Extracellular Adenosine Formation Research Articles

Evidence for control of adenosine metabolism in rat oxidative skeletal muscle by changes in pH.

1. We investigated the effects of pH elevation or depression on adenosine output from buffer-perfused rat gracilis muscle, and kinetic properties of adenosine-forming enzymes, 5'-nucleotidase (5'N) and non-specific phosphatase (PT), and adenosine-removing enzymes, adenosine kinase (AK) and adenosine deaminase (AD), in homogenates of muscle. 2. Depression of the perfusion buffer pH from 7.4 to 6.8, by addition of sodium acetate, reduced arterial perfusion pressure from 8.44 +/- 1.44 to 7.33 +/- 0.58 kPa, and increased adenosine output from 35 +/- 5 to 56 +/- 6 pmol min-1 (g wet wt muscle)-1 and AMP output from 1.8 +/- 0.3 to 9.1 +/- 3.9 pmol min-1 (g wet wt muscle)-1. 3. Elevation of the buffer pH to 7.8, by addition of ammonium chloride, reduced arterial perfusion pressure from 8.74 +/- 0.57 to 6.96 +/- 1.37 kPa, and increased adenosine output from 25 +/- 5 to 47 +/- 8 pmol min-1 (g wet wt muscle)-1 and AMP output from 3.7 +/- 1.1 to 24.6 +/- 6.8 pmol min-1 (g wet wt muscle)-1. 4. Activity of membrane-bound 5'N was an order of magnitude higher than that of either cytosolic 5'N or PT: pH depression reduced the K(m) of 5'N, which increased its capacity to form adenosine by 10-20% for every 0.5 unit decrease inpH within the physiological range. PT was only found in the membrane fraction: its contribution to extracellular adenosine formation increased from about 5% at pH 7.0 to about 15% at pH 8.0. 5. Cytosolic 5'N had a low activity, which was unaffected by pH; the rate of intracellular adenosine formation was an order of magnitude lower than the rate of adenosine removal by adenosine kinase or adenosine deaminase, which were both exclusively intracellular enzymes. 6. We conclude that (i) adenosine is formed in the extracellular compartment of rat skeletal muscle, principally by membrane-bound 5'N, where it is protected from enzymatic breakdown; (ii) adenosine is formed intracellularly at a very low rate, and is unlikely to leave the cell; (iii) enhanced adenosine formation at low pH is driven by an increased extracellular AMP concentration and an increased affinity of membrane-bound 5'N for AMP; (iv) enhanced adenosine formation at high pH is driven solely by the elevated extracellular AMP concentration, since the catalytic capacity of membrane 5'N is reduced at high pH.

Correlation Between Adenine Nucleotide-Induced Cyclic AMP Elevation and Extracellular Adenosine Formation in NG108-15 Cells

We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation in NG108-15 cells. This response was resistant to adenosine deaminase (ADA) and the ecto-5'-nucleotidase (CD73) inhibitor alpha,beta-methylene ADP (alpha,beta-MeADP), but was inhibited by both P1- and P2-receptor antagonists. In the present study, we investigated the relationship between adenine nucleotide-induced cyclic AMP elevation and extracellular adenosine formation. ATP, AMP and beta,gamma-methylene ATP (beta,gamma-MeATP) were time-dependently metabolized to adenosine in NG108-15 cells. Adenosine formations from ATP, AMP and beta,gamma-MeATP were not affected by alpha,beta-MeADP, but suppressed by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). A close correlation between extracellular adenosine formation and cyclic AMP increasing effects were obtained with several adenine nucleotide agonists in NG108-15 cells as well as their parent cell line C6Bu-1 and N18TG-2 cells, all of which possess functional adenosine A2 receptors. When NG108-15 cells were incubated with [3H]ATP or [3H]AMP in the presence of ADA, [3H]adenosine was found to distribute dominantly on the cell surface. NG108-15 cells expressed mRNA for the ecto-ATPase and nucleotide pyrophosphatase, but not for CD73. These results suggest that local adenosine formation by an ecto-enzyme distinct from CD73 is involved in adenine nucleotide-induced cyclic AMP formation in NG108-15 cells.

