Inosine

Objectives: Adenosine is an endogenous nucleoside that signals through G-protein coupled receptors. Extracellular adenosine is required for receptor activation and two pathways nave been identified for formation and cellular release of adenosine. The CLASSICAL pathway relies on intracellular formation of adenosine from adenine nucleotides and cellular efflux of adenosine via equilibrative nucleoside transporters (ENTs). The ALTERNATE pathway involves cellular release of adenine nucleotides, hydrolysis via ecto-5'-nucleotidases and extracellular formation of adenosine.

Methods: A rat model of cerebral ischemia and primary cultures of rat forebrain astrocytes and neurons were used.

Results: Using a rat model of cerebral ischemia, the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) significantly increased post-ischemic forebrain adenosine levels and significantly decreased hippocampal neuron injury relative to saline-treatment. NBMPR-induced increases in adenosine receptor activation were not detected, suggesting that altering the intracellular:extracellular distribution of adenosine can affect ischemic outcome.

Using primary cultures of rat forebrain astrocytes and neurons, adenosine release was evoked by ischemic-like conditions. Dipyridamole, an inhibitor of ENTs, was more effective at inhibiting adenosine release from neurons than from astrocytes. In contrast, α,β-methylene ADP, an inhibitor of ecto-5'-nucleotidase, was effective at inhibiting adenosine release from astrocytes, but not from neurons. Thus, during ischemic-like conditions, neurons released adenosine via the CLASSICAL pathway, while astrocytes released adenosine via the ALTERNATE pathway.

Discussion: These cell type differences in pathways for adenosine formation during ischemia may allow transport inhibitors to block simultaneously adenosine release from neurons and adenosine uptake into astrocytes. In principle, this could improve neuronal ATP levels without decreasing adenosine receptor activation. [Neurol Res 2005; 27: 153-160]

Keywords: Adenosine; inosine; nucleoside transport; 5'-nucleotidase

Adenosine is an endogenous nucleoside with receptor signaling functions. In the brain, adenosine acts as a neuromodulator. Although adenosine levels increase with synaptic activity, it is not considered a neurotransmitter, as evidence to support synaptic release of adenosine is lacking1. Several CNS effects including decreased alertness, decreased inflammation, and neuroprotection in conditions such as stroke or seizure are described for adenosine2. These effects of adenosine are mediated by adenosine receptors, of which the four subtypes A^sub 1^, A^sub 2A^, A^sub 2B^ and A^sub 3^ have been characterized and cloned.

In brief, adenosine is formed intracellularly by dephosphorylation of AMP, by the enzyme cytosolic 5'nucleotidase. Adenosine can also be formed from S-adenosylhomocysteine (SAH), by the enzyme SAH hydrolase although this pathway appears to be less important3. Once formed, adenosine can be metabolized to inosine, by the enzyme adenosine deaminase (ADA) or exported from cells via nucleoside transporters. AMP, SAH and inosine are not metabolically inert and their levels are also subject to enzymatic control. In addition to intracellular formation of adenosine, extracellular formation has also been described. Many cells are capable of releasing adenine nucleotides, which can be metabolized extracellularly to adenosine by soluble4 or membrane bound ecto-nucleotidases5. Extracellular metabolism of adenosine to inosine has also been described6. Purine metabolism is illustrated in Figure 1.

As indicated above, adenosine formation can occur both intra- and extracellularly. As adenosine receptors are located on plasma membranes and are activated by extracellular adenosine, nucleoside transporters play an important role in regulating the levels of adenosine in the vicinity of adenosine receptors. Two families of nucleoside transporters have been described: equilibrative (facilitated diffusion)7 and concentrative (secondary active)8 transporters. Equilibrative nucleoside transporters (ENTs) transport adenosine, and other nucleosides, down its concentration gradient. Thus, ENTs can facilitate adenosine influx or efflux depending on the direction of its concentration gradient. Under conditions of intracellular adenosine formation, ENTs are predicted to mediate adenosine efflux, while under conditions of extracellular formation, ENTs are predicted to mediate adenosine influx. In contrast, concentrative nucleoside transporters (CNTs) are symporters that transport adenosine, or other nucleosides, together with sodium ions into cells. These transporters normally facilitate nucleoside influx, although efflux has been demonstrated under conditions of experimentally-induced reversal of the trans-membrane sodium gradient9.

