Gene/Protein Disease Symptom Drug Enzyme Compound
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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 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

1. Adenosine modulates acetylcholine (ACh) release from the rat motor nerve terminals. Tonic activation of presynaptic A1 inhibitory and/or A2A facilitatory adenosine receptors is regulated by the concentration of the nucleoside at the synapse. The parameters (frequency, duration of pulses, train length) of nerve stimulation determine the amount of transmitter and/or modulator released, and have long been proposed as important features of synaptic control. This prompted us to investigate which was the prevailing response to adenosine on evoked [3H]-ACh release from rat phrenic nerve hemidiaphragm preparations in different stimulation conditions. 2. With low-frequency, short-duration pulses (5 Hz, 40 microseconds in duration), the adenosine inhibitory tonus (approximately 30%) predominates. The magnitude of the adenosine tonic inhibition was dependent on the number of pulses (250-750) delivered in each stimulation train, e.g., the facilitatory effect of adenosine deaminase (ADA, 0.5 U/ml) and the inhibitory effect of the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI, 5 microM) reached significance only when > 250 pulses were applied. Facilitation was only observed with high concentrations of either exogenous adenosine (> 100 microM) or NBTI (> 10 microM). 3. When the stimulation pulse duration was increased to 1 ms (5 Hz, 750 pulses), endogenously generated adenosine consistently facilitated evoked [3H]-ACh release. In these conditions, ADA (0.5 U/ml) decreased evoked [3H]-ACh release by 29 +/- 4% (mean +/- SE) (n = 3), and both NBTI (3-30 microM) and adenosine (10-500 microM), which had biphasic effects with pulses of 40 microseconds, facilitated transmitter release. 4. When high-frequency "trains" (50 Hz, 40 microseconds, 500 pulses) were applied, both ADA (0.5 U/ml) and NBTI (5 microM) failed to modify evoked [3H]-ACh release. To bypass putative feedforward inhibition of ecto-5'-nucleotidase induced by released ATP, which might reduce adenosine formation during high-frequency trains, experiments containing a series of five high-frequency "bursts" (50 Hz, 40 microseconds, 100 pulses) with variable interburst intervals (5-20 s) were performed. In such conditions, the prevailing tonic response to adenosine turned out to be facilitatory, because ADA (0.5 U/ml) inhibited and NBTI (5 microM) facilitated evoked [3H]-ACh release. The magnitude of the inhibitory effect of ADA (0.5 U/ml) ranged from -9 +/- 6% (n = 4) to -54 +/- 8% (n = 5) as the interburst interval changed from 5 to 20 s, respectively. 5. Prolongation of individual pulses from 40 microseconds to 1 ms (5 Hz frequency) or increasing the frequency of stimulation (1-50 Hz, 40 microseconds) did not significantly change the excitatory effect of the A2A receptor agonist 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680C). In contrast, the inhibitory effect of the A1 receptor agonist R-N6-phenylisopropyladenosine was significantly attenuated in both stimulation conditions. 6. In conclusion, the results suggest that high-intensity, high-frequency motor nerve stimulation critically influences endogenous adenosine formation and the A1/A2A receptor activation balance, i.e., it potentiates the tonic adenosine A2A-receptor-mediated facilitation of ACh release, whereas activation of the inhibitory A1 receptors becomes less effective. A model is proposed that attempts to further elucidate adenosine's involvement in synaptic transmission adaptation.
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PMID:Presynaptic A1 inhibitory/A2A facilitatory adenosine receptor activation balance depends on motor nerve stimulation paradigm at the rat hemidiaphragm. 898 88

