Presence Of Adenosine Deaminase Research Articles

Lysophosphatidylcholine induces adenosine release from macrophages via TRPM7-mediated mitochondrial activation.

Even though macrophages have the potential to harm tissues through excessive release of inflammatory mediators, they play protective roles to maintain tissue integrity. In this study, we hypothesized that lysophosphatidylcholine (LPC), via G2A and A2B receptors, puts brakes on macrophages by the induction of adenosine release which could contribute to termination of inflammation. Mechanistically, LPC-induced PGE2 production followed by the activation of cAMP/protein kinase A (PKA) pathway which results in the activation of LKB1/AMPK signaling pathway leading to increasing Mg2+ influx concomitantly with an increase in mitochondrial membrane potential (MMP, Δψm) and ATP production. Then, ATP is converted to adenosine intracellularly followed by efflux via ENT1. In a parallel pathway, LPC-induced elevation of cytosolic calcium was essential for adenosine release, and Ca2+/calmodulin signaling cooperated with PKA to regulate ENT1 permeation to adenosine. Pharmacological blockade of TRPM7 and antisense treatment suppressed LPC-induced adenosine release and magnesium influx in bone marrow-derived macrophages (BMDMs). Moreover, LPC suppressed LPS-induced phosphorylation of connexin-43, which may counteract TLR4-mediated inflammatory response. Intriguingly, we found LPC increased netrin-1 production from BMDMs. Netrin-1 induces anti-inflammatory signaling via A2B receptor. In the presence of adenosine deaminase which removes adenosine in the medium, the chemotaxis of macrophages toward LPC was significantly increased. Hypoxia and metabolic acidosis are usually developed in a variety of inflammatory situations such as sepsis. We found LPC augmented hypoxia- or acidosis-induced adenosine release from BMDMs. These results provide evidence of LPC-induced brake-like action on macrophages by adenosine release via cellular magnesium signaling.

Emerald Jewel Wasp Venom Adenosine Deaminase Antagonizes Purinergic Signaling in the Cockroach Brain

Ampulex compressa injects venom directly into the cerebral ganglia of the cockroach (Periplaneta americana) to induce a reversable, but long‐term compliant state of hypokinesia, marked by an increase in escape threshold and a decrease in spontaneous walking. The venom is not paralytic as stung cockroaches can still stand, groom, eat and drink, and can even run if aggressively provoked. The venom is also not necrotic or cytotoxic, and despite its long‐lasting duration the venom’s effects are reversible, as the escape response returns to normal after several days. Previous work involved generation of a comprehensive venom apparatus transcriptome and venom proteome or “venome” and described several interesting signaling pathways that appear to be targeted by venom components. This includes adenosine deaminase (ADA), which may act to antagonize purinergic signaling in the brain.The substrates of venom ADA, adenosine (Ado) and adenosine monophosphate (AMP), are also the activating ligands of the cockroach adenosine receptor (AdoR). We hypothesize that the presence of ADA at the site of injection can reduce cell damage‐induced signaling by clearing Ado and AMP released from compromised cells. We have cloned, expressed, and characterized both venom ADA and cockroach brain AdoR and show that the presence of venom ADA can attenuate Ado induced calcium signaling in vitro. Antagonizing ADA or AdoR in vivo did not alter locomotory behavior. Thus, the action of ADA in the venom may be more neuroprotective than inhibitory of specific behaviors as is the case with other venom components.

Reynoutria Japonica from Traditional Chinese Medicine: A Source of Competitive Adenosine Deaminase Inhibitors for Anticancer.