The effect of muscle contraction on the regulation of adenosine formation in rat skeletal muscle cells.

1. The present study examined the effect of muscle contraction on the rate of extracellular adenosine formation and on the distribution of 5' nucleotidase in primary rat skeletal muscle cells in culture. Experiments were also performed to determine whether the muscle cells release a metabolite upon contraction which may influence the extracellular production of adenosine. 2. Muscle contraction, induced by electrical stimulation, increased (P < 0.05) the rate of adenosine formation in the presence of physiological concentrations (2 and 5 microM) of adenosine monophosphate (AMP). Muscle contraction also led to an increase (P < 0.05) in the maximal rate of extracellular adenosine formation from 4.09 +/- 0.19 to 7.04 +/- 0.27 micromol (g protein)-1 min-1. Similarly, homogenates of contracted muscle cells had a higher (by 19.5 +/- 10.5 %; P < 0.05) AMP 5' nucleotidase activity than homogenates of control cells. 3. Addition of buffer from contracted cells to control cells induced an elevation (18.4 +/- 5.3 %; P < 0.05) in the rate of adenosine formation. The rate of adenosine formation was also increased with decreased intracellular adenylate charge (P < 0.05). 4. Cell homogenates treated with detergent had a higher (by 58.0 +/- 16.3 %; P < 0.05) AMP 5' nucleotidase activity than untreated homogenates, suggesting the existence of an enclosed pool of 5' nucleotidase within the muscle cells. The rate of adenosine formation in the detergent-treated homogenates was similar for electrically stimulated and non-electrically stimulated cells. 5. The present data show that muscle contraction induces an enhanced extracellular adenosine production via an increase in the activity of ecto AMP 5' nucleotidase. The activity of 5' nucleotidase can be elevated via a compound released by muscle cells during contraction and by alteration in intracellular adenylate charge. It is furthermore proposed that the extracellular adenosine formation is increased by translocation of 5' nucleotidase from an enclosed intracellular pool to the muscle membrane.

Inhibition by ATP of Hippocampal Synaptic Transmission Requires Localized Extracellular Catabolism by Ecto-Nucleotidases into Adenosine and Channeling to Adenosine A1Receptors

ATP analogs substituted in the gamma-phosphorus (ATPgammaS, beta, gamma-imido-ATP, and beta,gamma-methylene-ATP) were used to probe the involvement of P2 receptors in the modulation of synaptic transmission in the hippocampus, because their extracellular catabolism was virtually not detected in CA1 slices. ATP and gamma-substituted analogs were equipotent to inhibit synaptic transmission in CA1 pyramid synapses (IC50 of 17-22 microM). The inhibitory effect of ATP and gamma-phosphorus-substituted ATP analogs (30 microM) was not modified by the P2 receptor antagonist suramin (100 microM), was inhibited by 42-49% by the ecto-5'-nucleotidase inhibitor and alpha,beta-methylene ADP (100 microM), was inhibited by 74-85% by 2 U/ml adenosine deaminase (which converts adenosine into its inactive metabolite-inosine), and was nearly prevented by the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10 nM). Stronger support for the involvement of extracellular adenosine formation as a main requirement for the inhibitory effect of ATP and gamma-substituted ATP analogs was the observation that an inhibitor of adenosine uptake, dipyridamole (20 microM), potentiated by 92-124% the inhibitory effect of ATP and gamma-substituted ATP analogs (10 microM), a potentiation similar to that obtained for 10 microM adenosine (113%). Thus, the present results indicate that inhibition by extracellular ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases and channeling of the generated adenosine to adenosine A1 receptors.

Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices.

The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high-frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long-term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low-frequency (5 Hz) train stimulation, to mimic a long-term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high-frequency burst stimulation, whereas the release of [3H]adenosine was slightly greater at a more prolonged and low-frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used: alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low-frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [3H]-adenosine, whereas AOPCP inhibited [3H]adenosine outflow only marginally. In contrast, at high-frequency stimulation, AOPCP inhibited by 30% the evoked release of [3H]adenosine, whereas NBTI produced a 40% inhibition of [3H]adenosine outflow. At both frequencies, the kinetics of evoked [3H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [3H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [3H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high-frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.