The adenosine producing enzyme ecto-5'-nucleotidase (5'-NT) is not normally expressed during thymocyte development until the medullary stage. To determine whether earlier expression would lead to adenosine accumulation and/or be deleterious for thymocyte maturation, thymic purine metabolism, and T cell differentiation were studied in lckNT transgenic mice overexpressing 5'-NT in cortical thymocytes under the control of the lck proximal promoter. In spite of a 100-fold elevation in thymic 5'-NT activity, transgenic adenosine levels were unchanged and T cell immunity was normal. Inosine, the product of adenosine deamination, was elevated more than twofold, however, indicating that adenosine deaminase (ADA) can prevent the accumulation of adenosine, even with a dramatic increase in 5'-NT activity, and demonstrating the availability of 5'-NT substrates in the thymus for the first time. Thymic adenosine concentrations of mice treated with the ADA inhibitor 2'-deoxycoformycin (dCF) were elevated over 30-fold, suggesting that high ADA activity, rather than an absence of 5'-NT, is mainly responsible for low thymic adenosine levels. The adenosine concentrations in dCF-treated mice are sufficient to cause adenosine receptor-mediated thymocyte apoptosis in vitro, suggesting that adenosine accumulation could play a role in ADA-deficient severe combined immunodeficiency.
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PMID:Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'-nucleotidase (CD73). 904 70

Adenosine has potent immunosuppressive activity. Since the source of adenosine and the mechanism of its release in the immune system is largely unknown and may vary according to cell type, we have evaluated the relationship between adenosine metabolism and the enzymatic activities and mRNA levels of adenosine-metabolizing enzymes in myeloid and lymphoid cell lines. Induction of HL-60 cell differentiation along the macrophage lineage by PMA resulted in a reduction in the activities of adenosine deaminase (ADA), adenosine kinase (AK), and inosine monophosphate-specific cytosolic 5'-nucleotidase and an elevation of ecto-5'-nucleotidase (ecto-5'-NT). These changes were accompanied by an elevation of ecto-5'-NT mRNA and a decrease in ADA and AK mRNAs in a time-dependent fashion. Comparison of AK and ADA mRNA levels in several other leukemic cell lines revealed generally similar responses to PMA with much stronger suppression in immature T cells than in B cells. The metabolism of adenosine either through phosphorylation (AK) or deamination (ADA) was reduced in PMA-stimulated cells. Furthermore, the cumulative changes in enzyme expression resulted in a 2.5-fold increase in intracellular adenosine formation in PMA-stimulated cells. The inhibition of AK by 5'-iodotubercidin further increased adenosine formation by 6-fold over that in untreated cells. In accord with the increase in ecto-5'-NT activity, extracellular AMP dephosphorylation increased dramatically, but there was no increase in extracellular ATP degradation. These results indicate that a coordinated shift in adenosine-metabolizing enzyme levels during PMA-induced HL-60 cell differentiation is accompanied by a decrease in adenosine uptake and an increase in adenosine release.
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PMID:Adenosine metabolism during phorbol myristate acetate-mediated induction of HL-60 cell differentiation: changes in expression pattern of adenosine kinase, adenosine deaminase, and 5'-nucleotidase. 914 13

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.
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PMID:Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors. 948 85

The extracellular "cAMP-adenosine pathway" refers to the local production of adenosine mediated by cAMP egress into the extracellular space, conversion of cAMP to AMP by ectophosphodiesterase, and the metabolism of AMP to adenosine by ecto-5'-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway limits cardiac fibroblast growth. Studies were conducted in ventricular cardiac fibroblasts maintained in 3-dimensional cultures. Addition of exogenous cAMP to cardiac fibroblasts increased extracellular levels of AMP, adenosine, and inosine in a concentration-dependent and time-dependent manner. This effect was attenuated by blockade of total phosphodiesterase activity (3-isobutyl-1-methylxanthine), ectophosphodiesterase activity (high concentration of 1, 3-dipropyl-8-p-sulfophenylxanthine), or ecto-5'-nucleotidase (alpha, beta-methylene-adenosine-5'-diphosphate). Treatment with exogenous cAMP inhibited cell growth as assessed by DNA synthesis ((3)H-thymidine incorporation), cell proliferation (cell counts), and protein synthesis ((3)H-leucine incorporation). Antagonism of A(2) (KF17837) or A(1)/A(2) (low concentration of 1, 3-dipropyl-8-p-sulfophenylxanthine), but not A(1) (8-cyclopentyl-1, 3-dipropylxanthine), adenosine receptors blocked the growth-inhibitory effects of exogenous cAMP, but not the growth inhibitory effects of 8-bromo-cAMP (stable cAMP analogue). The growth-inhibitory effects of exogenous cAMP were enhanced by the combined inhibition of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl) adenine] and adenosine kinase (iodotubercidin). In conclusion, the extracellular cAMP-adenosine pathway exists in cardiac fibroblasts and attenuates cell growth. Pharmacological augmentation of this pathway could abate pathological cardiac remodeling in heart disease.
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PMID:Cardiac fibroblasts express the cAMP-adenosine pathway. 1098 61