Adenosine deaminase (ADA) is an important enzyme in purine metabolism and is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Traditional Chinese Herbal Medicine (TCHM) is widely used alone or in combination with chemotherapy to treat cancer, due to its ability to deliver a broad variety of bioactive secondary metabolites as promising sources of novel organic natural agents. In the present study, 29 varieties of medicinal plants were screened for the presence of ADA inhibitors. Extracts from Reynoutria japonica, Glycyrrhiza uralensis, Lithospermum erythrorhizon, Magnolia officinalis, Gardenia jasminoides, Stephania tetrandra, Commiphora myrrha, Raphanus sativus and Corydalis yanhusuo demonstrated strong ADA inhibition with rates greater than 50%. However, Reynoutria japonica possessed the highest ADA inhibitory activity at 95.26% and so was used in our study for isolating the ADA inhibitor to be further studied. Eight compounds were obtained and their structures were identified. The compound H1 had strong ADA inhibitory activity and was deduced to be emodin by 1H and 13C-NMR spectroscopic analysis with an IC50 of 0.629 mM. The molecular docking data showed that emodin could bind tightly to the active site of ADA. Our results demonstrated that emodin displayed a new biological activity which is ADA inhibitory activity with high cytotoxic activity against K562 leukemia cells. The bioactivity of cordycepin was significantly increased when used in combination with emodin. Emodin may represent a good candidate anti-cancer therapy and adenosine protective agent.

A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition.

Adenosine deaminase (ADA), able to catalyze the irreversible deamination of adenosine into inosine, can be found in almost all tissues and plays an important role in several diseases. In this work, we developed a label-free fluorescence method for the detection of adenosine deaminase activity and inhibition. In the presence of ADA, ATP has been shown to be hydrolyzed. The ATP aptamer was shown to form a G-quadruplex/thioflavin T (ThT) complex with ThT and exhibited an obvious fluorescence signal. However, the ATP aptamer could bind with ATP and exhibited a low fluorescence signal because of the absence of ADA. This assay showed high sensitivity to ADA with a detection limit of 1 U/L based on an SNR of 3 and got a good linear relationship within the range of 1–100 U/L with R2 = 0.9909. The LOD is lower than ADA cutoff value (4 U/L) in the clinical requirement and more sensitive than most of the reported methods. This technique exhibited high selectivity for ADA against hoGG I, UDG, RNase H and λexo. Moreover, this strategy was successfully applied for assaying the inhibition of ADA using erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and, as such, demonstrated great potential for the future use in the diagnosis of ADA-relevant diseases, particularly in advanced drug development.

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission.

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

Modulation of cGMP accumulation by adenosine A1 receptors at the hippocampus: Influence of cGMP levels and gender

Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100μM) and Bay 60-7550 (10μM), and cAMP accumulation, induced by Forskolin (20μM) and Rolipram (50μM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2U/ml) unmasked a CPA (0.03–300nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100μM) abolished the CPA (100nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP.

P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat cortical astrocytes.

Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca(2+) ]i . Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca(2+) levels through the PLC-PKC pathway. GABA transporters are a substrate for a number of Ca(2+) -related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial-enriched cultures expressed functional P2Y receptors, as evaluated through Ca(2+) imaging, being P2Y1 the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT-1 or GAT-3 transporters, decreasing the Vmax kinetic constant. ATP-induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine-mediated effect. This, was mimicked by a specific agonist for the P2Y1,12,13 receptor (2-MeSADP). The effect of 2-MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y1 selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2-MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA-AM) or where calcium stores were depleted (α-cyclopiazonic acid, CPA). In conclusion, P2Y1 receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y1 -mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA.

Ultrastructural and Enzymatic Activity of Membranous Vesicles Isolated from Canine Seminal Plasma

The objectives of this study were to verify the presence of membranous vesicles (MV) in canine seminal plasma by mean of transmission electron microscopy (TEM), to describe the ultrastructural characteristics and to identify some enzymatic activity associated with them. Semen samples, collected by digital manipulation from dogs with proven fertility, were pooled and used for membrane vesicles preparation according to conventional procedures. TEM observations showed the existence of vesicular membranous structures of more or less spherical shape with different sizes. These vesicles were surrounded by a single-, double- or multiple-layered laminar membranes. The mean vesicle diameter was 117.6 ± 86.9 nm ranging from 24.4 to 716.6 nm. Enzyme activity determinations showed the presence of adenosine deaminase, 5'-nucleotidase, ADPase, ATPase, dipeptilpeptidase IV, alkaline phosphatase, total acid phosphatase and prostatic acid phosphatase, while the aminopeptidase activity was absent. In conclusion, results of this study, compatible with results from other mammals, showed for the first time the presence of MV, their ultrastructural and enzymatic characteristics in dog seminal plasma.

