Metabolism of adenosine and deoxyadenosine by human erythrocytes and CCRF-CEM leukemia cells

Human lymphocytes lacking adenosine deaminase die and T-cell leukemias are killed by deoxycoformycin (dCf), an inhibitor of adenosine deaminase, due to impaired metabolism of dAdo. The initial metabolism of exogenous adenosine (Ado) and deoxyadenosine (dAdo) has been compared in human erythrocytes and CCRF-CEM leukemia cells and the data obtained have been simulated using kinetic constants obtained in vitro for the enzymes involved. Cells were mixed with ³H-labelled Ado or dAdo, samples were taken at 3 sec intervals and progress curves for the ³H-labelled metabolites formed were determined by quantitative two-dimensional thin layer chromatography. Erythrocytes rapidly take up Ado and the predominant metabolite after 60 sec is hypoxanthine (Hyp), while for dAdo, deoxyinosine (dIno) predominates. By contrast, leukemia cells convert Ado predominantly to AMP, while dAdo is converted first to Hyp and then to AMP. The presence of dCf had little effect upon Ado metabolism but induced accumulation of dAdo. Erythrocytes rapidly degrade Ado and dAdo to Hyp, although the phosphorolysis of dIno is relatively slow. Human CCRF-CEM leukemia cells convert most of the Ado or dAdo to AMP after 60 sec. For dAdo, the sequence of reactions would be dAdo→dIno→Hyp→IMP→sAMP→AMP. dCf does not significantly affect the conversion of Ado→AMP, but dCf blocks AMP accumulation from dAdo, consistent with the reaction sequence shown above. A computer model has been developed for the metabolism of Ado and dAdo, but some of the kinetic constants determined in vitro for this model do not pertain to intact cells.     

Chronic hypoxia enhances adenosine release in rat PC12 cells by altering adenosine metabolism and membrane transport

Acute exposure to hypoxia causes a release of adenosine (ADO) that is inversely related to the O2 levels in oxygen-sensitive pheochromocytoma (PC12) cells. In the current study, chronic exposure (48 h) of PC12 cells to moderate hypoxia (5% O2) significantly enhanced the release of ADO during severe, acute hypoxia (1% O2). Investigation into the intra- and extracellular mechanisms underpinning the secretion of ADO in PC12 cells chronically exposed to hypoxia revealed changes in gene expression and activities of several key enzymes associated with ADO production and metabolism, as well as the down-regulation of a nucleoside transporter. Decreases in the enzymatic activities of ADO kinase and ADO deaminase accompanied by an increase in those of cytoplasmic and ecto-5'-nucleotidases bring about an increased capacity to produce intra- and extracellular ADO. This increased potential to generate ADO and decreased capacity to metabolize ADO indicate that PC12 cells shift toward an ADO producer phenotype during hypoxia. The reduced function of the rat equilibrative nucleoside transporter rENT1 also plays a role in controlling extracellular ADO levels. The hypoxia-induced alterations in the ADO metabolic enzymes and the rENT1 transporter seem to increase the extracellular concentration of ADO. The biological significance of this regulation is unclear but is likely to be associated with modulating cellular activity during hypoxia.     

Enzymes of adenosine metabolism in Hymenolepis diminuta (Cestoda)

Gaitanaki C. and Beis I. 1985. Enzymes of adenosine metabolism in Hymenolepis diminuta (Cestoda). International Journal for Parasitology15: 651–654. The activities of 5-nucleotidase (E.C. 3.1.3.5), adenosine deaminase (E.C. 3.5.4.4), adenosine kinase (E.C. 2.7.1.20), AMP deaminase (E.C. 3.5.4.6) and adenylate kinase (E.C. 2.7.4.3) were demonstrated in homogenates of Hymenolepis diminuta. The K_m values for adenosine and AMP of the above enzymes were determined. The importance of these enzymes in the maintenance of adenosine concentration on a steady state in H. diminuta is discussed     

Uptake and metabolism of adenosine mediate a meiosis‐arresting action on mouse oocytes