Based on experimental observations in our laboratory and many others, there are clearly two distinct pathways for adenosine formation and release, and we have termed these the CLASSICAL and ALTERNATE pathways.

The CLASSICAL view of adenosine is that it is a 'retaliatory metabolite'10. It is produced from ATP during conditions that deplete ATP and it stimulates A^sub 1^ adenosine receptors to depress cellular activity11 and A2 receptors to increase blood flow12,13. Thus, adenosine is produced as a consequence of ATP depletion and its receptor-mediated effects facilitate recovery of ATP levels11. The following represents the CLASSICAL pathway of adenosine release, uptake and metabolism in brain: intracellular formation from ATP [arrow right] efflux from cells [arrow right] receptor activation [arrow right] uptake into cells [arrow right] intracellular metabolism (Figure 2).

This CLASSICAL view of adenosine as a 'retaliatory metabolite' fails to explain many features of adenosine signaling. Some examples include the following:

* Adenosine receptor activity can be measured in preparations thought to represent physiological conditions and not just during conditions of metabolic stress14.

* The adenosine receptor antagonist caffeine is widely consumed for its stimulatory properties, indicating that adenosine exerts a tonic, inhibitory tone15.

* Excitatory adenosine A^sub 2A^ receptors are found on neurons in striatum and superior colliculus and A^sub 2A^ receptor knock-out mice show reduced ischemic brain injury than controls, indicating that ischemiainduced adenosine formation has effects in addition to inhibition of neuronal activity, and promotion of cerebral blood flow16.

* Adenine nucleotides can be released from a variety of cell types and metabolized to adenosine and, thus, can provide an extracellular source of adenosine available to interact with adenosine receptors17.

These data indicate that while adenosine may play an important role in neuroprotection during ischemia or seizures, adenosine also has important functions during physiological conditions. Furthermore, it is clear that ATP has signaling functions in addition to its role as an energy molecule. ATP is a co-transmitter and undergoes synaptic release together with classical neurotransmitters, such as acetylcholine, norepinephrine, and serotonin18,19. In addition, ATP is released from non-neuronal cells. The mechanism(s) responsible for this ATP release are still under dispute as ATP binding cassette proteins, connexin hemichannels, volume-regulated anion channels and multi-drug resistance proteins are all processes that have been reported to mediate ATP release17,20-24. In addition to ATP, it appears that cAMP can be released from cells and multidrug resistance protein 4 has been identified as an efflux mechanism for this nucleotide25,26. Release of cAMP and ATP from astrocytes and release of ATP from neurons has been demonstrated27,28. Catalytic conversion of ATP or cAMP to adenosine can occur in the interstitium17,28,29. This documentation of adenine nucleotide release and ectonucleotidase activity has challenged the CLASSICAL pathway of adenosine release, uptake and metabolism, and has indicated the presence of an ALTERNATE pathway: release of cAMP or ATP [arrow right] metabolism to adenosine [arrow right] activation of adenosine receptors [arrow right] cellular uptake of adenosine [arrow right] intracellular metabolism (Figure 3). It has been suggested that this extracellular pathway, initiated by ATP release, is the more important pathway for adenosine formation evoked by physiological, as opposed to pathophysiological, conditions18.

As described, it may appear that with either the CLASSICAL or ALTERNATE pathways adenosine production simply follows a stoichiometric relationship with AMP levels. However, there are several factors that can affect adenosine formation. For example, the ALTERNATE pathway is dependent upon the presence and abundance of ecto-nucleotidases. Cell type differences and treatment-dependent up-regulation of ecto5'-nucleotidase have been described30. Furthermore, several reports have demonstrated that adenosine kinase activity is suppressed during hypoxic conditions31-34. Substrate inhibition of adenosine kinase by adenosine occurs at concentrations that are only moderately elevated above its K^sub m^ value35. This provides an amplification mechanism for conditions that evoke adenosine formation and release via the CLASSICAL pathway.