A detailed understanding of adenosine metabolism of vascular smooth muscle cells (VSMC) is highly desirable to critically evaluate possible autocrine effects of adenosine in this cell species. Therefore, this study quantified intra- and extracellular adenosine flux rates, the transmembrane concentration gradient, and the adenosine surface concentration in porcine VSMC and, for comparison, aortic endothelial cells (PAEC). Cell-covered microcarrier beads packed in a chromatography column were superfused with a HEPES buffer. With the use of specific inhibitors of adenosine kinase (iodotubericidine, 10 microM), adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)-adenine, 5 microM], ecto-5'-nucleotidase (alpha,beta-methylene-adenosine 5'-diphosphate, 50 microM), and adenosine membrane transport (n-nitrobenzylthioinosine, 1 microM), total production rates of 12.3 +/- 2.7 and 7.5 +/- 1.3 pmol x min(-1) x microl cell volume(-1) were obtained for VSMC and PAEC, respectively. Despite prevailing intracellular adenosine production (76 and 70% of total production, respectively), transmembrane concentration gradients under control conditions were directed toward the cytosol as a result of rapid intracellular adenosine rephosphorylation and continuous extracellular hydrolysis from 5'-AMP. Surface concentrations were approximately 18 nM in VSMC and PAEC under control conditions and increased to approximately 60 nM during partial inhibition of adenosine metabolism. Simultaneously, the transmembrane adenosine concentration gradient was reversed. We conclude that adenosine flux rates in VSMC and PAEC are quantitatively similar and that VSMC may influence the interstitial adenosine concentration under basal steady-state conditions.
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PMID:Significance of adenosine metabolism of coronary smooth muscle cells. 1112 25

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.
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PMID:Correlation between adenine nucleotide-induced cyclic AMP elevation and extracellular adenosine formation in NG108-15 cells. 1113 34

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 +/- 6 and 21 +/- 2 nmol Pi mg(-1) min(-1) for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 0.13 nmol adenosine mg(-1) min(-1), respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.
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PMID:Ectonucleotidase activities in Sertoli cells from immature rats. 1159 98

We recently demonstrated that extracellular ATP effectively activates adenosine (Ade) A(2B) receptors indirectly through a localized rapid conversion to Ade by ectonucleotidases on the membrane surface of C6Bu-1 rat glioma cells. These responses were observed even in the presence of adenosine deaminase (ADA). Here, we demonstrate that such responses indeed occur in A(2B) receptor-expressing Xenopus laevis oocytes, which possess endogenous ectonucleotidase activity. In oocytes coexpressing the A(2B) receptor and cystic fibrosis transmembrane conductance regulator (CFTR), Ade induced a concentration-dependent increase in a cyclic AMP-activated CFTR current, a response that was inhibited by the P1 antagonist xanthine-amine congener (XAC). A brief application of ATP and beta,gamma-methylene ATP (beta,gamma-MeATP) also induced the CFTR current in a manner similar to that seen with Ade. Among several nucleotide agonists, ADP, AMP, and adenosine-5'-O-(3-thio)triphosphate induced the CFTR current. Although adenine nucleotide-induced CFTR currents were inhibited by XAC, they were highly resistant to ADA treatment; 5 U/ml ADA was required for inhibition of adenine nucleotide-induced CFTR current, whereas 1 U/ml ADA was sufficient to abolish the Ade-induced response. In addition, the ecto-5'-nucleotidase inhibitor alpha,beta-methylene ADP markedly inhibited the beta,gamma-MeATP-induced response but not the Ade-induced one. These results support our hypothesis that adenine nucleotides are rapidly and locally converted into Ade on the membrane surface, resulting in the activation of A(2B) receptors.
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PMID:Adenine nucleotide-induced activation of adenosine A(2B) receptors expressed in Xenopus laevis oocytes: involvement of a rapid and localized adenosine formation by ectonucleotidases. 1185 41


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