This study was carried out to evaluate the possible role of adenosine uptake and metabolism in mediating the inhibitory actions of this nucleoside on spontaneous mouse oocyte maturation. Uridine blocked ³H‐adenosine uptake by oocyte–cumulus cell complexes (OCCs) and cumulus cell–enclosed oocytes (CEOs) by 82–85%, whereas uptake by denuded oocytes (DOs) was suppressed by 97%. Uridine had no effect on germinal vesicle breakdown (GVB) in CEOs when meiotic arrest was maintained with hypoxanthine or hypoxanthine plus adenosine but reversed the combined inhibitory action of these purines in DOs. Five of six adenosine analogs that bind to purinoceptors demonstrated meiosis‐arresting activity but not in relation to their relative affinities for inhibitory or stimulatory adenosine receptors and only at high concentrations. Moreover, in DOs, uridine reversed the inhibitory effect of 2‐chloroadenosine and 5′‐N‐ethylcarboxamidoadenosine, two receptor agonists that are poor substrates for adenosine‐metabolizing enzymes. Results of experiments with adenosine kinase inhibitors showed that methylmercaptopurine riboside (MMPR) and tubercidin, but not 5′‐amino‐5′‐deoxyadenosine, reversed meiotic arrest maintained by hypoxanthine ± adenosine, but this required an additional inhibitory action on de novo purine synthesis. Inhibition of de novo purine synthesis alone was not sufficient because azaserine failed to reverse meiotic arrest. MMPR was a very potent meiosis‐inducing agent, completely reversing meiotic arrest in CEOs and DOs in the presence of a variety of meiotic inhibitors. The adenosine deaminase inhibitor deoxycoformycin had opposite effects on oocyte maturation depending on the presence or absence of adenosine: the inhibitory action of hypoxanthine alone was bolstered, but the meiosis‐arresting action of adenosine was reversed. These data therefore indicate that at low adenosine concentrations phosphorylation predominates, but at higher adenosine concentrations deaminated products contribute to the meiotic inhibition. This idea was borne out by the ability of inosine to mimic the synergistic interaction of adenosine with hypoxanthine. The action of adenosine is not due to deamination to inosine and conversion to nucleotides through the hypoxanthine salvage pathway because adenosine‐mediated inhibition was not compromised in oocytes from mutant mice unable to salvage hypoxanthine. Mol. Reprod. Dev. 53:208–221, 1999. © 1999 Wiley‐Liss, Inc.     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

The aspartate‐derived amino‐acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non‐intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration.     

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Adenosine is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in these cultures. Our aim in the present work was to study the participation of the adenosine metabolism as well as the ERK pathway on adenosine uptake in different types of retinal cultures (mixed and purified glial cultures). Kinetic analysis in both cultures revealed that the uptake reached equilibrium after 30 min and presented two components. Incubation of cultures with S-(p-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole, different inhibitors of equilibrative nucleoside transporters (ENTs), produced a significant and concentration-dependent uptake reduction in both cultures. However, while dipyridamole presented similar maximal inhibitory effects in both cultures (although in different concentrations), the inhibition by NBTI was smaller in glial cultures than in mixed cultures, suggesting the presence of different transporters. Moreover, pre-incubation of [³H]-adenosine with adenosine deaminase (ADA) or adenosine kinase (ADK) inhibition with iodotubercidin promoted significant uptake inhibition in both cultures, indicating that the uptake is predominantly for adenosine and not inosine, and that taken up adenosine is preferentially directed to the synthesis of adenine nucleotides. In both cultures, the MEK inhibitors PD98059 or UO126, but not the inactive analog U0124, induced a significant and concentration-dependent uptake decrease. We have not observed any change in adenosine metabolism induced by MEK inhibitors, suggesting that this pathway is mediating a direct effect on NTs. Our results show the expression of different NTs in retinal cells in culture and that the activity of these transporters can be regulated by the ERK pathway or metabolic enzymes such as ADK which are then potential targets for regulation of Ado levels in normal or pathological conditions.     

Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

The ribose-modified chromophoric and fluorescent analog of ATP 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5′-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635–647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293–297). In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties. Their visible absorption and fluorescent properties were found to be quite similar. Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity. TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively. TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems. The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.     

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K_I (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Kinetic,thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37°C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37°C and the inhibition constants are 42.8 and 96.8 μM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27°C and uncompetitive at 37°C with inhibition constants of 56.4 and 30.0 μM, at respectively. The binding constant and enthalpy of binding, at 27°C are 45 μM, ? 64.5 kJ/mol and 61 μM, ? 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

An in vitro comparison of a new vinyl chalcogenide and sodium selenate on adenosine deaminase activity of human leukocytes

Selenium (Se) is a dietary essential trace element with important biological roles. Sodium selenate (Na₂SeO₄) is an inorganic Se compound used in human and animal nutrition that acts as precursor for selenoprotein synthesis. The organoselenium 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one (C₂₁H₂HOSe) is an α,β-unsaturated ketone functionalized vinyl chalcogenide that has been found as a potential tool in organic synthesis. Adenosine deaminase (ADA) is an important enzyme in the degradation of adenine nucleotides. In this study, we investigated the in vitro effects of both Se compounds on ADA activity and cell viability in leukocyte suspension (LS) of healthy donors (n = 12). We first observed an inhibition of ADA activity using 0.1 μM of 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one, and an increase in cellular viability when 30 μM were used. However, we did not observe alterations in the presence of sodium selenate. Moreover, both Se compounds did not alter lactate dehydrogenase activity and thiobarbituric acid reactive substance levels. These results suggest that the inhibition of ADA activity caused by α,β-unsaturated ketone may affect the adenosine levels in LS and modulate cell viability, attenuating conditions that involve the activation of the immune system.     

