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Query: EC:3.5.4.4 (adenosine deaminase)
 5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion. 836 41

When human umbilical vein endothelial cells were prelabeled with [14C]-adenine and then exposed to xanthine oxidase (40 mU/ml) and hypoxanthine (100 microM) for 4 h, cellular adenine nucleotides were depleted (18 +/- 3% of total radioactivity vs. 61 +/- 10% in controls), nucleotides appeared in the culture medium (8 +/- 3% vs. 4 +/- 3%) together with the catabolic products inosine, hypoxanthine, and uric acid (74 +/- 4% vs. 35 +/- 11%). In the presence of H2O2 (100 microM) for 30 min, cellular nucleotides were depleted (46 +/- 25%) and catabolic products appeared in the medium (40 +/- 26%), but radioactive nucleotides in the medium were unaltered. In the presence of an inhibitor of ecto-5'-nucleotidase [alpha, beta-methylene-adenosine 5'-diphosphate (ADP), 0.5 mM], exposure to xanthine oxidase and hypoxanthine resulted in the appearance of three times more nucleotides in the culture medium than in the absence of the inhibitor, but there was no change in medium nucleotides after H2O2 exposure. In the presence of an inhibitor of adenosine deaminase (2-deoxycoformycin, 2 microM), both exposures caused an accumulation of adenosine in the medium, calculated to represent a minimum of 25% of nucleotide catabolism. We conclude that exposure to both a superoxide-generating system (hypoxanthine plus xanthine oxidase) and H2O2 induce catabolism of adenine nucleotides, which mainly takes place through adenosine 5'-monophosphate (AMP) deaminase. However, superoxide but not H2O2 also causes membrane damage and leakage of nucleotides into the medium.
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PMID:Mechanisms of adenine nucleotide depletion from endothelial cells exposed to reactive oxygen metabolites. 838 Nov 5

Exogenous adenosine triphosphate (ATP) added to brush-border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto-nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto-adenosine deaminase and ecto-adenosine monophosphate (AMP) nucleotidase was shown. The ecto-adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush-border membrane vesicles. Using orthovanadate, levamisole, and alpha, beta-methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto-AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto-AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine.
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PMID:Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes. 840 44

A1 adenosine receptors in the rat prepiriform cortex play an important role in the inhibition of bicuculline methiodide-induced convulsions. In the present study we evaluated manipulation of endogenous adenosine in this brain area as a strategy to effect seizure suppression. All compounds evaluated were unilaterally microinjected into the rat prepiriform cortex. Administration of exogenous adenosine afforded a dose-dependent protection (ED50 = 48.1 +/- 8.4 nmol) against bicuculline methiodide-induced seizures, and these anticonvulsant effects were significantly potentiated by treatment with an adenosine kinase inhibitor, 5'-amino-5'-deoxyadenosine; by the adenosine transport blockers, dilazep or nitrobenzylthioinosine 5'-monophosphate; and by an adenosine deaminase inhibitor, 2'-deoxycoformycin. When administered alone, 5'-amino-5'-deoxyadenosine, 5'-iodotubercidin and dilazep were found to be highly efficacious as anticonvulsants with respective ED50 values of 2.6 +/- 0.8, 4.0 +/- 2.7 and 5.6 +/- 1.5 nmol. In contrast, 2'-deoxycoformycin was both less potent and less efficacious. These results suggest that accumulation of endogenous adenosine may contribute to seizure suppression, and that adenosine kinase and adenosine transport may play a pivotal role in the regulation of extracellular levels of adenosine in the central nervous system. The adenosine antagonist, 8-(p-sulfophenyl)theophylline, increased markedly the severity of bicuculline methiodide-induced seizures. Moreover, reduction of extracellular adenosine formation by a focal injection of an ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine diphosphate, produced generalized seizures (ED50 = 37.3 +/- 22.7 nmol). Together the proconvulsant effect of an adenosine receptor antagonist and the convulsant action of an ecto-5'-nucleotidase inhibitor further support the role of endogenous adenosine as a tonically active antiepileptogenic substance in the rat prepiriform cortex.
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PMID:Manipulation of endogenous adenosine in the rat prepiriform cortex modulates seizure susceptibility. 845 Apr 75

