Control of basal extracellular adenosine concentration in rat cerebellum

To re-examine how the basal extracellular concentration of adenosine is regulated in acutely isolated cerebellar slices we have combined electrophysiological and microelectrode biosensor measurements. In almost all cases, synaptic transmission was tonically inhibited by adenosine acting via A₁ receptors. By contrast, in most slices, the biosensors did not measure an adenosine tone but did record a spatially non-uniform extracellular tone of the downstream metabolites (inosine and hypoxanthine). Most of the extracellular hypoxanthine arose from the metabolism of inosine by ecto-purine nucleoside phosphorylase (PNP). Adenosine kinase was the major determinant of adenosine levels, as its inhibition increased both adenosine concentration and A₁ receptor-mediated synaptic inhibition. Breakdown of adenosine by adenosine deaminase was the major source of the inosine/hypoxanthine tone. However adenosine deaminase played a minor role in determining the level of adenosine at synapses, suggesting a distal location. Blockade of adenosine transport (by NBTI/dipyridamole) had inconsistent effects on basal levels of adenosine and synaptic transmission. Unexpectedly, application of NBTI/dipyridamole prevented the efflux of adenosine resulting from block of adenosine kinase at only a subset of synapses. We conclude that there is spatial variation in the functional expression of NBTI/dipyridamole-sensitive transporters. The increased spatial and temporal resolution of the purine biosensor measurements has revealed the complexity of the control of adenosine and purine tone in the cerebellum.     

Striatal adenosine levels measured 'in vivo' by microdialysis in rats with unilateral dopamine denervation

Adenosine, inosine, hypoxanthine and guanosine were measured in perfusates collected from the right and left striatum of halothane-anaesthetized naive and 6-hydroxydopamine-denervated rats by using microdialysis. Samples were taken under basal and KCl-stimulated conditions. Dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were simultaneously measured. Purines and monoamines were assayed by HPLC-UV and HPLC-EC respectively. In naive rats, basal adenosine (1 microM), inosine (2 microM), hypoxanthine (4 microM), guanosine (0.5 microM) and dopamine (DA, 0.02 microM) levels (corrected by using the in vitro % recovery of each probe) were increased by the inclusion of 100 mM of KCl into the perfusion medium (2.5-, 3-, 3.5-, 1.5- and 30-fold, respectively, while DOPAC (6 microM), HVA (5 microM) and 5-HIAA (3 microM) levels were (72%, 68% and 45% respectively). DA was strongly diminished in the denervated striatum, but when detectable, could be increased (1- to 12-fold) by KCl stimulation. Adenosine, inosine, hypoxanthine and guanosine were, however, largely unaffected by the DA denervation and could be enhanced by KCl stimulation (2-, 3-, 4- and 1.5-fold respectively). The present results indicate that: (1) as in the case for DA, there is a pool of striatal adenosine which is releasable by high concentrations of extracellular K+; however, (2) this pool of adenosine seems not to be significantly modified by mesencephalic dopamine deafferentation.     

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.     

Trophic effects of purines in neurons and glial cells

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.     

Purine‐related metabolites and their converting enzymes are altered in frontal,parietal and temporal cortex at early stages of Alzheimer's disease pathology

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5′‐nucleotidase (5′‐NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer''s disease (AD) and in age‐matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5′‐NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β‐amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines.     

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.     

The availability of purines influences both the number of parasites and the splenocyte levels of purine-metabolizing enzymes in trypanosome-infected mice.

Growth on Trypanosoma musculi in the murine host was limited by the availability of host purines. A portion of the spleen cells of infected mice (many of them granulocytes) displayed high levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase, probably as a compensatory response to extracellular purine deficiency. Injections of adenosine or 2-deoxycoformycin stimulated significant increases in the growth of parasites. 2-Deoxycoformycin treatment also diminished parasite-induced splenomegaly. Treatment of mice with polyethylene glycol-modified ADA, a slowly catabolized form of ADA, had no effect on the course of T. musculi infection, indicating that the parasites can utilize purines other than adenosine. The apparent competition between parasites and host cells for available purines suggests that depletion of extracellular purines should be considered as an approach to treating extracellular trypanosome infections.     

Interstitial lactate,inosine and hypoxanthine in rat kidney during normothermic ischaemia and recirculation*

The aim of this study was to determine the potential value of extracellular fluid (ECF) lactate, inosine and hypoxanthine for monitoring the disturbance in energy metabolism associated with kidney ischaemia and recirculation, using intrarenal microdialysis as sampling technique. Normothermic ischaemia was produced in rats by clamping of the left renal pedicle. Microdialysis probes were implanted into the renal cortex and the medulla, respectively. Dialysates were collected in 10-minute fractions before, during 20 (Group A) or 40 minutes (Group B) ischaemia and 2 hours of recirculation. Samples were analysed by HPLC for lactate, inosine and hypoxanthine. Ischaemia caused a dramatic increase of extracellular fluid lactate, inosine and hypoxanthine in both groups, reflecting the disturbance of energy metabolism. The basal extracellular fluid level of lactate as well as that during ischaemia was markedly higher in the medulla compared to cortex, whereas the relative change in lactate concentration was similar (i. e. about 4-fold). In group A all three metabolites returned to the pre-ischaemic level within 20 minutes after reperfusion. However, while inosine and hypoxanthine returned promptly to base line in Group B, recovery of lactate varied dramatically between animals suggesting a persistent metabolic disturbance in some rats. Our results indicate that extracellular fluid lactate, inosine and hypoxanthine, measured by intrarenal microdialysis, may be useful for monitoring of the energy state of the kidney during normothermic ischaemia and that extracellular fluid lactate may be a sensitive indicator of post-ischaemic disturbances in energy metabolism.     