Studies on the adenosine deaminase-catalyzed conversion of adenosine and nucleoside prodrugs by different capillary electrophoresis modes

Four kinds of fast and efficient capillary electrophoresis modes, i.e., immobilized enzymatic reactor (IER), electrophoretically mediated microanalysis (EMMA), capillary zone electrophoresis (CZE), and micellar electrokinetic chromatography (MEKC), were first developed to study the adenosine deaminase (ADA)-catalyzed conversion of adenosine and nucleoside prodrugs, which is critical for releasing prodrugs into the intracellular compartment for phosphorylation. The enzyme-activated prodrug approach is a strategy that has been successfully employed to improve physicochemical and pharmacokinetic properties of potential therapeutic agents, especially in the search for antiviral nucleoside analogues. Adenosine, amino-ddG, and amino-D4G could be converted by ADA to different extents under our experimental conditions. Steady-state parameters K_m, V_max, and k_cat were also determined. The substrate efficiencies (k_cat/K_m) of adenosine, amino-ddG, and amino-D4G were 0.19 ± 0.01, 0.047 ± 0.005, and 0.017 ± 0.010 μM⁻¹ s⁻¹, respectively. The enzymatic reaction could be performed at a nanoliter scale and all manipulation steps were combined into a fully automated assay in on-line modes, which opened the possibilities of high-throughput screening of large libraries of synthetic nucleoside analogues for biological activity and a relative mechanism study of nucleoside and its analogues.     

Anti-inflammatory effects of purine nucleosides, adenosine and inosine, in a mouse model of pleurisy: evidence for the role of adenosine A2 receptors

Adenosine and its metabolite, inosine, have been described as molecules that participate in regulation of inflammatory response. The aim of this study was to investigate the effect of adenosine and inosine in a mouse model of carrageenan-induced pleurisy as well as the participation of adenosine receptors in this response. Injection of carrageenan into the pleural cavity induced an acute inflammatory response characterized by leukocyte migration, pleural exudation, and increased release of interleukin-1β and tumor necrosis factor-α in pleural exudates. The treatment with adenosine (0.3–100 mg/kg, i.p.) and inosine (0.1–300 mg/kg, i.p.) 30 min before carrageenan injection reduced significantly all these parameters analyzed. Our results also demonstrated that A_2A and A_2B receptors seem to mediate the adenosine and inosine effects observed, since pretreatment with selective antagonists of adenosine A_2A (ZM241385) and A_2B (alloxazine) receptors, reverted the inhibitory effects of adenosine and inosine in pleural inflammation. The involvement of A₂ receptors was reinforced with adenosine receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment, since its anti-inflammatory effects were reversed completely and partially with ZM241385 and alloxazine injection, respectively. Moreover, the combined treatment with subeffective dose of adenosine (0.3 mg/kg) and inosine (1.0 mg/kg) induced a synergistic anti-inflammatory effect. Thus, based on these findings, we propose that inosine contributes with adenosine to exert anti-inflammatory effects in pleural inflammation, reinforcing the notion that endogenous nucleosides play an important role in controlling inflammatory diseases. This effect is likely mediated by the activation of adenosine A₂ subtype receptors and inhibition of production or release of pro-inflammatory cytokines.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

L-[3H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.     

Sequestration of adenosine in crude extract from mouse liver and other tissues

Adenosine (1 μM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Sæbø, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333–340). These data point to a role of this protein in the sequestration of adenosine in crude extract.The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed.The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 μM.The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract.The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.     

Identification and characterization of human ribokinase and comparison of its properties with E. coli ribokinase and human adenosine kinase

The gene responsible for ribokinase (RK) in human/eukaryotic cells has not yet been identified/characterized. Blast searches with E. coli RK have identified a human protein showing significant similarity to the bacterial RK. The cDNA for this protein was expressed in E. coli and the recombinant protein efficiently phosphorylated ribose to ribose-5-phosphate using ATP, confirming its identity as RK. In contrast to ribose, the enzyme exhibited very little to no phosphorylation of D-arabinose, D-xylose, D-fructose and D-galactose. The catalytic activity of human RK was dependent upon the presence of inorganic phosphate, as observed previously for E. coli RK and mammalian adenosine kinases (AK). A number of activators and inhibitors of human AK, produced very similar effects on the human and E. coli RKs, indicating that the catalytic mechanism of RK is very similar to that of the AKs.     

2''-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery

2'-Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 microM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 micrograms/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the greater than 95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5-50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68-78% of control values in brain regions with normally high levels of activity and by 44-59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.     

Heme oxygenase-1-derived carbon monoxide stimulates adenosine triphosphate generation in human hepatocyte

Heme oxygenases cleave the pro-oxidant heme molecule into carbon monoxide, ferrous iron, and biliverdin, which is subsequently converted to bilirubin. Increasing the enzymatic activities of heme oxygenase by expression of its inducible isoform, heme oxygenase-1, protects hepatocyte from apoptosis. In the present study, we investigated the mechanisms involving in heme oxygenase-1-mediated cytoprotection. Heme oxygenase-1 could induce the expression of anti-apoptotic protein-Bcl-xL in human hepatocyte. This effect is associated with the activation of p38 MAPK signaling pathway. Carbon monoxide derived from heme oxygenase activities significantly increased adenosine triphosphate levels in hepatocyte that was essential for potentiation of the activation of p38 MAPK signaling. Our demonstration of the importance of the energy status to maximize an anti-apoptotic response provides a new insight into HO-mediated cytoprotection.     

Activation of adenosine A1 and A2A receptors modulates dopamine D2 receptor-induced responses in stably transfected human neuroblastoma cells

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.