Inhibition of rabbit cardiac adenylate cyclase by theophylline.

Theophylline inhibits basal adenylate cyclase activity as well as cyclase stimulated by sodium chloride, sodium fluoride, GTP or 5'-guanylimidodiphosphate. This inhibition, is dose-dependent and shows non-competitive inhibition, with respect to MgATP. The presence of adenosine deaminase does not alter the effect of theophylline. The inhibition produced by theophylline is not additive with that due to 2'-deoxyadenosine 3'-monophosphate (a P-site agonist). It is suggested that theophylline may act at the P-site to reduce adenylate cyclase activity.

Adenosine Potentiates Human Lung Mast Cell Tissue Plasminogen Activator Activity

We investigated whether adenosine, a potent contributor to the regulation of pulmonary function, can modulate human lung mast cell (HLMC) fibrinolytic activity. Tissue plasminogen activator (tPA) activity and tPA transcript expression levels from a human mast cell line (HMC-1) and HLMC were monitored following adenosine application. Adenosine potentiated mast cell tPA activity and tPA gene expression in a dose-dependent manner. Adenosine effects were abolished in the presence of adenosine deaminase. HMC-1 cells and HLMC predominantly expressed adenosine A(2A) and A(2B) receptor transcripts (A(2B) ≈ A(2A) > A(3) >> A(1)). Pharmacological and signaling studies suggest that the A(2A) receptor is the major subtype accounting for adenosine-induced mast cell tPA activity. Finally, the supernatant from HMC-1 cells and HLMC treated with adenosine (for 24 h) significantly increased fibrin clot lysis, whereas ZM241385, an A(2A) receptor antagonist, abolished this effect. To our knowledge, this study provides the first data to demonstrate the potentiating effect of adenosine on mast cell tPA activity and fibrin clot lysis.

P182 Adenosine potentiates human mast cell fibrinolytic activity

We investigated whether adenosine, a potent contributor to the regulation of pulmonary function, can modulate human lung mast cell (HLMC) fibrinolytic activity. Tissue plasminogen activator (tPA) activity and tPA transcript...

Resistance to the antilipolytic effect of insulin in adipocytes of African-American compared to Caucasian postmenopausal women

High fatty acid (FA) flux is associated with systemic insulin resistance, and African-American (AA) women tend to be more insulin resistant. We assessed possible depot and race difference in the antilipolytic effect of insulin in adipocytes isolated from abdominal (Abd) and gluteal (Glt) subcutaneous (sc) adipose tissue of overweight, postmenopausal AA and Caucasian (C) women. Percent body fat, fasting insulin, visceral adiposity, and adipocyte size was higher in AA women. Disinhibited lipolysis (presence of adenosine deaminase) per unit adipocyte surface area was similar in Abd and Glt and in AA and C. However, rates of 'basal' [submaximal phenylisopropyl adenosine (PIA)-suppressed] and insulin-suppressed lipolysis were higher in Abd of AA compared with C women even after adjustment for percent fat and visceral fat area. The race difference in rates of PIA- and insulin-suppressed lipolysis in AA were correlated with their hyperinsulinemia, but AA race, independent of fasting insulin, was associated with lower responsiveness (percent suppression) to submaximal insulin concentrations, although sensitivity (ED50) was not affected. Overall, these data are consistent with the hypothesis that decreased responsiveness of Abd adipocytes to antilipolytic effectors may contribute to higher FA availability and thereby to racial differences in insulin resistance.

