar to the actions of L-PIA and NECA, the ulcer potentiating effect is probably acting through adenosine A1 and A2 receptors in anaesthetised rats.     

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.