We have proposed the existence of yet another pathway for adenosine formation and release36. It appears that intracellular metabolism of adenosine can be regulated by signal transduction mechanisms leading to an intracellular source of adenosine that is not tightly coupled to the energy charge. Energy charge is defined as [ATP + ½ ADP]/[ATP + ADP + AMP] and is a measure of ATP utilization35. Protein kinase C activators appear to increase adenosine efflux from cells by inhibiting adenosine kinase36 or by activating nucleoside transporters37-39. This NOVEL pathway for adenosine production parallels the CLASSICAL pathway by placing adenosine formation within cells but at the same time challenges the CLASSICAL pathway by minimizing the importance of ATP depletion (Figure 4). This NOVEL pathway may explain the ability of various receptor agonists and signal transduction modulators to induce or enhance adenosine release from brain preparations40-43.

At present, the relative importance of CLASSICAL, ALTERNATE and NOVEL pathways as sources of adenosine for the receptor-mediated effects of adenosine is not known. The relative contributions of these pathways are likely to vary with different conditions that evoke adenosine release. Cell type differences add another layer of complexity to stimulus-evoked adenosine release.

In recent studies, we have investigated adenosine formation and release in vivo and in vitro during basal and ischemic, or ischemic-like conditions. Our expectation was that these conditions would evoke adenosine release via the CLASSICAL pathway, but our data indicated that even under ischemic conditions the ALTERNATE pathway can be an important source of adenosine.

In the first set of studies, we tested the hypothesis that inhibition of nucleoside transport can enhance ischemia-induced adenosine levels, potentiate adenosine receptor activation and provide neuroprotection. Propentofylline, an inhibitor of both nucleoside transporters and adenosine receptors had previously been shown to be neuroprotective44. We tested nitrobenzylthioinosine (nitrobenzylmercaptopurine ribonucleoside, NBMPR), a more potent and selective inhibitor of ENT1 than propentofylline, for effects on ischemic neuronal injury in rats. Dipyridamole and dilazep are also inhibitors of both ENT1 and ENT2 transporters; however, their use is complicated by their lack of specificity. Dipyridamole inhibits phosphodiesterases, dilazep inhibits calcium channels, and both can scavenge reactive oxygen species and inhibit glucose transport45-50. Because of these additional actions of dipyridamole and dilazep, we chose to use NBMPR, which is highly selective for inhibition of nucleoside transport. Rats were given intracerebroventricular injections of saline or 50 mol of the phosphorylated prodrug form of NBMPR (NBMPR-P). Previously, it has been demonstrated that phosphorylated drug is rapidly metabolized in vivo to the active form by alkaline phosphatases and 5'-nucleotidase51. Thirty minutes later rats were subjected to 10 minutes of carotid occlusion plus controlled hypotension (45 ± 5 mmHg). Rats were killed 0-30 minutes post-ischemia to measure adenosine levels or 7 or 28 days post-ischemia to assess neuronal injury. Using the method of microwave irradiation, which provides rapid inactivation of purine metabolizing enzymes52, adenosine was quantified in frontal cortex, striatum and hippocampus. In shamtreated rats adenosine levels ranged from 50 pmol/mg protein in frontal cortex to 650 pmol/mg protein in the CA1 region of the hippocampus. After 10 minutes of ischemia, adenosine levels increased 8-85-fold and reached 3.5-5.4 nmol/mg protein. By 15 minutes of reperfusion, adenosine levels had returned to the levels measured in sham-treated animals. Only at 7 minutes post-reperfusion, the earliest post-ischemic time point that could be tested reliably, was a significant effect of NBMPR to increase adenosine levels observed (p

* rapid redistribution from brain to other tissues;

* decreased 'free' NBMPR as a result of extensive binding to ENT1 transporters;

* metabolism of NBMPR to 6-thioinosine51.