Interstitial lactate, inosine and hypoxanthine in rat kidney during normothermic ischaemia and recirculation.

The aim of this study was to determine the potential value of extracellular fluid (ECF) lactate, inosine and hypoxanthine for monitoring the disturbance in energy metabolism associated with kidney ischaemia and recirculation, using intrarenal microdialysis as sampling technique. Normothermic ischaemia was produced in rats by clamping of the left renal pedicle. Microdialysis probes were implanted into the renal cortex and the medulla, respectively. Dialysates were collected in 10-minute fractions before, during 20 (Group A) or 40 minutes (Group B) ischaemia and 2 hours of recirculation. Samples were analysed by HPLC for lactate, inosine and hypoxanthine. Ischaemia caused a dramatic increase of extracellular fluid lactate, inosine and hypoxanthine in both groups, reflecting the disturbance of energy metabolism. The basal extracellular fluid level of lactate as well as that during ischaemia was markedly higher in the medulla compared to cortex, whereas the relative change in lactate concentration was similar (i.e. about 4-fold). In group A all three metabolites returned to the pre-ischaemic level within 20 minutes after reperfusion. However, while inosine and hypoxanthine returned promptly to base line in Group B, recovery of lactate varied dramatically between animals suggesting a persistent metabolic disturbance in some rats. Our results indicate that extracellular fluid lactate, inosine and hypoxanthine, measured by intrarenal microdialysis, may be useful for monitoring of the energy state of the kidney during normothermic ischaemia and that extracellular fluid lactate may be a sensitive indicator of post-ischaemic disturbances in energy metabolism.     

Ergometer exercise in myoadenylate deaminase deficient patients

Summary Three patients with primary myoadenylate deaminase deficiency were subjected to exercise on a bicycle ergometer at 125 W for 30 minutes. Blood samples prior to, during, and at the end of exercise were analyzed for lactate, ammonia, and hypoxanthine. In addition, urinary hypoxanthine excretion was measured. In these patients the serum lactate level increased to concentrations between 7.9 and 9.0 mmol/1 at the end of exercise whereas the mean lactate level in nine control subjects at the end of exercise was 3.3 mmol/l (range 1.1–8.1 mmol/l). There was no difference to control subjects in the exercise-induced increase in plasma levels of ammonia and hypoxanthine or in the increase in urinary hypoxanthine excretion. The findings support the hypothesis of a reduced substrate supply to the citric acid cycle in myoadenylate deaminase deficiency. The normal formation of ammonia and hypoxanthine excludes a marked loss of adenine nucleotides in working muscles in these patients.Abbreviations ATP adenosine triphosphate - AMP adenosine monophosphate - IMP inosine monophosphate - MAD myoadenylate deaminase - NCP non-collagen protein Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

Interaction of adenosine and its phosphorylated derivatives with putative purinergic receptors in the gill vascular bed of rainbow trout

1. The haemodynamic responses of trout gill to pulses of adenosine and related nucleotides were recorded in isolated trout head preparations. 2. Pulses of adenosine and related nucleotides induced a vasoconstriction of arterial gill vessels. Theophylline antagonized the resonse to adenosine but had not influence on its metabolism. 3. Dipyridamole and two adenosine deaminase inhibitors [deoxycoformycin and erythro-9(2-hydroxy-3-nonyl) adenine] had no effect on either the haemodynamic response of adenosine or its deamination and its uptake by gill tissues. 4. The adenosine response was neither mediated by cholinergic nor adrenergic receptors. 5. These results suggest the existence of extracellular "purinergic receptors" in the gills of trout.     

Adenosine Deaminase and Thymocyte Maturation

The congenital absence of adenosine deaminase in humans results in severe combined immunodeficiency. To clarify the process whereby thymocytes are destroyed in the absence of adenosine deaminase activity, we induced a parallel condition in mice through the injection of an inhibitor of adenosine deaminase, deoxycoformycin. We have observed that deoxycoformycin, in addition to maintaining high levels of dATP in thymocytes, blocks the progression of thymocyte differentiation at two points. As a result of the first block, the cortex is depleted of immature cortical thymocytes while CD4+CD8+ thymocytes with functionally rearranged T-cell receptors survive. As a result of the second block, the CD4+CD8+ thymocytes are prevented from further differentiation to mature CD4+CD8- or CD4-CD8+ T lymphocytes and accumulate at the corticomedullar junction and in the medulla. These observations suggest that the maintenance of dNTP pools by adenosine deaminase is critical to at least two stages of thymocyte differentiation.     