Allium sativum L. extract prevents methyl mercury-induced cytotoxicity in peripheral blood leukocytes (LS)

Adenosine deaminase (ADA) is involved in purine metabolism and plays a significant role in the immune system. The focus of this investigation was to examine the effects of low concentrations of organic mercury on ADA activity in human leukocytes and to investigate the relationship between these effects and cell death. We have examined the protective potential effects of Allium sativum extract (GaE) against Methylmercury (MeHg)-induced cytotoxic effects on human leucocytes under in vitro conditions. MeHg (0.05–10 μM) significantly decreased leukocyte viability (58.97% for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and 51.67% for Alamar Blue (AB) and this decrease was positively correlated to the MeHg-induced inhibition of ADA activity. N-acetylcysteine (NAC) and GaE prevented both the MeHg-induced cytotoxic effects on leukocytes according to MTT and AB assays and the effects on the ADA activity. The present results suggest that the protective effects of GaE against MeHg-induced leukocyte damage is related to the removal of oxidant species generated in the presence of MeHg due to the antioxidant efficacy of garlic constituents. It is important to point out that the intense presence of ADA in Leukocyte suspension (LS) highlights the relevant effects in the immune system and in vitro cytotoxicity of MeHg exposure.

Evidence of a new phosphoryl transfer system in nucleotide metabolism

Crude rat liver extract showed AMP-AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20). The activity was duplicated using the same enzymes purified from recombinant sources. The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the presence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis. The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interactions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP. The proposed mechanism suggests a physiological role for the enzymes and for the AMP-AMP phosphotransferase reaction under conditions of extreme energy drain (such as hypoxia or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency.

Adenosine analogs and electromagnetic fields inhibit prostaglandin E 2 release in bovine synovial fibroblasts

To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E(2) (PGE(2)) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N6-cyclohexyladenosine (CHA, A(1)), 2-[p-(2-carboxyethyl)-phenetyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, A(2A)), 5'-N-ethylcarboxamidoadenosine (NECA, non-selective), N6-(3-iodobenzyl)2-chloroadenosine-5'-N-methyluronamide (Cl-IB-MECA, A(3)). SFs were treated with TNF-alpha (10 ng/ml) and lipopolysaccharide (LPS) (1 microg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-alpha- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75 Hz, 1.5 mT) during the period in culture (24h). PGE(2) release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. TNF-alpha and LPS stimulated PGE(2) release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE(2) production. EMFs inhibited PGE(2) production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE(2) release induced by CHA, CGS and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE(2) levels were associated to modification of COX-2 expression. This study supports anti-inflammatory activities of A(1) and A(2A) adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A(2A) receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine

During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A(1) receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A(1) and A(2A) receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O(2)) caused a significant decrease in adenosine A(1) receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A(2A) receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1alpha expression in C6 cells. However, HIF-3alpha, CREB and CREM were decreased. Adenosine A(1) and A(2A) receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A(1) receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A(2A) receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A(1) receptor activation is involved.

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor‐positive and ‐negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Different roles of adenosine A 1, A 2A and A 3 receptors in controlling kainate-induced toxicity in cortical cultured neurons

Adenosine is a neuromodulator that can control brain damage through activation of A 1, A 2A and A 3 receptors, which are located in both neurons and other brain cells. We took advantage of cultured neurons to investigate the role of neuronal adenosine receptors in the control of neurotoxicity caused by kainate and cyclothiazide. Both A 1, A 2A and A 3 receptors were immunocytochemically identified in cortical neurons. Activation of A 1 receptors with 100 nM CPA did not modify the extent of neuronal death whereas the A 1 receptor antagonist, DPCPX (50 nM), attenuated neurotoxicity by 28 ± 5%, and effect similar to that resulting from the removal of endogenous adenosine with 2 U/ml of adenosine deaminase (27 ± 3% attenuation of neurotoxicity). In the presence of adenosine deaminase, DPCPX had no further effect and CPA now exacerbated neurotoxicity by 42 ± 4%. Activation of A 2A receptor with 30 nM CGS21680 attenuated neurotoxicity by 40 ± 8%, an effect prevented by the A 2A receptor antagonists, SCH58261 (50 nM) or ZM241385 (50 nM), which by themselves were devoid of effect. Finally, neither A 3 receptor activation with Cl-IB-MECA (100–500 nM) nor blockade with MRS1191 (5 μM) modified neurotoxicity. These results show that A 1 receptor activation enhances and A 2A receptor activation attenuates neurotoxicity in cultured cortical neurons, indicating that these two neuronal adenosine receptors directly control neurodegeneration. Interestingly, the control by adenosine of neurotoxicity in cultured neurons is similar to that observed in vivo in newborn animals and is the opposite of what is observed in adult brain preparations where A 1 receptor activation and A 2A receptor blockade are neuroprotective.