Significant protection from neuronal death in the CA1 region of the hippocampus was observed 7 days post-ischemia, suggesting that NBMPR enhanced extracellular adenosine levels and adenosine receptor activation. However, sulfophenyltheophylline, an adenosine receptor antagonist, did not block the neuroprotective effects of NBMPR and expression of TNFa, which is negatively regulated by adenosine receptors, was not affected by NBMPR . Therefore, our data suggest that the neuroprotective effects of NBMPR result from inhibition of adenosine release via the CLASSICAL pathway and facilitation of recovery of cellular ATP levels. However, we cannot completely rule out the possibility that NBMPR treatment may have blocked adenosine uptake, perhaps following adenosine formation and release via the ALTERNATE pathway, leading to enhanced extracellular adenosine levels and receptor activation.

In separate experiments, NBMPR-P or saline was administered by intraperitoneal injection. Protection of CA1 pyramidal neurons in the hippocampus and elevated post-reperfusion adenosine levels were observed in NBMPR-P treated rats54. As NBMPR and NBMPR-P cross the blood-brain barrier weakly55, these data suggested that loss of adenosine from brain to blood following ischemia contributes to neuronal injury. This was also the conclusion of an earlier study, which reported that intraperitoneal injections of dipyridamole enhanced hypoxia-evoked brain adenosine levels56, despite the inability of dipyridamole to cross the blood-brain barrier57.

Identifying pathways of adenosine formation and release in vivo preparations is problematic because of the potential for non-uniform drug distribution and also because of the presence of multiple cell types, which may express different pathways. For this reason, we used primary cultures of rat forebrain astrocytes and neurons to explore further the pathways for adenosine formation and release. Initial experiments were performed to characterize nucleoside transporter expression in these cells. Cells were incubated with [3H]adenosine for 1 minute in the absence or presence of graded concentrations of NBMPR or dipyridamole. As NBMPR is a potent and selective inhibitor of ENT1, inhibition of [^sup 3^H]adenosine uptake by concentrations of NBMPR ≤100nM indicate the presence of this transporter. Dipyridamole is a non-selective inhibitor of ENT1 and ENT2, thus, the component of [^sup 3^H]adenosine uptake that is inhibited by dipyridamole, but not by NBMPR indicates the presence of ENT2 transporters. From our results (Figure 5) it appears that in neurons about 22% of [^sup 3^H]adenosine uptake was inhibited by NBMPR with an IC^sub 50^ value of 0.4 nM, whereas 85% of [^sup 3^H]adenosine uptake was inhibited by dipyridamole with an IC^sub 50^ value of 390 nM. Results with astrocytes were similar: NBMPR inhibited 33% of uptake with an IC^sub 50^ value of 0.2 nM and dipyridamole inhibited 80% of uptake with an IC^sub 50^ value of 460 nM (Figure 5). These data indicate the presence of both ENT1 and ENT2 in both cell types, with ENT2 contributing about 2-3-fold more adenosine uptake than ENT1. As CNTs are generally insensitive to NBMPR and dipyridamole35, under the conditions tested CNTs appear to account for a minor component of adenosine uptake.

Cells were incubated with [^sup 3^H]adenine to radiolabel intracellular ATP then treated with buffer, 10 mM 2deoxyglucose (2-DG) or 2 μg/ml oligomycin for 10 minutes at 37°C or with oxygen-glucose deprivation (OGD) for 1 hour at 37°C. The rationale for the different durations of treatments was that ischemic changes occur rapidly in vivo; however, chemical treatments are required to produce similarly rapid changes in vitro. Conversely, while OGD does not reproduce the rate of changes seen with ischemia it may more closely represent the types of changes that occur with ischemia. Tritiated purines released from the cells were separated by thin layer chromatography and quantified by scintillation spectroscopy. The separation method used produced clear bands for adenine nucleotides, adenosine, inosine, hypoxanthine, xanthine and adenine.