Follicular oocytes of Xenopus laevis possess P1 purinoceptors where, seemingly, both adenosine (Ado) and ATP are agonists. The basis of ATP agonism at this P1 purinoceptor was investigated using electrophysiological and biochemical procedures. Ado and ATP activated an outward K+ current that reversed at -90 mV, was reduced by TEA and was inhibited by theophylline and 8-(p-sulphophenyl)-theophylline but not by suramin. Outward K+ current to ATP and Ado also was inhibited by alpha, beta-methylene ATP. The affinity constants for Ado and ATP were identical, although ATP was a partial agonist. The potency order of nucleosides/nucleotides was 5'-N-ethylcarboxamide- adenosine > Ado > AMP > CGS-21680 > beta, gamma-methylene ATP = ATP > ADP > R-N6 phenylisopropyl-adenosine, whereas 2-methylthioadenosine, ATP-O-(3-thiotriphosphate), uridine 5'-triphosphate and alpha, beta-methylene ATP were inactive. Outward K+ current to ATP and nondegradable Ado analogs was unaffected by adenosine deaminase (although this enzyme prevented Ado agonism), which suggests that ATP is not broken down to Ado before activating K+ channels. The activity of oocyte ecto-ATPase was determined by HPLC analysis of ATP breakdown and by the production of inorganic phosphate. Oocyte ecto-ATPase showed a low rate of ATP hydrolysis and was incapable of generating sufficient Ado/AMP to activate P1 purinoceptors. The results show that a P1 purinoceptor that is not typical of other known Ado receptors (and ATP receptors) is present in the follicle cell layer of Xenopus oocytes and represents a novel purinoceptor subtype where both Ado and ATP are agonists in their own right.
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PMID:A novel P1 purinoceptor activates an outward K+ current in follicular oocytes of Xenopus laevis. 855 61

The effect of human platelets on chemoattractant-induced generation of oxygen metabolites in neutrophils was investigated, using luminol-enhanced chemiluminescence (CL). Resting platelets inhibited the extracellular, but not the intracellular, production of oxygen radicals in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils. Maximal effect was obtained at the physiological neutrophil/platelet ratio of 1/50. Similar results were acquired by adding supernatants of platelets, indicating a role for a soluble factor. Removal of extracellular adenosine by adenosine deaminase (ADA), or blocking of adenosine-receptors by theophylline, antagonized the inhibitory effects of platelets (or the equivalent supernatant) on the neutrophil respiratory burst. In contrast, accumulation of adenosine by apyrase enhanced the inhibition. Exogenous adenosine mimicked the effects of platelets on the fMet-Leu-Phe-induced respiratory burst. To further assess the role of platelet-derived adenosine, the platelets were fixed with paraformaldehyde. We found that fixed platelets, as well as their supernatant, inhibited the fMet-Leu-Phe-induced CL-response to the same extent as viable cells. These effects were also reversed by ADA and theophylline, respectively. A prior removal of adenosine in the platelet suspension by ADA, followed by treatment with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) to inactivate ADA, did not reverse the inhibitory action of platelets on the fMet-Leu-Phe-induced CL-response in neutrophils. However, if adenosine receptors of neutrophil at the same time were blocked with theophyline, the inhibition was significantly reduced. Platelets markedly increased the generation of adenosine in a neutrophil suspension. The effect was antagonized by S-(4-Nitrobenzyl)-6-thioguanosine (NBTG), but unaffected by alpha, beta-methyl-eneadenosine5'diphosphate (AMP-CP), indicating that the platelet-dependent accumulation of adenosine is due to an increased release of endogenous adenosine from neutrophils and not to a degradation of extracellular AMP. In correlation, NBTG, but not AMP-CP, reversed the platelet-mediated inhibition of the fMet-Leu-Phe-induced CL-response in neutrophils. Consequently, these data suggest that a platelet-derived factor increases the release of endogenously formed adenosine from neutrophils, terminating the production of oxygen radicals. The inhibition of oxidase activity was also associated with a platelet-induced polymerization of actin in the margin of the neutrophils. Treatment of neutrophils with cytochalasin B reversed the effects of platelets, both on F-actin content and CL-response. In summary, resting platelets limit the release of oxygen radicals from chemoattractant-stimulated neutrophils, thus preventing excessive damage to host tissues in the vascular space. This effect is suggested to be associated with an increase generation of neutrophil-derived adenosine enhancing an autoregulatory inhibitory pathway, and a peripheral accumulation of actin filaments forming a barrier for extracellular release of reactive oxygen radicals.
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PMID:Release of oxygen metabolites from chemoattractant-stimulated neutrophils is inhibited by resting platelets: role of extracellular adenosine and actin polymerization. 863 3

