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Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine.

ABSTRACT: Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-? production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

SUBMITTER: Pettengill M

PROVIDER: S-EPMC3779727 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine.

Pettengill Matthew M Robson Simon S Tresenriter Megan M Millán José Luis JL Usheva Anny A Bingham Taiese T Belderbos Mirjam M Bergelson Ilana I Burl Sarah S Kampmann Beate B Gelinas Laura L Kollmann Tobias T Bont Louis L Levy Ofer O

The Journal of biological chemistry 20130729 38

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood ...[more]

PMID: 23897810

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Project description:ObjectiveAdenosine deaminase (ADA) plays an important role in immune response, which includes two isoenzymes: ADA1 and ADA2. This study aims to explore the roles of ADA1 and ADA2 in cancers.MethodsHuman Protein Atlas (HPA) and Gene Expression Profiling Interactive Analysis (GEPIA2) databases were used to analyze the mRNA expression of ADA1 and ADA2 in human normal cells and tumor tissues. The enzyme assay was used to detect the ADA1 and ADA2 activities in serum from cancer patients. The Kaplan-Meier (KM) plotter was used to analyze the prognostic value of ADA1 and ADA2. TIMER2.0 was used to explore how ADA1 and ADA2 correlate with immune infiltration and immune checkpoints. cBioPortal database was used to investigate the mutations of ADA1 and ADA2. LinkedOmics was used to screen the ADA1 and ADA2 expression-related genes.ResultsADA1 was significantly increased in several tumor tissues, including cholangiocarcinoma (CHOL), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), thymoma (THYM), and uterine carcinosarcoma (UCS). ADA2 expression was significantly increased in esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), acute myeloid leukemia (LAML), OV, PAAD, skin cutaneous melanoma (SKCM), and stomach adenocarcinoma (STAD). There were no significant changes in serum ADA1 activities in most cancers, while serum ADA2 activities were increased in most cancers. For prognosis, high ADA1 expression was associated with the poor survival in several cancers, including esophageal squamous cell carcinoma (ESCC), HNSC, KIRC, kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and uterine corpus endometrial carcinoma (UCEC). However, high ADA2 expression showed a favorable prognosis in breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), HNSC, KIRC, KIRP, LUAD, OV, PAAD, sarcoma, and THYM. ADA1 showed a moderate positive correlation with multiple infiltrating immune cells in most cancers. ADA2 was positively correlated with B cells, CD8 T cells, monocytes/macrophages, and dendritic cells (DCs) and was strongly negatively correlated with myeloid-derived suppressor cells. Function analysis showed that ADA1 expression-related genes were mainly enriched in cell division biological progression. However, ADA2-related genes were mainly associated with immune response.ConclusionAs isoenzymes, ADA1 and ADA2 showed opposite prognostic values and different correlative patterns with immune infiltrating. These data demonstrated the distinct roles of ADA1 and ADA2 in cancer. ADA2 might act as a protective factor in cancer.

Project description:Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment.

Project description:RATIONALE:Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. OBJECTIVES:1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. METHODS AND RESEARCH:Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (?80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4?mol/L vs 12.9?mol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9?mol/L vs 2.7?mol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). CONCLUSIONS:Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to impaired capacity to clear adenosine monophosphate. AP supplementation improves serum clearance of adenosine monophosphate to adenosine. These findings represent a potential therapeutic mechanism for alkaline phosphatase infusion during cardiac surgery. NEW AND NOTEWORTHY:We identify alkaline phosphatase (AP) as the primary soluble ectonucleotidase in infants undergoing cardiopulmonary bypass and show decreased capacity to clear AMP when AP activity decreases post-bypass. Supplementation of AP ex vivo improves this capacity and may represent the beneficial therapeutic mechanism of AP infusion seen in phase 2 studies.

Project description:Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1? and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 ?M of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1?, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Project description:RationaleHypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood.ObjectiveWe sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression.Methods and resultsUsing high-throughput quantitative reverse-transcription polymerase chain reaction profiling, we discovered that the expression level of 5'-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II-infused mice, an animal model of hypertensive nephropathy. Genetic and pharmacological studies in mice revealed that elevated CD73-mediated excess renal adenosine preferentially induced A2B adenosine receptor (ADORA2B) production and that enhanced kidney ADORA2B signaling contributes to angiotensin II-induced hypertension. Similarly, in humans, we found that CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared with normal individuals and were further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 production in a hypoxia-inducible factor-α-dependent manner and underlies the pathogenesis of the disease. Finally, we revealed that hypoxia-inducible factor-α is an important factor responsible for angiotensin II-induced CD73 and ADORA2B expression at the transcriptional level.ConclusionsOverall, our studies reveal that angiotensin II-induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling-mediated endothelin-1 induction in a hypoxia-inducible factor-α-dependent manner. The inhibition of excess adenosine-mediated ADORA2B signaling represents a novel therapeutic target for the disease.

Dataset Information

Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine.

Publications

Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine.

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