Our results indicated several differences between astrocytes and neurons:

* Under control conditions, astrocytes released quantitatively more adenosine and less inosine and hypoxanthine than neurons (Figure 6).

* 2-DG, oligomycin and OGD increased total purine release from neurons, but only 2-DG increased total purine release from astrocytes (Figure 6).

* Dipyridamole inhibited adenosine and inosine release from neurons but only inhibited inosine release from astrocytes treated with 2-DG (Figure 7).

* α,β-Methylene ADP, an inhibitor of ecto-5'nucleotidase, inhibited adenosine release from astrocytes but not neurons (Figure 7).

* The combination of EHNA + ITU, which inhibit adenosine deaminase and adenosine kinase, respectively, consistently enhanced adenosine release from neurons but only enhanced adenosine release from buffer- and OGD-treated astrocytes (Figure 7].

* AMP deaminase activity, but not adenosine deaminase or purine nucleoside phosphorylase activity, was three-fold greater in astrocytes than in neurons

From this reductionist approach we conclude that astrocytes and neurons respond differently when exposed to ischemia-like conditions. Whether these cell type differences faithfully reproduce differences that exist in vivo or arise as a product of cell culture conditions, remains to be verified. Our data indicate that cultured rat neurons and astrocytes produce adenosine via the CLASSICAL and ALTERNATE pathways, respectively, during ischemia-like conditions. A caveat to this conclusion is that intracellular ATP levels were significantly lower in neurons than in astrocytes treated with 2-DG, oligomycin or OGD58; thus, the CLASSICAL pathway may become more important in astrocytes exposed to more severe ischemia-like conditions. Nevertheless, our data suggest that during in vivo exposure to ATP-depleting conditions, neurons may release adenosine per se, while astrocytes may release adenine nucleotides. By maintaining low intracellular concentrations of adenosine, astrocytes may maintain an inwardly directed concentration gradient for adenosine, and take up both adenosine released by neurons and adenosine formed from adenine nucleotides released by astrocytes. An attractive strategy to enhance adenosine receptormediated ischemic neuroprotection would be to inhibit adenosine uptake by astrocytes. However, data accumulated to date indicate similar ENT subtype expression in rat astrocytes and neurons. Thus, it seems unlikely that an ENT inhibitor can be identified that will selectively block adenosine uptake into astrocytes without simultaneously blocking adenosine release from neurons.

Perhaps cell type selectivity of transport inhibitors is not necessary given cell type differences in adenosine release pathways. Our experimental treatment of ischemic rats with intracerebroventricular injections of NBMPR resulted in small, but significant increases in adenosine levels and reduced neuronal injury with no evidence of increased adenosine receptor activation. NBMPR may have reduced adenosine release from neurons, and facilitated ATP recovery in these cells. Simultaneously, NBMPR may have reduced adenosine uptake into astrocytes, compensating for the reduced adenosine release from neurons, and maintained extracellular adenosine levels and adenosine receptor activation. Further experiments are required to explore these issues.

ACKNOWLEDGEMENTS

Research in the authors' laboratory was supported by an operating grant to FEP from the Canadian Institutes of Health Research and a grant-in-aid from the Heart and Stroke Foundation of Canada. FEP would like to thank Dr. John Phillis for many stimulating and thought provoking discussions over the past 11 years.

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Fiona E. Parkinson*, Wei Xiong and Christina R. Zamzow

Department of Pharmacology and Therapeutics, University of Manitoba, A203-753 McDermot Avenue, Winnipeg MB Canada R3E 0T6

Correspondence and reprint requests to: F. E. Parkinson, Department of Pharmacology and Therapeutics, University of Manitoba, A203-753 McDermot Avenue, Winnipeg MB Canada R3E 0T6. [parkins@ms. umanitoba.ca] Accepted for publication December 2004.

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