We have shown previously that a soluble factor(s) released by the myenteric plexus promotes neurite outgrowth from postnatal striatal neurons, and that this effect was abolished by tetrodotoxin. We have now investigated the possible involvement of purines in the mediation of this neuritogenic response, by examining their effect on neurite length of striatal neurons both in co-culture with myenteric plexus explants and cultured alone. Both ATP and 2-chloroadenosine partially reversed the inhibitory effect of tetrodotoxin in co-cultures with whole myenteric plexus, while the stable ATP analogue, alpha, beta-methylene ATP, had no effect, suggesting that ATP was being broken down to adenosine before exerting its action. Further support for this view was that the ATP (P2) purinoceptor antagonist suramin did not reverse the effects of ATP, while the adenosine (P1) purinoceptor antagonist 8-(p-sulphophenyl)theophylline did antagonize the effects of ATP in tetrodotoxin-treated co-cultures. Further, both 8-(p-sulphophenyl)theophylline and adenosine deaminase reduced the effect of the myenteric plexus on striatal neurons in the absence of tetrodotoxin, and the adenylate cyclase activator forskolin completely reversed the effect of tetrodotoxin in our co-culture system. The neurite outgrowth-promoting effect of 2-chloroadenosine in tetrodotoxin-treated co-cultures was not further enhanced by a combination of neuropeptides. Serotonin and GTP were without effect on striatal neurons in the presence or absence of myenteric plexus explants. In experiments without myenteric plexus, both 2-chloroadenosine and forskolin caused a slight increase in striatal neurite length; ATP and GTP were ineffective. Basic fibroblast growth factor, nerve growth factor, neurotrophin-3 or neurotrophin-4/5 had no effect on neurite outgrowth in postnatal striatal cultures after two days in vitro. When these growth factors were added in combination with 2-chloroadenosine, the observed increase in mean neurite length did not exceed that induced by 2-chloroadenosine alone. Both 2-chloroadenosine and the ganglioside mix AGF1 increased neurite elongation of striatal neurons after two days in vitro, but an inhibition of enhanced neurite outgrowth was observed when both substances were added together. Both laminin and fibronectin were not neuritogenic for postnatal striatal neurons under our culture conditions. These observations suggest that a factor other than the growth factors tested here is involved in the promotion of striatal neurite outgrowth in co-culture with myenteric plexus explants. In summary, adenosine (probably acting through the A2 subclass of the P1 purinoceptor) leads to increased striatal neurite outgrowth in co-culture with myenteric plexus and we propose that it does so either (1) by triggering the release of a neuritogenic factor, possibly from enteric glial cells, or (2) by acting synergistically with such a growth factor. Adenosine acts via P1 purinoceptors, which leads to changes in cyclic AMP, and the response to forskolin suggests that cyclic AMP is probably involved in the events leading to increased striatal neurite outgrowth.
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PMID:Neurite outgrowth of striatal neurons in vitro: involvement of purines in the growth-promoting effect of myenteric plexus explants. 888 77

1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings.
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PMID:Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides. 888 6