Potentiation of excitatory amino acid-evoked adenosine release from rat cortex by inhibitors of adenosine kinase and adenosine deaminase and by acadesine

Endogenous extracellular adenosine provides some protection against excitotoxicity in the central nervous system, but it appears to be incomplete. Potentiating the formation of extracellular adenosine that occurs when excitatory amino acid receptors are activated might provide additional protection. We studied the effects of AICAR (AICA riboside, acadesine) and of inhibitors of adenosine metabolism on the release of adenosine from rat cortical slices. AICAR had no effects on basal N-methyl- d-aspartate (NMDA)- or ( RS)-α-amino-3-hydroxy-5-methyl-4-isoxasole propionic acid (AMPA)-evoked adenosine release, but it increased kainate-evoked adenosine release 1.4-fold. This selective action of AICAR may make it useful for treating kainate receptor-mediated excitotoxicity. Inhibition of adenosine kinase with either 20 μM 5′-amino-5′-deoxyadenosine or 5′-iodotubercidin had a much greater effect on excitatory amino acid-evoked adenosine release than on basal adenosine release. Inhibition of adenosine kinase increased excitatory amino acid-evoked adenosine release 3–7-fold whereas inhibition of adenosine deaminase only increased evoked adenosine release 2–2.5-fold. Finally, 0.2 μM 5′-iodotubercidin and 200 μM 2′-deoxycoformycin caused similar increases in the basal rates of extracellular adenosine formation, but 5′-iodotubercidin produced over twice as much potentiation of the rate of NMDA-evoked adenosine formation than did 2′-deoxycoformycin. These findings suggest that adenosine kinase inhibitors may produce an event-specific potentiation of evoked adenosine formation, i.e. more effect on evoked formation than on basal formation. If so, adenosine kinase inhibitors may prove useful for preventing/treating diseases associated with excessive excitation in the brain, such as seizures, excitotoxicity and neurodegeneration.

Adenosine uptake and metabolism in human endothelial cells.

The handling of adenosine by the endothelium is crucial for the effects of this nucleoside in the vascular system. Strategic location of endothelial cells at the vessel wall border indicate its important role in the turnover of adenosine. High endothelial activity of ecto 5’nucleotidase1 implies predominant role in the extracellular formation of adenosine from secreted nucleotides. However, the capacity to release intracellularly produced adenosine, phosphorylation of extracellular adenosine and to some extent adenosine transit between interstitial and intravascular space depend on the membrane transport capacity in this type of cells.

Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion.

In this study, the basal and evoked release of [3H]- and endogenous adenosine, inosine and hypoxanthine from rat hippocampal slices, labelled with [3H]adenine, was investigated. Evoked release was brought about by either electrical stimulation or energy depletion. The aim was to determine whether adenosine is formed intracellularly, and released as adenosine or extracellularly, from sequential extracellular hydrolysis of released ATP. All measurements were made in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit the enzyme adenosine deaminase. It was found that electrical field stimulation (5 min) increased the release of endogenous adenosine from hippocampal slices 10-fold and increased the proportion of [3H]-label associated with adenosine from approx 7% of the total released to 13% after the first stimulation and 20% after the second stimulation. Removal of oxygen and glucose from the superfusion medium (energy depletion) increased the release rate of endogenous adenosine 16-fold and increased the proportion of [3H]-label associated with [3H]adenosine from approx 10% of the total released to 50%. In order to prevent extracellular formation of adenosine, experiments were carried out in the presence of 50 microM alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase. AOPCP was found to be without effect on either the basal or evoked release of adenosine. In contrast, L-homocysteine thiolactone (0.1-1.0 mM) which was used to "trap" intracellular adenosine reduced both the basal and evoked release of adenosine by 70-85%. This effect of L-homocysteine thiolactone also occurred in the presence of adenosine uptake inhibitors. It is concluded from these results that adenosine is formed predominantly intracellularly in hippocampal slices and is released as adenosine as a result of either tissue depolarisation or energy depletion. Furthermore, the finding that during energy depletion there is a proportionally greater release of adenosine than other ATP breakdown products, such as inosine and hypoxanthine, indicates that energy depletion is both a potent and selective stimulus for adenosine formation and release.