Uptake and supply of purine compounds by the liver.

We have analyzed for purine compounds entering and leaving the liver in lightly anesthetized rabbits and rats and for the export of utilizable purine from liver perfused with oxypurine. The in vivo results indicate that roughly 80% of hypoxanthine, xanthine, and urate is removed in a single passage of blood through liver. Conversely, the adenosine concentration of hepatic venous blood is increased 10-fold over portal or arterial levels. When the liver is isolated and perfused with hypoxanthine there is significant release of adenosine, whether measured quantitatively by microbiological assay or qualitatively by analysis of the radioactive purines released from liver that has been prelabeled with [14C]hypoxanthine. These results provide direct evidence for the clearance of hydroxylated purines and the release of utilizable adenine derivatives by liver.     

Is adenosine deaminase involved in adenosine transport?

The hypothesis that adenosine metabolizing enzymes may have a key role in the transport of adenosine is discussed. The enhancement of adenosine transport by inhibitors of adenosine deaminase (the enzyme which deaminates adenosine to inosine) and the ecto-localization of adenosine deaminase suggest a contribution of the enzyme in taking up nucleosides. Two possible mechanisms are suggested: 1) transport and deamination of adenosine as a coupled process, or 2) uptake of inosine after cleavage of adenosine by ecto-adenosine deaminase. In both cases, the so-called adenosine deaminase binding protein which is a membrane protein could be the real nucleoside transporter. This behaviour of adenosine deaminase as an ectoenzyme anchored to a membrane protein remembers the behaviour of periplasmic binding proteins of bacteria. Thus, adenosine deaminase as well as, for instance, adenosine kinase would be a kind of 'periplasmic proteins' of eukaryotic cells. The function of adenosine deaminase and adenosine kinase would then be to take adenosine and give it to the true transporters.     

Purine metabolizing enzymes of Plasmodium lophurae and its host cell, the duckling (Anas domesticus) erythrocyte

Adenosine kinase, adenosine deaminase, hypoxanthine phosphoribosyltransferase, inosine-nucleoside phosphorylase, 5'-AMP deaminase and 5'-IMP nucleotidase were identified in cell-free extracts of duckling erythrocytes; no evidence for 5'-AMP nucleotidase and xanthine oxidase activity was found. The Km values for the duckling red cell enzymes were similar to those reported for human erythrocytes. Plasmodium lophurae extracts demonstrated similar enzyme activities except for 5'-AMP deaminase and 5'-IMP nucleotidase which were absent. It is proposed that during infection erythrocytic AMP is catabolized to IMP, inosine and hypoxanthine; the hypoxanthine is taken up by the plasmodium, utilized to form IMP, and this in turn is converted into adenine and guanine nucleotides.     

Tissue content of adenosine,inosine and hypoxanthine in the rat kidney after ischemia and postischemic recirculation

Summary The effect of renal ischemia of 15 s to 60 min duration on the tissue levels of adenosine, inosine and hypoxanthine was investigated in Sprague Dawley rats. A sharp increase in the tissue levels of adenosine from 5.13±0.56 to 31.3±2.96 nmol/g wet weight after 1 min of ischemia was found. The tissue levels of inosine and hypoxanthine in the controls were 3.62±0.51 and 3.19±0.76 nmol/g wet weight, respectively. Maximal levels of adenosine (38.1±6.3 nmol/g wet weight) were reached after 10 min of ischemia. The hypoxanthine levels rose steadily up to 922±183 nmol/g wet weight after 60 min of ischemia. Recirculation of 15 min after 60 min ischemia resulted in a fall of adenosine and inosine levels to values comparable to controls, whereas hypoxanthine was elevated above control values. In a second experimental series with tracing of renal blood flow (RBF) by a means of an electromagnetic flow meter a transient marked reduction of RBF after occlusion of the renal artery for 30 s was observed. The 3-fold increase of adenosine tissue levels within 30 s of renal artery occlusion and the inhibition of the postocclusive RBF reduction by theophylline (3.3 mol/100 g body weight) make it likely that this phenomenon may be caused by intrarenal adenosine.Parts of this investigation were presented at the 46th Meeting of the German Physiological Society in Regensburg, March 15–20, 1976.Supported by the Deutsche Forschungsgemeinschaft Os 42/2     

Comparative aspects of purine metabolism in some African trypanosomes

Some enzymes of purine salvage were detected in the cell-free preparations from bloodstream forms of African trypanosomes: Trypanosoma vivax; T. brucei and T. congolense. Extracts of trypanosomes cleave adenosine and inosine hydrolytically except in T. congolense where adenosine cleavage was mediated by a phosphorylase. All the trypanosomes apparently lacked adenosine deaminase. Adenine aminohydrolase was found only in T. vivax while adenosine monophosphate deaminase was detected in T. brucei and T. congolense. There was no detectable adenosine kinase activity in T. brucei. A pathway is proposed for the metabolism of purines in these trypanosomes.