In this study we determined whether cAMP is metabolized to adenosine in vascular smooth muscle cells and whether cAMP-derived adenosine modulates vascular smooth muscle cell growth. Confluent smooth muscle cells were exposed to cAMP (0.01 to 30 mumol/L) in the presence and absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/L; an inhibitor of both extracellular and intracellular phosphodiesterase), alpha, beta-methyleneadenosine 5'-diphosphate (AMP-CP, 100 mumol/L; an ecto-5'-nucleotidase inhibitor), and 1,3-dipropyl-8-p-sulfophenyl-xanthine (DPSPX, 100 mumol/L; a xanthine that can inhibit extracellular phosphodiesterase) for 0 to 60 minutes. Medium was then sampled and assayed for AMP, adenosine, and inosine. cAMP increased the amount of AMP, adenosine, and inosine in the medium in a time- and concentration-dependent manner. The conversion of cAMP to adenosine and inosine was inhibited by blockade of phosphodiesterase with IBMX, of ecto-phosphodiesterase with DPSPX, and of ecto-5'-nucleotidase with AMP-CP. To evaluate the physiological relevance of cAMP-derived adenosine in vascular smooth muscle cell proliferation, we studied the inhibitory effects of cAMP (10(-4) mol/L) and 8-bromo-cAMP (10(-4) mol/L) on fetal calf serum-induced DNA synthesis ([3H]thymidine incorporation) in the presence and absence of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, an inhibitor of adenosine deaminase), dipyridamole (a blocker of adenosine transport), KF17837 (a selective A2 adenosine receptor antagonist), and DPSPX (a nonselective adenosine receptor antagonist). cAMP inhibited DNA synthesis, and both EHNA and dipyridamole enhanced this effect. Both KF17837 and DPSPX significantly reduced the inhibitory effects of cAMP on DNA synthesis; however, they did not reduce the inhibitory effects of 8-bromo-cAMP on DNA synthesis. These results indicate that vascular smooth muscle cells metabolize cAMP to adenosine via the sequential action of ecto-phosphodiesterase and ecto-5'-nucleotidase and provide the first evidence that cAMP-derived adenosine can inhibit vascular smooth muscle cell growth. Hence, this cAMP-adenosine pathway may importantly contribute to the regulation of vascular biology.
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PMID:Cyclic AMP-adenosine pathway inhibits vascular smooth muscle cell growth. 890 21

PC12 pheochromocytoma cells have P2 purinoceptors which are activated by ATP and coupled to Ca2+ influx and catecholamine release. Also PC12 cells have adenosine receptors coupled positively to adenylyl cyclase, and cyclic AMP regulates cell functions such as catecholamine release. The effects of ATP and ATP analogs on cyclic AMP accumulation in PC12 cells were investigated in this study. ATP and adenosine 5'-0-(3-thiotriphosphate) stimulated cyclic AMP accumulation at low concentrations up to 300 microM but showed inhibitory effects above this concentration. 2',3'-O-(4-Benzoyl)benzoyl ATP and 2-methylthio ATP showed similar effects, although the responses were very limited. Addition of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) or beta, gamma-methylene ATP, but not alpha, beta-methylene ATP, stimulated cyclic AMP accumulation markedly without causing an inhibitory phase. The effects of ATP, ADP beta S and beta, gamma-methylene ATP were not inhibited by adenosine deaminase or specific antagonists to A1 and A2 adenosine receptors. Neither ADp beta S nor beta, gamma-methylene ATP showed any effect on Ca2+ influx or noradrenaline release. Suramin, a P2 receptors antagonists, had no inhibitory effect against ATP analog-stimulated cyclic AMP accumulation, although reactive blue 2 inhibited the beta, gamma-methylene ATP-stimulated reaction but not that up-regulated by ADP beta S. These findings suggest that the pharmacological characteristics of these ATP receptors coupled to adenylyl cyclase are clearly different from those of ligand-gated ion channels defined by P2X purinoceptors, which have been cloned and shown to be coupled to Ca2+ influx and catecholamine release in PC12 cells. The existence of a new type of P2 purinoceptor-mediating stimulation of adenylyl cyclase is proposed in PC12 cells.
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PMID:P2 purinoceptor-mediated stimulation of adenylyl cyclase in PC12 cells. 895 42


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