Hippocampal localization of 5′-nucleotidase as revealed by immunocytochemistry

The distribution of binding sites for an antibody against ecto-5′-nucleotidase was investigated in the mouse hippocampus by light microscopical immunocytochcmistry. The antibody selectively labels a band corresponding to the innervation area of mossy fibre terminals within area CA3. Area CA1 as well as the dendate gyms are negative. In area CA3 only the proximal but not the distal parts of the apical dendrites of pyramidal cells are labelled. Labelling is in the form of large dots around dendrites of pyramidal cells suggesting that mossy fibre terminals are immunopositive. In contrast, an antibody against the ubiquitous synaptic vesicle protein SV2 labels the large mossy fibre terminals as well as fine and punctate structures in the dendritic and somatic regions throughout the hippocampus. Labelled astrocytes can be found in the entire hippocampus and are frequent in the stratum radiatum and stratum oriens of the CA1 region. Immunopositive astrocytic processes can be found in association with capillary walls. Our results suggest that ecto-5′-nucleotidase may play a crucial role in the hydrolysis of AMP to adenosine at the mossy fibre synapses. Thus, at these synapses, 5′-nucleotidases could function both in completing the extracellular hydrolysis of synaptically released ATP as well as in the extracellular formation of adenosine.

Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine.

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.

5′-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart

Changes in 5'-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5'-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5'-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5'-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5'-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5'-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.

Adenosine in cerebral homeostatic role: appraisal through actions of homocysteine, colchicine, and dipyridamole.

Mammalian neocortical tissues were incubated in [14C]adenine-containing fluids and their newly-synthesized adenine derivatives examined after periods of superfusion. Increased [K+] released adenine derivatives from the tissues, a release diminished by homocysteine. Homocysteine acted also to diminish the tissue content of adenosine plus its metabolites hypoxanthine and inosine, while increasing that of S-adenosylhomocysteine. Hypoxia also increased the tissue content and the output of adenosine plus its metabolites, and again homocysteine augmented the S-adenosylhomocysteine. Glutamic acid also increased tissue content and output of adenosine and derivatives, an action diminished by homocysteine and associated with augmented S-adenosylhomocysteine. Colchicine or dipyridamole did not prevent augmentation of S-adenosylhomocysteine by the reagents described; the sequence from adenosine phosphates to S-adenosylhomocysteine is concluded to be intracellular and not to involve extracellular formation of precursor adenosine. Adenosine displayed properties consistent with its being involved in two distinct categories of homeostasis, and also with its exerting an inhibitory tone in normal cerebral systems.

Degradation of perfused adenine compounds up to uric acid in isolated rat heart

Cells are generally impermeable to nucleotides like ATP, ADP and AMP while nucleosides and bases readily cross the plasma membrane. A release of adenosine and of its catabolic derivatives by the myocardium of different animal species has been demonstrated in physiological and physiopathological conditions [2, 4, 13]. Furthermore there are many studies on the uptake of adenosine and inosine by the myocardial cells and their incorporation into tissue nucleotides [7–9, 15]. Taking into account the extracellular localization of adenosine receptors [5] and the role of this nucleoside as regulator of coronary blood flow and modulator of the positive inotropic effects of catecholamines [4] it was of interest to study a possible extracellular formation of adenosine from adenine nucleotides. Most mammalian tissues like muscle, liver and adipose tissue and cells like platelets, leukocytes and lymphocytes possess some enzyme activities associated with the cell surface membrane. Ecto-ATPase (EC 3.6.1.3/15) and ecto-5′-nucleotidase (EC 3.1.3.5) activities have been demonstrated in myocardial, smooth-muscle and endothelial cell membranes [3,4,8,12]. In the present paper we report a comparative study on the ability of the isolated rat heart to breakdown exogenous adenine nucleotides, nucleosides and bases: we have determined in the coronary perfusate all the degradation products in basal conditions and after perfusion with ATP, ADP, AMP, adenosine, inosine and hypoxanthine. All the perfused compounds were degraded up to uric acid that amounted to about 10%. Nucleotides were catabolized at a higher rate than nucleosides and so adenosine may accumulate outside the cells.