331. Lentivirus-Mediated Ex Vivo Gene Therapy in ADA-Deficient SCID Mice

Adenosine deaminase (ADA) deficiency is a severe combined immunodeficiency (SCID) characterized by systemic toxicity and profoundly impaired lymphoid cell development and functions due to the accumulation of purine metabolites. ADA-deficient (ADA KO) mice die within 3 weeks of age due to metabolic toxicity and can be rescued by enzyme replacement therapy with PEG-ADA. The aim of our study was to investigate the efficacy of gene therapy (GT) in comparison to bone marrow transplantation (BMT) in the ADA KO mouse model. A SIN HIV-1-based lentiviral vector encoding human ADA cDNA under the control of PGK promoter was constructed. ADA KO mice were maintained short-term on PEG-ADA and then transplanted, after non-myeloablative conditioning either with WT BM cells or with ADA KO BM cells transduced ex vivo. We first showed that BMT with donor WT cells rescued mice from lethality, restored T-cell and B-cell counts in the peripheral blood and led to normal proliferation after T or B cells specific stimulation. Similarly, GT with transduced BM cells rescued ADA KO mice from lethality and restored their growth to normal levels. Quantitative PCR for donor cells (Y chromosome in sex-mismatched transplant) and vector-positive cells showed the presence of ADA transduced cells in the peripheral circulation of transplanted ADA KO mice (30|[ndash]|40% 5 months post-GT). Two months after GT, ADA activity in red blood cells reached normal levels and dAXP toxic metabolites decreased to undetectable levels. GT resulted in normal lymphoid differentiation in primary and secondary lymphoid organs, as well as restoration of T-cell and B-cell in vitro proliferative responses after stimulation with anti-CD3/anti-CD28 and anti-IgM antibody, respectively. Finally, serum levels of IgM and IgG were comparable between GT and BMT mice. These studies demonstrate that lentiviral-mediated ADA gene transfer associated with non-myeloablative conditioning corrects both the metabolic and the immunological defects in the ADA KO animal model. We are currently evaluating the long-term safety related to the use of hematopoietic stem/progenitor cells transduced with lentiviral vectors.

Modulating effect of adenosine deaminase on function of adenosine A1 receptors1

To study the modulating effect of adenosine deaminase (ADA) on the adenosine A1 receptor (A1R) in HEK293 cells stably expressing the human A1R. cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A1R was studied by using [3H]DPCPX binding assay and an intracellular calcium assay. HEK293 cells stably expressing human A1R were obtained. Saturation studies showed that the K(D) value and B(max) value of [3H]DPCPX were 1.6+/-0.2 nmol/L and 1.819+/-0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3+/-0.2 nmol/L and 1.992+/-0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that the K(D) value and B(max) value of [3H]DPCPX were unaffected by ecto-ADA. In the case of [3H]DPCPX competition curves obtained from intact cells or membranes, A1R agonist CCPA/[3H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K(H) value of 0.74 (0.11+/-4.8) nmol/L (intact cells) or 1.8 (0.25+/-10) nmol/L (membrane) and a K(L) value of 0.94 (0.62+/-1.41) micromol/L (intact cells) or 0.77 (0.29+/-0.99) micromol/L (membrane). The K(L) value is not significantly different from the IC50 value of 0.84(0.57+/-1.23) micromol/L (intact cells) or 0.84 (0.63+/-1.12) micromol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[3H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC50 value of CPA were 10 (5+/-29) nmol/L and 94 (38+/-229) nmol/L in the presence or absence of ecto-ADA, respectively. A1R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A1 receptor in HEK293 cells.